Newborn screening for primary immunodeficiencies, cystinosis, and wilson disease

Abstract

To provide multiplexed assays that can be used to screen newborns for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson disease (WD).SOLUTION: The current disclosure describes peptides associated with each of disorders that can be reliably detected and quantified using peptide immunoaffinity enrichment coupled to selected reaction monitoring mass spectrometry (immuno-SRM). The current disclosure also provides high affinity antibodies that can be used to enrich for the peptides, as well as methods of tagging the peptides to increase the throughput of an assay.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 742,161, filed October 5, 2018, which is incorporated by reference in its entirety as if fully set forth herein.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. 1R21HD069890-01A1, Grant No. AI106784-01A1, and Grant No. R01AI123135, all awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] SEQUENCE LISTING STATEMENT The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The text file containing the sequence listing is named 2698490_ST25.txt. The text file is 146KB, was created on October 1, 2019, and has been submitted electronically via EFS-Web.

[0004] Field of Disclosure The current disclosure provides newborn screening for primary immunodeficiencies, cystinosis, and Wilson's disease. Newborn screening may detect these disorders from dried blood spots routinely collected already at birth. Early detection of these disorders would significantly improve patient outcomes, as each can be fatal once symptoms become evident. [Background technology]

[0005] Background of the Disclosure There are many diseases with effective treatments available. However, for many of these illnesses, once symptoms appear, the disease is already fatal or has caused irreversible damage. Examples of such diseases include primary immunodeficiency syndrome (PIDD), cystinosis, and Wilson's disease (WD).

[0006] PIDD is a group of life-threatening genetic or inherited congenital disorders characterized by a deficiency, impairment, or dysfunction of the immune system. Examples of PIDD include severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked agammaglobulinemia (XLA).

[0007] SCID is a group of rare disorders caused by mutations in different genes involved in the development and function of infection-fighting immune cells such as T cells and B cells. SCID patients are usually affected by severe bacterial, viral, or fungal infections early in life and often suffer from lung scarring, chronic diarrhea, and growth failure. The condition is fatal, usually within the first 1-2 years of life, unless the infant receives immune-restoring treatments such as hematopoietic stem cell transplants, gene therapy, or enzyme therapy.

[0008] WAS is an immune deficiency that mainly affects men and is characterized by a decreased ability to form platelets due to a decrease in the number and size of platelets. This platelet abnormality is usually present from birth and also leads to easy bruising and symptoms of prolonged bleeding following minor trauma, which can be life-threatening in some cases. Individuals with WAS are also at increased risk of developing infections, autoimmune disorders, and certain types of cancer. In WAS patients, immunoglobulin infusions and antibiotics can be used to prevent infections, and in severe cases, stem cell transplants can provide a cure. Gene therapy is also being explored as another alternative treatment.

[0009] XLA is an inherited immunodeficiency disorder that mainly affects men. With XLA, the body may produce the antibodies needed to defend against bacteria, viruses, and other foreign substances. Children with XLA are usually healthy for the first one to two months of life, as they are protected by maternal antibodies acquired before birth. However, after this time, the maternal antibodies are eliminated from the body, and the affected child suffers from recurrent infections that lead to organ damage. Once the disease is detected, standard treatment is offered to strengthen the immune system by immunoglobulin infusions, and antibiotics are administered to fight bacterial infections. However, by the time a diagnosis is made, organ damage may already have occurred.

[0010] Cystinosis is a rare metabolic disorder in which the amino acid cystine enters cells but cannot exit due to a defect in a cystine-specific transporter called cystinosin. Due to the defect in cystine transport, cells accumulate cystine in crystals in lysosomes, organelles within the cells, causing premature cell death. Cystinosis slowly destroys organs in the body, including the kidneys, liver, eyes, muscles, and brain. Children with cystinosis in infancy appear normal at birth, but by 9 to 10 months of age, symptoms such as excessive thirst and urination, as well as failure to thrive, appear. Cysteamine is a treatment that slows the progression of cystinosis by removing cystine from cells. However, if cysteamine treatment is not started early enough in the disease's progression, kidney transplantation is usually required.

[0011] WD is a copper transport disorder in which copper accumulates in vital organs such as the liver, kidneys, and brain. The progression of WD is slow and the range of clinical symptoms is wide, making it difficult to diagnose. Thus, despite the fact that treatment is available, many patients still exhibit irreversible multi-organ damage at the time of diagnosis.

[0012] Treatment of each of the above diseases would be significantly enhanced if a diagnosis could be made before clinical symptoms appear. Newborn screening (NBS) is the standard public preventive mandatory screening test for the 4 million infants born each year in the United States. NBS typically involves a blood test performed 24-48 hours after birth. Screening uses a few drops of blood from the heel of the newborn that are placed on filter paper. The paper containing the dried blood spot (DBS) may be stored until testing is performed.

[0013] To perform an NBS evaluation, a punch of dried blood is taken from the DBS and laboratory tests are performed to detect the presence or absence of certain substances in the blood (called markers or biomarkers) that indicate disorders that are not evident at birth but that may cause serious health problems later in life. The disorders screened for vary from condition to condition, but most conditions screen for phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease. NBS has proven highly effective in improving patient outcomes and avoiding long-term disability for affected individuals while also reducing health care costs.

[0014] SCID has been recently added to the NBS panel; however, current molecular testing for SCID does not reliably identify all patients with SCID-like disorders such as late-onset and late-onset adenosine deaminase (ADA) deficiency, ZAP70 deficiency, and MHC class II deficiency. Unfortunately, there are currently no widespread, cost-effective screening methods available for most of the other life-threatening but treatable "non-SCID" immunodeficiencies.

[0015] One reason that there are many disorders that would be beneficial to screen for is that clinical tests have not been able to reliably measure the marker associated with the disorder due to a lack of available tests. One reason this cannot be done is that the disorder is associated with the absence of a marker or very low levels of the marker. In this scenario, it may be difficult to reliably detect the absence of a marker due to the "background noise" associated with many testing techniques that may mask clinically relevant results.

[0016] Peptide immunoaffinity enrichment combined with selected reaction monitoring mass spectrometry (immuno-SRM) is a method that allows accurate quantification of low abundance markers. The use of immuno-SRM generally involves the following steps: (i) selection of a marker that indicates the presence or absence of a disorder; (ii) if the marker is present, treating the biological sample containing the marker with an enzyme to digest all proteins in the biological sample into smaller fragments called peptides; (iii) enrichment of peptides derived from the selected marker, and (iv) analysis and quantification of the enriched peptides of interest in a mass spectrometer.

[0017] Recently, Jung et al. (J. Proteome Res. 2017;16:862-871) described the use of immuno-SRM to quantify the protein marker ATP7B associated with WD from DBS. This is the first demonstration that immuno-SRM can be used to reliably detect low-abundance markers from preserved DBS, opening the possibility of using this approach to screen a broader panel of disorders.

[0018] Although Jung and colleagues demonstrated the feasibility of using DBS in an immuno-SRM assay to distinguish between WD affected and unaffected individuals, many aspects of such an assay will depend on the disorder being diagnosed, the biomarkers available for each disorder, the ability to develop molecular entities that can enrich for peptides of interest, and the behavior of each peptide of interest in a mass spectrometer. All of these and other aspects will need to be carefully considered and experimented with to realize an assay that can reliably detect NBS panel disorders using DBS before clinical symptoms appear. [Prior art documents] [Non-patent literature]

[0019] [Non-Patent Document 1] Jung et al. (J. Proteome Res. 2017;16:862-871) Summary of the Invention

[0020] Summary of the Disclosure The current disclosure describes the development of a multiplexed assay that can be used to screen newborns for Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), X-linked Agammaglobulinemia (XLA), Cystinosis, and Wilson's Disease (WD). The assay may significantly improve outcomes for affected individuals by reliably diagnosing these disorders before the devastating and often fatal clinical symptoms manifest. The assay may detect the presence or absence of markers associated with these disorders using dried blood spots (DBS) that are already routinely collected as part of existing newborn screening (NBS) procedures.

[0021] The present disclosure describes peptides associated with each disorder that can be reliably detected and quantified using peptide immunoaffinity enrichment combined with selected reaction monitoring mass spectrometry (immuno-SRM). The present disclosure also provides high affinity antibodies that can be used to enrich the peptides, and methods for tagging the peptides to increase assay throughput. [Brief description of the drawings]

[0022] [Figure 1-1] Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 1-2] Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 1-3]Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 1-4] Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 1-5] Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 1-6]Protein targets and peptide sequences used for peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM-MS) to diagnose severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and Wilson's disease (WD). Total mass, parent ion mass, and daughter ion mass are also shown. ++ indicates a doubly charged parent ion. The ion type of the daughter ion is in brackets. *Total mass and parent ion mass when detecting irregular terminal sequences with a leading K amino acid residue. [Figure 2A] Figures 2A-2E. Response curves for peptides measured by a multiplexed immunomultiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in one mass spectrometry (MS) run, this type of analysis is called multiple reaction monitoring (MRM). With MRM analysis, multiple proteins and multiple regions of a protein (signature peptides) can be monitored in one mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of the heavy peptide concentration measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve allows the linear range and sensitivity of the assay to be determined. Each data point is plotted as a grey box and the linear regression is plotted as a line. The regression fit parameters are reported in the corners of each plot. Each plot is weighted 1 / x. (Figure 2A) BTK 407; (Figure 2B) BTK 545; (Figure 2C) CD3ε 197; (Figure 2D) WASp 274; (Figure 2E) WASp289. [Figure 2B]Figures 2A-2E. Response curves for peptides measured by a multiplexed immunomultiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in one mass spectrometry (MS) run, this type of analysis is called multiple reaction monitoring (MRM). With MRM analysis, multiple proteins and multiple regions of a protein (signature peptides) can be monitored in one mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of the heavy peptide concentration measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve allows the linear range and sensitivity of the assay to be determined. Each data point is plotted as a grey box and the linear regression is plotted as a line. The regression fit parameters are reported in the corners of each plot. Each plot is weighted 1 / x. (Figure 2A) BTK 407; (Figure 2B) BTK 545; (Figure 2C) CD3ε 197; (Figure 2D) WASp 274; (Figure 2E) WASp289. [Figure 2C] Figures 2A-2E. Response curves for peptides measured by a multiplexed immunomultiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in one mass spectrometry (MS) run, this type of analysis is called multiple reaction monitoring (MRM). With MRM analysis, multiple proteins and multiple regions of a protein (signature peptides) can be monitored in one mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of the heavy peptide concentration measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve allows the linear range and sensitivity of the assay to be determined. Each data point is plotted as a grey box and the linear regression is plotted as a line. The regression fit parameters are reported in the corners of each plot. Each plot is weighted 1 / x. (Figure 2A) BTK 407; (Figure 2B) BTK 545; (Figure 2C) CD3ε 197; (Figure 2D) WASp 274; (Figure 2E) WASp289. [Figure 2D]Figures 2A-2E. Response curves for peptides measured by a multiplexed immunomultiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in one mass spectrometry (MS) run, this type of analysis is called multiple reaction monitoring (MRM). With MRM analysis, multiple proteins and multiple regions of a protein (signature peptides) can be monitored in one mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of the heavy peptide concentration measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve allows the linear range and sensitivity of the assay to be determined. Each data point is plotted as a grey box and the linear regression is plotted as a line. The regression fit parameters are reported in the corners of each plot. Each plot is weighted 1 / x. (Figure 2A) BTK 407; (Figure 2B) BTK 545; (Figure 2C) CD3ε 197; (Figure 2D) WASp 274; (Figure 2E) WASp289. [Figure 2E] Figures 2A-2E. Response curves for peptides measured by a multiplexed immunomultiple reaction monitoring (MRM) assay. When multiple parent ions are monitored in one mass spectrometry (MS) run, this type of analysis is called multiple reaction monitoring (MRM). With MRM analysis, multiple proteins and multiple regions of a protein (signature peptides) can be monitored in one mass spectrometry run. The response curve plots the heavy:light peak area ratio as a function of the heavy peptide concentration measured in a background matrix of digested proteins extracted from dried blood spots (DBS). This curve allows the linear range and sensitivity of the assay to be determined. Each data point is plotted as a grey box and the linear regression is plotted as a line. The regression fit parameters are reported in the corners of each plot. Each plot is weighted 1 / x. (Figure 2A) BTK 407; (Figure 2B) BTK 545; (Figure 2C) CD3ε 197; (Figure 2D) WASp 274; (Figure 2E) WASp289. [Figure 3A]Figures 3A-E. MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) CD3ε 197; (Figure 3B) BTK 407; (Figure 3C) BTK 545; (Figure 3D) WASp 274; (Figure 3E) WASp 289. [Figure 3B] Figures 3A-E. MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) CD3ε 197; (Figure 3B) BTK 407; (Figure 3C) BTK 545; (Figure 3D) WASp 274; (Figure 3E) WASp 289. [Figure 3C] Figures 3A-E. MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) CD3ε 197; (Figure 3B) BTK 407; (Figure 3C) BTK 545; (Figure 3D) WASp 274; (Figure 3E) WASp 289. [Figure 3D] Figures 3A-E. MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) CD3ε 197; (Figure 3B) BTK 407; (Figure 3C) BTK 545; (Figure 3D) WASp 274; (Figure 3E) WASp 289. [Figure 3E] Figures 3A-E. MRM traces of internal standard (left panel) and endogenous (right panel) signature peptides: (Figure 3A) CD3ε 197; (Figure 3B) BTK 407; (Figure 3C) BTK 545; (Figure 3D) WASp 274; (Figure 3E) WASp 289. [Figure 4A] Figures 4A-D. Inter-laboratory correlation of measured PIDD peptide concentrations. (Figure 4A) WASp 274; (Figure 4B) CD3ε 197; (Figure 4C) BTK 407; (Figure 4D) ATP7B 1056. [Figure 4B] Figures 4A-D. Inter-laboratory correlation of measured PIDD peptide concentrations. (Figure 4A) WASp 274; (Figure 4B) CD3ε 197; (Figure 4C) BTK 407; (Figure 4D) ATP7B 1056. [Figure 4C]Figures 4A-D. Inter-laboratory correlation of measured PIDD peptide concentrations. (Figure 4A) WASp 274; (Figure 4B) CD3ε 197; (Figure 4C) BTK 407; (Figure 4D) ATP7B 1056. [Figure 4D] Figures 4A-D. Inter-laboratory correlation of measured PIDD peptide concentrations. (Figure 4A) WASp 274; (Figure 4B) CD3ε 197; (Figure 4C) BTK 407; (Figure 4D) ATP7B 1056. [Diagram 5] Interlaboratory analytical validation of WASp289. [Figure 6A] Figures 6A-E. Differences in signature peptide levels (Pt) between patients: (Figure 6A) BTK 545; (Figure 6B) BTK 407; (Figure 6C) CD3ε 197; (Figure 6D) WASp 274; (Figure 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). ****p<0.0001, *p<0.05. [Figure 6B] Figures 6A-E. Differences in signature peptide levels (Pt) between patients: (Figure 6A) BTK 545; (Figure 6B) BTK 407; (Figure 6C) CD3ε 197; (Figure 6D) WASp 274; (Figure 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). ****p<0.0001, *p<0.05. [Figure 6C] Figures 6A-E. Differences in signature peptide levels (Pt) between patients: (Figure 6A) BTK 545; (Figure 6B) BTK 407; (Figure 6C) CD3ε 197; (Figure 6D) WASp 274; (Figure 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). ****p<0.0001, *p<0.05. [Figure 6D]Figures 6A-E. Differences in signature peptide levels (Pt) between patients: (Figure 6A) BTK 545; (Figure 6B) BTK 407; (Figure 6C) CD3ε 197; (Figure 6D) WASp 274; (Figure 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). ****p<0.0001, *p<0.05. [Figure 6E] Figures 6A-E. Differences in signature peptide levels (Pt) between patients: (Figure 6A) BTK 545; (Figure 6B) BTK 407; (Figure 6C) CD3ε 197; (Figure 6D) WASp 274; (Figure 6E) WASp 289 (SCID: n=3, WAS: n=11, BTK: n=26) and normal controls (NC, n=40). ****p<0.0001, *p<0.05. [Figure 7-1] ATP7B1056 signature peptide concentrations. [Figure 7-2] ATP7B1056 signature peptide concentrations. [Figure 7-3] ATP7B1056 signature peptide concentrations. [Figure 8-1] Quantification of signature peptides in normal controls from a blinded cohort study. [Figure 8-2] Quantification of signature peptides in normal controls from a blinded cohort study. [Figure 9-1] Signature peptide concentrations in a blinded patient cohort study. [Figure 9-2] Signature peptide concentrations in a blinded patient cohort study. [Figure 9-3] Signature peptide concentrations in a blinded patient cohort study. [Figure 10A] Receiver operating characteristic (ROC) plots showing the diagnostic performance of immuno-SRM for PIDD. (FIG. 10A) ROC plots for BTK 407, BTK 545, CD3ε 197, WASp 274, and WASp 289. True and false positive rates are plotted for increasingly stringent cutoff values. Lines of identity indicate tests unable to distinguish between cases and controls. [Figure 10B] Receiver operating characteristic (ROC) plots showing the diagnostic performance of immuno-SRM for PIDD. (FIG. 10B) Area under the curve (AUC) values ​​and p-values ​​for peptides shown in FIG. 10A. [Figure 11-1] Signature peptide levels in DBS obtained from the Washington State Newborn Screening Laboratory. [Figure 11-2] Signature peptide levels in DBS obtained from the Washington State Newborn Screening Laboratory. [Figure 12-1] Proportions of signature peptides to ATP7B peptides and patient diagnoses in a blinded cohort study. [Figure 12-2] Proportions of signature peptides to ATP7B peptides and patient diagnoses in a blinded cohort study. [Figure 13] Concentrations of CTNS and SHPK proteins in patient and normal control samples. Samples outlined in black have confirmed mutation status, whereas samples outlined in grey are predicted to be homozygous for the 57 kb deletion. Samples CTNS00001-CTNS00013 were blinded and predicted genotypes are based on protein levels demonstrated by immuno-SRM. [Figure 14] Comparison of signal-to-noise (S / N) ratios achieved using both the microflow ion key detection method and the new nanoflow method of CTNS 115. Nanoliter / minute flow rates boost the signal generated by the CTNS115 target peptide. [Figure 15] DBS CTNS 115 and SHPK 363 concentrations were measured and genotypes were predicted by immuno-SRM. Peptides were considered not detected (ND) if their signal-to-noise ratio was less than 10. * indicates currently unknown mutations. [Figure 16]Figures 16A-D. Internal standard peptide peak areas generated during peptide capture of both WASp 274 (Figures 16B and 16D) and BTK 407 (Figures 16A and 16C) using polyclonal and monoclonal antibodies. Peak areas were measured in blank control samples only (i.e., no patient blood, Figures 16C, 16D), and in a compilation set of both patient and blank control samples (Figures 16A and 16B). [Figure 17] Signature CTNS115 and SHPK363 peptide levels in patient and normal control (NC) samples in screening for cystinosis. This study is an extension of the study shown in FIG. 13 and Table 8. [Figure 18A] Figures 18A-C. Study of 16 WD patients (NC: normal controls; Pt: Wilson's disease patients). (Figure 18A) ATP7B 1056 concentrations in NC and WD patients. 50 NCs in the first row were run individually to establish reference ranges in the laboratory. The 3 NCs in the middle row were run together with all 16 WD patients as quality control. (Figure 18B) ATP7B214 concentrations in NC and WD patients. (Figure 18C) ATP7B887 concentrations in NC and WD patients. [Figure 18B] Figures 18A-C. Study of 16 WD patients (NC: normal controls; Pt: Wilson's disease patients). (Figure 18A) ATP7B 1056 concentrations in NC and WD patients. 50 NCs in the first row were run individually to establish reference ranges in the laboratory. The 3 NCs in the middle row were run together with all 16 WD patients as quality control. (Figure 18B) ATP7B214 concentrations in NC and WD patients. (Figure 18C) ATP7B887 concentrations in NC and WD patients. [Figure 18C] Figures 18A-C. Study of 16 WD patients (NC: normal controls; Pt: Wilson's disease patients). (Figure 18A) ATP7B 1056 concentrations in NC and WD patients. 50 NCs in the first row were run individually to establish reference ranges in the laboratory. The 3 NCs in the middle row were run together with all 16 WD patients as quality control. (Figure 18B) ATP7B214 concentrations in NC and WD patients. (Figure 18C) ATP7B887 concentrations in NC and WD patients. [Figure 19-1] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-2] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-3] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-4] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-5] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-6] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-7] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-8] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-9] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-10] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-11] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-12] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-13] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-14] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-15] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-16] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-17] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-18]Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-19] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-20] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-21] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-22] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-23] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-24] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-25] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). [Figure 19-26] Sequences supporting the present disclosure (SEQ ID NOs: 22-76 and 78-141). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description There are many diseases with effective treatments available. However, for many of these diseases, once symptoms appear, the disease is already fatal or has caused irreversible damage. Examples of such diseases include primary immune deficiency syndrome (PIDD), cystinosis, and Wilson's disease (WD).

[0024] PIDD is a diverse collection of congenital disorders characterized by a deficient or impaired immune response, including: autosomal recessive CD3ε-related severe combined immunodeficiency (SCID, OMIM#615615); X-linked Wiskott-Aldrich syndrome (WAS, OMIM#301000); and X-linked agammaglobulinemia (XLA, OMIM#300755). Although genetically and clinically heterogeneous, these disorders lead to fatal infections unless detected and treated early. Early detection of PIDD can be life-saving, but unfortunately, most affected infants are diagnosed only after they have developed a serious infection.

[0025] SCID is a group of rare disorders caused by mutations in different genes involved in the development and function of infection-fighting immune cells such as T cells and B cells. More than a dozen genes have been implicated in SCID. In most cases, SCID is inherited in an autosomal recessive pattern, where both copies of a particular gene (one inherited from the mother and one from the father) contain defects. The best-known form of autosomal recessive SCID is caused by adenosine deaminase (ADA) deficiency, in which infants lack the ADA enzyme required for T cell survival. X-linked SCID, caused by mutations in a gene on the X chromosome, primarily affects males. Boys with this type of SCID have white blood cells that grow and develop abnormally. As a result, they have low numbers of T cells and natural killer cells, and their B cells do not function. SCID patients are usually affected by severe bacterial, viral, or fungal infections early in life and often suffer from lung scarring, chronic diarrhea, and growth disorders. The condition is fatal, usually within the first 1 to 2 years of life, unless the infant receives immune-restoring treatment such as hematopoietic stem cell transplantation, gene therapy, or enzyme therapy.

[0026] WAS is an immunodeficiency disorder characterized by a decrease in the number and size of platelets. WAS is caused by mutations in the WAS gene, which produces the WAS protein (WASp), and is often considered part of a disease spectrum with two other disorders: X-linked thrombocytopenia and severe congenital neutropenia. These conditions have overlapping signs and symptoms and share the same genetic causes.

[0027] The reduction in the number and size of platelets associated with WAS results in a reduced ability to form blood clots. This leads to easy bruising and episodes of prolonged bleeding following minor trauma, which can be life-threatening in some cases. Individuals with WAS are also more susceptible to infections, autoimmune disorders (e.g., eczema), and certain cancers (such as lymphoma). Once diagnosed, treatments for WAS are available. Exemplary treatments include immunoglobulin infusions, antibiotics, and stem cell transplants. Gene therapy is also being considered as a treatment option for WAS.

[0028] XLA is an inherited immunodeficiency disorder that prevents B cells from developing normally. XLA is caused by a mutation in a gene called Bruton's tyrosine kinase (BTK). With XLA, the body is unable to produce the antibodies needed to defend against bacteria, viruses, and other foreign substances. Children with XLA are usually healthy for the first one to two months of life because they are protected by maternal antibodies acquired before birth. However, after this time, the maternal antibodies are cleared from the body and affected children develop recurrent infections. Recurrent infections can lead to organ damage. Once diagnosed, treatments in the form of antibody infusions and antibiotics are available to treat XLA.

[0029] Cystinosis is a rare metabolic disorder in which the amino acid cystine enters cells but cannot exit due to a defect in a cystine-specific transporter called cystinosin. Because cystine transport is defective, cells accumulate cystine in crystals in lysosomes, organelles within the cell, causing premature cell death. Over 90 mutations have been reported in the cystinosin (CTNS) gene or region. Approximately half of the cystinosis variants in Western populations are caused by a large chromosomal deletion that spreads from part of the CTNS gene through the neighboring SHPK and the first part of the TRPV1 (capsaicin receptor) genes. Cystinosis slowly destroys the body's organs, including the kidneys, liver, eyes, muscles, and brain. Infant cystinosis children appear normal at birth, but by 9-10 months of age, symptoms such as excessive thirst and urination, as well as failure to thrive, appear. If diagnosed early, it can be treated with cysteamine, a cystine-depleting drug that removes cystine from cells and slows the progression of cystinosis. If it is not diagnosed early enough, kidney transplantation is often required.

[0030] Wilson's disease (WD) is a slow, progressive copper transport disorder in which excess copper is not properly eliminated and accumulates in vital organs such as the liver, kidneys, and brain. WD is caused by a genetic defect in the ATP7B gene. If not diagnosed and treated early, WD leads to irreversible neurological damage and liver cirrhosis. Unfortunately, WD remains challenging due to its slow progression and wide clinical spectrum of symptoms. Thus, many patients still present with irreversible, sometimes fatal, multi-organ damage at the time of diagnosis. If diagnosed early enough, available treatments include D-penicillamine, trientine, and zinc salts.

[0031] Treatment of each of the above diseases would be significantly enhanced if a diagnosis could be made before clinical symptoms appear. Newborn screening (NBS) is the standard public preventive mandatory screening test for the 4 million infants born annually in the United States. NBS typically involves a blood test performed 24-48 hours after birth. Screening uses a few drops of blood from the heel of the newborn that are placed on filter paper. The paper containing the dried blood spot (DBS) may be stored until testing is performed.

[0032] To perform an NBS evaluation, a punch of dried blood is taken from the DBS and laboratory tests are performed to detect the presence or absence of certain substances in the blood (called markers or biomarkers) that indicate disorders that are not evident at birth but that cause serious health problems later in life. The disorders screened for vary from condition to condition, but most conditions screen for phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease. NBS has proven highly effective in improving patient outcomes and avoiding long-term disability for affected individuals while also reducing health care costs.

[0033] T-cell receptor excision circle (TREC) analysis and kappa deletion element recombination circle (KREC) screening from dried blood spots (DBS) on filter paper have been recently introduced for SCID and some X-linked or autosomal recessive agammaglobulinemias, although no NBS methods exist for other PIDDs.

[0034] Tandem mass spectrometry (MS / MS) was first applied to NBS in the 1990s, paving the way for rapid screening of multiple metabolites, and therefore several diseases, from DBS samples collected at birth. Chace J Mass Spectrom. Wiley-Blackwell; 2009; 44: 163-170; Millington et al. J. Inherit. Metab. Dis. 1990; 13: 321-324; Sweetman et al. Pediatrics. 2006; 117: S308-S314; Almannai et al. Curr. Opin. Pediatr. 2016; 28: 694-699; Watson et al. Genet. Med. Nature Publishing Group; 2006. pp. 1S-252S; Chace et al. Clin. Chem. 1993; 39: 66-71. Selected reaction monitoring mass spectrometry (SRM-MS) performed on triple quadrupole mass spectrometers has further enabled accurate, high-throughput, analytically robust quantification of specific biomarkers and is therefore now the standard of care in clinical NBS laboratories worldwide. Chace DH J Mass Spectrom. Wiley-Blackwell; 2009; 44: 163-170; Chace & Kalas. Clinical Biochemistry. 2005; 38: 296-309. Dott et al. American Journal of Medical Genetics Part A. Wiley Subscription Services, Inc., A Wiley Company; 2006; 140: 837-842.

[0035] MS / MS relies on the measurement of enriched upstream metabolites to detect various inborn errors of metabolism with specific enzyme deficiencies. This precludes application to diseases such as PIDD, where accumulated metabolites are absent or not currently validated. For this reason, protein-based assays such as flow cytometry or Western blotting have been used as the first-line investigative method for diseases such as WAS and its milder phenotype, X-linked thrombocytopenia (XLT), where most mutations are absent or have reduced protein products. Qasim et al. Br. J. Haematol. 2001;113:861-865; Jin et al. Blood. American Society of Hematology; 2004;104:4010-4019. These approaches require the availability of intact blood samples or cells from patients, making population-based screening or testing of patients from resource-poor areas impossible.

[0036] SRM-MS utilizes proteolytically generated signature peptides as stoichiometric surrogates for a protein of interest. This can then be used to estimate the number of specific cell types expressing that protein in a sample (i.e., quantification of CD3ε to indicate the amount of CD3+ T cells in blood). The high specificity of MS for each signature peptide is provided by three physicochemical properties: its mass, its retention time during high performance liquid chromatography (HPLC) separation, and the resulting target-specific fragmentation pattern. Kennedy et al. Nat. Methods. 2014;11:149-155. Despite these advances, there are limits of quantification ranging from 100-1000 ng protein / mL, and the use of complex matrices such as blood or plasma often precludes accurate quantification of low abundance targets by SRM-MS-based assays. This limits their application to many PIDDs such as SCID, WAS, and XLA, where target proteins are absent or at reduced levels only expressed intracellularly. de Saint Basile et al. J. Clin. Invest. American. Society for Clinical Investigation;2004;114:1512-1517.

[0037] Peptide immunoaffinity enrichment combined with SRM (immuno-SRM), also known as Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA), enhances the sensitivity of SRM-MS assays by utilizing anti-peptide antibodies to purify and enrich peptides of interest from complex biological samples prior to SRM-MS analysis. Zhao et al. J Vis Exp. 2011;53:2812;Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2010;9:184-196;Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2012;11:M111.015347;Kuhn et al. Clin. Chem. 2009;55:1108-1117;Anderson et al. J Proteome Res. 2004;3(2):235-244. This additional peptide enrichment step, in combination with SRM-MS, lowers the detection limit from 1 mL of plasma into the low pg protein / mL range, which is suitable for accurate quantification of very low abundance proteins in complex matrices such as DBS. Whiteaker et al.Mol.Cell Proteomics.American Society for Biochemistry and Molecular Biology;2010;9:184-196;Hoofnagle et al.Clin.Chem.2008;54:1796-1804;Netzel et al.Clin.Chem.2016;62:297-299;Razavi et al.Bioanalysis.Future Science Ltd London,UK;2016;8:1597-1609.

[0038] The ability of LC-MS / MS to detect signature peptides from CD3ε, WASp, and BTK in proteolytically digested human peripheral blood mononuclear cell (PMBC) lysates has been previously demonstrated. In a blinded study, peptide levels were quantified in normal control PMBCs but were nearly undetectable by disease-specific methods in affected patients. Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402. The same proteomic method was applied to show elevated levels of α-aminoadipic semialdehyde antiquitin (α-AASA) and piperidine-6-carboxylate (P6C) in DBS of patients with pyridoxine-dependent seizures, revealing its potential application in NBS. Jung et al. Mol. Genet. Metab. 2013;110:237-240. To improve the sensitivity and reproducibility of the assay, we utilized the immuno-SRM platform to enable quantification of very low abundance peptides in DBS, such as surrogate peptides of the ATP7B protein in Wilson's disease (WD) patients. The results demonstrated the ability of immuno-SRM to detect ATP7B peptides in the low picomolar (pmol) range and reproducibly distinguish between WD patients and unaffected individuals. Jung et al. 2017, (supra).

[0039] Although the feasibility of using DBS in immuno-SRM assays to distinguish affected from unaffected individuals has been established, many aspects of the assay depend on the disorder being diagnosed, the biomarkers available for each disorder, the ability to develop molecular entities that can enrich for peptides of interest, and the behavior of each peptide of interest in a mass spectrometer. All of these and other aspects require careful consideration and experimentation to achieve a reliable assay that can reliably detect NBS panel disorders using DBS before clinical symptoms appear.

[0040] The present disclosure provides a multiplex immuno-SRM method for reliably diagnosing SCID, WAS, XLA, cystinosis, and WD from dried blood spots (DBS). The multiplex immuno-SRM assays disclosed herein may utilize anti-peptide antibodies generated against peptides of proteins that are reduced or absent in SCID, WAS, XLA, cystinosis, and WD.

[0041] The following aspects of the present disclosure are now described in more detail: (I) DBS collection and processing; (II) peptide markers for SCID, WAS, XLA, cystinosis, and Wilson's disease; (III) enzymatic digestion of proteins in DBS; (IV) antibodies for enriching peptide markers; (V) peptide enrichment strategies; (VI) liquid chromatography (LC); (VII) mass spectrometry (MS); (VIII) kits; (IX) methods of use; (X) exemplary embodiments; and (XI) experimental examples.

[0042] (I) Collection and processing of DBS. In certain embodiments, the sample used in the method of the present disclosure is DBS. In certain embodiments, whole blood from a subject can be prepared by placing blood on a filter paper card and drying the blood.

[0043] In certain embodiments, whole blood from a subject may be collected in an ACD (acid citrate dextrose) tube, and DBS may be prepared by pipetting 50-100 μL (e.g., 70 μL) of blood / spot onto a filter paper card (e.g., Protein Saver™ 903® Card, Whatman Inc, Piscataway, NJ) and allowed to dry at room temperature. In certain embodiments, blood is allowed to dry on the filter paper card overnight. DBS may be stored, for example, in a sealed plastic bag at −80° C. until use. In certain embodiments, whole DBS may be used in the immuno-SRM assays of the present disclosure. In certain embodiments, one or more 3 mm punches from DBS may be used in the immuno-SRM assays of the present disclosure.

[0044] (II) Peptide markers for SCID, WAS, XLA, cystinosis, and WD. There are many theoretical proteolytic peptides derived from the target protein. They can be potential candidates for monoclonal antibody production. Nevertheless, the best potential candidate peptides were selected after screening their features by MS / MS. These signature peptides with the highest sensitivity and specificity were selected to develop corresponding monoclonal antibodies and validated using clinical samples. In certain embodiments, multiple peptides and antibodies may be included in the multiplex analysis to maximize the sensitivity and specificity of the SRM assay.

[0045] Typically, one or two signature proteotypic peptides that are unique to the protein of interest and consistently observed in MS experiments are selected to stoichiometrically represent the protein of interest. Mallick et al. Nat Biotechnol 2007;25:125-131. Signature peptides may be selected by detection in previous MS experiments, by using computational tools to predict peptides most likely to be observable in MS, or a combination of both. In certain embodiments, tryptic peptides of 8-22 amino acids in length with moderate hydrophobicity may be selected. Very hydrophilic and very hydrophobic peptides may have reduced stability due to variable retention times on HPLC and loss to surfaces. In certain embodiments, methionine residues (oxidation), N-terminal glutamine (cyclization), glycine or proline following asparagine (prone to deamidation), and dibasic termini (e.g., adjacent lysine or arginine residues such as KK, KR, RR, RK, etc. may result in variable digestion efficiency) may be undesirable. Whiteaker and Paulovich Clin Lab Med. 2011;31(3):385-396. Shorter peptides and peptides containing proline residues may be better targets for SRM. Lange et al. Molecular Systems Biology 2008;4:222.

[0046] In certain embodiments, the peptide comprises a portion of CD3ε, WASp, BTK, CTNS, SHPK and / or ATP7B. In certain embodiments, the peptide comprises SEQ ID NOs: 1-21.

[0047] In certain embodiments, peptides of the disclosure include: CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 120 for cystinosis; CTNS 194 for cystinosis; CTNS 360 for cystinosis; SHPK 44 for cystinosis; SHPK 363 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 325 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; ATP7B 1056 for WD; and ATP7B 1061 for WD. In certain embodiments, proteolytically generated signature peptides useful in the methods of the disclosure include the peptides of Figure 1: CD3ε 197 (DLYSGLNQR, SEQ ID NO:1); WASp 274 (AGISEAQLTDAETSK, SEQ ID NO:2); WASp 289 (LIYDFIEDQGGLEAVR, SEQ ID NO:3); BTK 407 (ELGTGQFGVVK, SEQ ID NO:4); BTK 545 (YVLDDEYTSSVGSK, SEQ ID NO:5); CTNS 115 (FLVIR, SEQ ID NO:6); CTNS 360 (RPGYDQLN (SEQ ID NO:7) and KRPGYDQLN (SEQ ID NO:8)); SHPK 363 (DTHLTITPTVLGER, SEQ ID NO:9); ATP7B 325 (VSLPDGAEGSGTDHR, SEQ ID NO:10); and ATP7B 1056 (VLAVVGTAEASSEHPLGVAVTK, SEQ ID NO:11). In certain embodiments, additional peptides useful in the methods of the disclosure include the following from Table 1:

[0048] [Table 1]

[0049] (III) Enzymatic digestion of proteins in DBS. Proteins in DBS may be subjected to proteolysis to generate peptides, which may be further selected by immunoaffinity purification before analysis by LC-SRM-MS. Proteolysis may be achieved using site-specific endoproteases such as pepsin, arg-C proteinase, asp-N endopeptidase, BNPS-skatole, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, chymotrypsin, clostripain (clostridiopeptidase B), enterokinase, factor Xa, glutamyl endopeptidase, granzyme B, lysC, proline endopeptidase, proteinase K, staphylococcal peptidase I, thermolysin, thrombin and trypsin. Chemicals that specifically cleave sites may also be used. A combination of enzymes and / or chemicals may be used to obtain the desired analyte.

[0050] In certain embodiments, proteins in DBS may be digested into peptides with trypsin. Trypsin may be the preferred choice for generating peptides because it exclusively cleaves the C-terminus of arginine and lysine residues, the mass of the generated peptides is compatible with the detection capabilities of most mass spectrometers (up to 2000 m / z), and there are efficient algorithms available for generating a database of theoretical tryptic generated peptides. High cleavage specificity, availability, and cost are other advantages of trypsin. Peptides formed by treating proteins with trypsin are known as tryptic peptides.

[0051] (IV) Antibodies for enriching peptide markers. Antibodies include polypeptide ligands substantially encoded by immunoglobulin gene(s), or fragments thereof, whether natural or partially or wholly synthetically produced. Antibodies specifically (or selectively) bind and recognize an epitope (e.g., an antigen). Antibodies may include any protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin class, including any of the human classes, such as IgG, IgM, IgA, IgD, and IgE. Recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon, and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes. The "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that includes one or more heavy chain constant region domains, CH1, CH2, and CH3, but does not include the heavy chain variable region.

[0052] An intact antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (referred to herein as VH or V H Each light chain is composed of a light chain variable region (abbreviated herein as VL or VL) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL or VL) and a heavy chain constant region. LThe heavy and light chain variable regions are composed of a heavy chain constant region (abbreviated as FR1, CDR2, CDR3, CDR4) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0053] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of several well-known schemes, including those described by Kabat et al. (1991) "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat numbering scheme); Al-Lazikani et al. (1997) J Mol Biol 273:927-948 (Chothia numbering scheme); Maccallum et al. (1996) J Mol Biol 262:732-745 (Contact numbering scheme); Martin et al. (1989) Proc. Natl. Acad. Sci., 86:9268-9272 (AbM numbering scheme); Lefranc MP et al. (2003) Dev Comp Immunol 27(1):55-77 (IMGT numbering scheme); and Honegger and Pluckthun (2001) J Mol Biol 309(3):657-670 ("Aho" numbering scheme). The boundaries of a given CDR or FR may differ depending on the scheme used to identify it. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the length of the sequences of the most common antibody regions, with insertions corresponding to inserted letters such as "30a" and deletions being indicated in some antibodies. The two schemes place certain insertions and deletions ("indels") in different positions and number them differently. The contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme. In certain embodiments, the antibody CDR sequences disclosed herein are according to the Kabat numbering scheme.

[0054] Antibody fragments include any derivative or portion of an antibody that is less than full-length. In certain embodiments, an antibody fragment retains at least a substantial portion of the specific binding ability of the full-length antibody as a binding partner. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, single-chain variable fragment (scFv), Fv, dsFv diabody, and Fd fragments, and / or any biologically effective fragment of an immunoglobulin that specifically binds to an epitope described herein. Antibodies or antibody fragments include all or part of polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, synthetic antibodies, chimeric antibodies, bispecific antibodies, minibodies, and linear antibodies.

[0055] Single chain variable fragments (scFv) are fusion proteins of the variable regions of the heavy and light chains of immunoglobulins connected to a short linker peptide. Fv fragments contain the VL and VH domains of a single arm of an antibody. The two domains of an Fv fragment, VL and VH, are encoded by separate genes, but they may be linked by a synthetic linker that allows them to be produced as a single protein chain, for example using recombinant methods, where the VL and VH regions pair to form a monovalent molecule (single chain Fv (scFv)). For additional information regarding Fv and scFv, see, e.g., Bird, et al., Science 242 (1988) 423-426; Huston, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Plueckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), Springer-Verlag, New York), (1994) 269-315; WO1993 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458.

[0056] A Fab fragment is a monovalent antibody fragment containing the VL, VH, CL and CH1 domains. A F(ab')2 fragment is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region. For a discussion of Fab and F(ab')2 fragments with extended in vivo half-lives, see U.S. Pat. No. 5,869,046. Diabodies contain two epitope binding sites that may be bivalent. See, e.g., European Patent No. 0404097; WO 1993 / 01161; and Holliger, et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448. Dual affinity retargeting antibodies (DART™; based on the diabody format but featuring a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)) may also be used. Antibody fragments may also include isolated CDRs. For a review of antibody fragments, see Hudson, et al., Nat. Med. 9 (2003) 129-134.

[0057] Antibody fragments can be produced by any means. For example, they can be enzymatically or chemically produced by fragmentation of an intact antibody, or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively, they can be wholly or partially produced synthetically. Antibody fragments can include single-chain antibody fragments. In another embodiment, the fragments can include multiple chains linked together, for example, by disulfide bonds. The fragments can also include multimolecular complexes. Functional antibody fragments can typically include at least 50 amino acids, more typically at least 200 amino acids.

[0058] In certain embodiments, recombinant immunoglobulins can be produced, see Cabilly, U.S. Patent No. 4,816,567, and Queen et al., Proc Natl Acad Sci USA, 86:10029-10033 (1989).

[0059] As shown, in certain embodiments, the binding domains of the engineered antibody or antigen-binding fragment may be linked via a linker. A linker is an amino acid sequence that may provide flexibility and room for conformational movement between the binding domains or antigen-binding fragments of the engineered antibody. Any suitable linker may be used. Examples of linkers may be found in Chen et al., Adv Drug Deliv Rev. 2013 Oct15;65(10):1357-1369. Linkers may be flexible, rigid, or semi-rigid, depending on the presentation of the desired functional domain to the target. Commonly used flexible linkers include Gly-Ser linkers such as GGSGGGSGGSG (SEQ ID NO: 142), GGSGGGSGSG (SEQ ID NO: 143), and GGSGGGSG (SEQ ID NO: 144). Additional examples include: GGGGSGGGGS (SEQ ID NO: 145); GGGSGGGS (SEQ ID NO: 146); and GGSGGS (SEQ ID NO: 147). Linkers comprising one or more antibody hinge regions and / or immunoglobulin heavy chain constant regions, such as a CH3 alone or a CH2CH3 sequence, may also be used.

[0060] In some circumstances, flexible linkers may not be able to maintain the distance or arrangement of binding domains required for a particular use. In these instances, fixed or semi-fixed linkers may be useful. Examples of fixed or semi-fixed linkers include proline-rich linkers. In certain embodiments, proline-rich linkers are peptide sequences that have more proline residues than would be expected based on chance alone. In certain embodiments, proline-rich linkers are linkers that have at least 30%, at least 35%, at least 36%, at least 39%, at least 40%, at least 48%, at least 50%, or at least 51% proline residues. Particular examples of proline-rich linkers include fragments of proline-rich salivary proteins (PRPs).

[0061] It will also be understood by those skilled in the art that antibodies may undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the antibody. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation.

[0062] Monoclonal antibodies include antibodies obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody (such variants are generally present in small amounts). In contrast to polyclonal antibody preparations, which usually contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. This type of antibody is produced by daughter cells of a single antibody-producing hybridoma. A monoclonal antibody usually exhibits a single binding affinity for any epitope to which it binds.

[0063] The modifier "monoclonal" indicates the character of the antibody as being obtained from a homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. A monoclonal antibody recognizes only one type of antigen. Monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to corresponding sequences of antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to corresponding sequences of antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies. Techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999; Tickle et al. JALA: Journal of the Association for Laboratory Automation. 2009; 14(5): 303-307; Babcook et al. Proc. Natl. Acad. Sci. USA 1996; 93: 7843-7848; and U.S. Patent No. 5,627,052.

[0064] In certain embodiments, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and a peptide). The affinity of a molecule X for its partner Y is generally determined by the dissociation constant (K D ) or binding constant (K A Affinity can be measured by common methods known in the art.

[0065] In certain embodiments, "binding" refers to the binding domain of an antibody being more than 10 -8 A dissociation constant (KD) of less than or equal to M, and in certain embodiments, 10 -5 M~10 -13 M, in certain embodiments, 10 -5 M~10 -10 M, in certain embodiments, 10 -5 M~10 -7 M, in certain embodiments, 10 -8 M~10 -13 M, or in certain embodiments, 10 -9 M~10 -13 This means that the binding domain associates with its target peptide with a dissociation constant (KD) of 10 M. This term may also be used to indicate that the binding domain does not bind to other biomolecules that are present (e.g., it has a dissociation constant of 10 -4 M or more, in certain embodiments 10 -4 The dissociation constant (K D ) to bind to other biomolecules that have the same structure.

[0066] In certain embodiments, "binding" refers to the binding domain of an antibody being more than 10 7 M -1 The affinity constant (i.e., the association constant, K A ), in certain embodiments 10 5 M -1 ~10 13 M -1 , and in certain embodiments, 10 5 M -1 ~10 10 M -1 , and in certain embodiments, 10 5 M -1 ~10 8 M -1 , and in certain embodiments, 10 7 M -1 ~10 13 M -1 or in certain embodiments, 10 7 M -1 ~10 8 M -1This means that the binding domain associates with its target peptide with an affinity constant of 10. This term may also be used to indicate that the binding domain does not bind to other biomolecules present (e.g., it has an affinity constant of 10 4 M -1 The following association constant (K A ), in certain embodiments 10 4 M -1 ~1M -1 It binds to other biomolecules with an association constant of 0.1.

[0067] The antibodies of the present disclosure may be used for immunoaffinity enrichment of peptides described herein detected in SRM assays for the diagnosis of SCID, WAS, XLA, cystinosis, and WD. Particular embodiments of high affinity antibodies include anti-CD3ε 197, anti-WASp 274, anti-WASp 289, anti-BTK 407, anti-BTK 545, anti-CTNS 115, anti-CTNS 120, anti-CTNS 194, anti-CTNS 194, anti-CTNS 360, anti-SHPK 44, anti-SHPK 363, anti-SHPK 388, anti-ATP7B 214, anti-ATP7B 325, anti-ATP7B 466, anti-ATP7B 589, anti-ATP7B 621, anti-ATP7B 887, anti-ATP7B 1056, and anti-ATP7B 1061.

[0068] In certain embodiments, exemplary antibodies include the CDRs presented in Table 2.

[0069] [Table 2]

[0070] In certain embodiments, exemplary antibodies comprise the variable heavy and variable light domains presented in Table 3.

[0071] [Table 3] TIFF2025063133000004.tif142168

[0072] In certain embodiments, the amino acid sequences of exemplary antibodies include: anti-CD3ε 197 heavy chain variable domain amino acid sequence with a leader sequence (SEQ ID NO:79); anti-CD3ε 197 light chain variable domain amino acid sequence with a leader sequence (SEQ ID NO:81); anti-WASp 274 heavy chain amino acid sequence with a leader sequence (SEQ ID NO:83); anti-WASp 274 heavy chain variable domain amino acid sequence with a leader sequence (SEQ ID NO:85); anti-WASp 274 heavy chain amino acid sequence without a leader sequence (SEQ ID NO:86); anti-WASp 274 light chain amino acid sequence with a leader sequence (SEQ ID NO:88); anti-WASp 274 light chain variable domain amino acid sequence with a leader sequence (SEQ ID NO:90); anti-WASp 274 light chain amino acid sequence without a leader sequence (SEQ ID NO:91); anti-BTK 407 heavy chain amino acid sequence with a leader sequence (SEQ ID NO:93); anti-BTK 407 heavy chain variable domain amino acid sequence with a leader sequence (SEQ ID NO:95); anti-BTK without a leader sequence 407 heavy chain amino acid sequence (SEQ ID NO:96); anti-BTK 407 light chain amino acid sequence with leader sequence (SEQ ID NO:98); anti-BTK 407 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO:100); anti-BTK 407 light chain amino acid sequence without leader sequence (SEQ ID NO:101); anti-CTNS 115 heavy chain amino acid sequence with leader sequence (SEQ ID NO:103); anti-CTNS 115 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO:105); anti-CTNS 115 heavy chain amino acid sequence without leader sequence (SEQ ID NO:106); anti-CTNS 115 light chain amino acid sequence with leader sequence (SEQ ID NO:108); anti-CTNS 115 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO:110); anti-CTNS 115 light chain amino acid sequence without leader sequence (SEQ ID NO:111); anti-CTNS 360 heavy chain amino acid sequence with leader sequence (SEQ ID NO:113); anti-CTNS 360 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO:115); anti-CTNS without leader sequence 360 heavy chain amino acid sequence (SEQ ID NO:116); anti-CTNS 360 light chain amino acid sequence with leader sequence (SEQ ID NO:118);Anti-CTNS 360 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO:120); Anti-CTNS 360 light chain amino acid sequence without leader sequence (SEQ ID NO:121); Anti-SHPK 363 heavy chain amino acid sequence with leader sequence (SEQ ID NO:123); Anti-SHPK 363 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO:125); Anti-SHPK 363 heavy chain amino acid sequence without leader sequence (SEQ ID NO:126); Anti-SHPK 363 light chain amino acid sequence with leader sequence (SEQ ID NO:128); Anti-SHPK 363 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO:130); Anti-SHPK 363 light chain amino acid sequence without leader sequence (SEQ ID NO:131); Anti-ATP7B 1056 heavy chain amino acid sequence with leader sequence (SEQ ID NO:133); Anti-ATP7B 1056 heavy chain variable domain amino acid sequence with leader sequence (SEQ ID NO:135); Anti-ATP7B 1056 heavy chain amino acid sequence without leader sequence (SEQ ID NO:136); Anti-ATP7B with leader sequence 1056 light chain amino acid sequence (SEQ ID NO:138); anti-ATP7B 1056 light chain variable domain amino acid sequence with leader sequence (SEQ ID NO:140); and anti-ATP7B 1056 light chain amino acid sequence without leader sequence (SEQ ID NO:141).

[0073] In certain embodiments, exemplary antibody coding sequences include: anti-CD3ε 197 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO:78); anti-CD3ε 197 light chain variable domain coding sequence with leader sequence (SEQ ID NO:80); anti-WASp 274 heavy chain coding sequence with leader sequence (EB0603-2F8-H2) (SEQ ID NO:82); anti-WASp 274 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO:84); anti-WASp 274 light chain coding sequence with leader sequence (EB0603-2F8-K2) (SEQ ID NO:87); anti-WASp 274 light chain variable domain coding sequence with leader sequence (SEQ ID NO:89); anti-BTK 407 heavy chain coding sequence with leader sequence (EB0602-1G5-H2) (SEQ ID NO:92); anti-BTK 407 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO:94); anti-BTK with leader sequence 407 light chain coding sequence (EB0602-1G5-K1) (SEQ ID NO: 97); anti-BTK 407 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 99); anti-CTNS 115 heavy chain coding sequence with leader sequence (EB0606-3H8-H5) (SEQ ID NO: 102); anti-CTNS 115 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 104); anti-CTNS 115 light chain coding sequence with leader sequence (EB0606-3H8-K7) (SEQ ID NO: 107); anti-CTNS 115 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 109); anti-CTNS 360 heavy chain coding sequence with leader sequence (EB0604-4E3-H4) (SEQ ID NO: 112); anti-CTNS 360 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 114); anti-CTNS with leader sequence 360 light chain coding sequence (EB0604-4E3-K1) (SEQ ID NO: 117); anti-CTNS 360 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 119); anti-SHPK 363 heavy chain coding sequence with leader sequence (EB0605B-6G12-H1) (SEQ ID NO: 122); anti-SHPK 363 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 124); anti-SHPK 363 light chain coding sequence with leader sequence (EB0605B-6G12-K1) (SEQ ID NO: 127);Anti-SHPK 363 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 129); Anti-ATP7B 1056 heavy chain coding sequence with leader sequence (EB0601-H3-1) (SEQ ID NO: 132); Anti-ATP7B 1056 heavy chain variable domain coding sequence with leader sequence (SEQ ID NO: 134); Anti-ATP7B 1056 light chain coding sequence with leader sequence (EB0601-K3-2) (SEQ ID NO: 137); and Anti-ATP7B 1056 light chain variable domain coding sequence with leader sequence (SEQ ID NO: 139).

[0074] (V) Peptide Enrichment (Enrichment) Strategies. Enrichment of desired peptide targets prior to SRM can be accomplished by any means known in the art. Many enrichment procedures are available, including immunoadsorption-based depletion of abundant protein species from the sample, precipitation, chromatography, electrophoresis, solvent partitioning, immunoprecipitation, immunoelectrophoresis, and immunochromatography. In certain embodiments, the SISCAPA method for specific antibody-based capture of individual tryptic peptides from a sample digest may be used. Anderson et al., J. Proteome Research 2004;3:235-244; U.S. Patent No. 7,632,686.

[0075] In certain embodiments, antibodies that bind to peptide markers, such as the antibodies disclosed herein, may be attached to a solid support. Certain embodiments use affinity columns in which the antibodies are covalently attached to a chromatographic medium. In certain embodiments, POROS (Applied Biosystems, Foster City, CA) nanocolumns may be used in SISCAPA enrichment, characterized by high binding capacity, relatively high concentrations of antibodies that allow for rapid enrichment of target peptides, and the ability to prepare columns with a variety of functionalization groups. Alternatively, antibodies may be attached to beads, magnetic beads, or other solid particles. One means of attachment is the binding of the antibody to a protein coated on the bead. For example, particles coated with protein G provide for the binding of the antibody in a preferred orientation. Other means of attachment may be used, such as coating the beads directly with the antibody. Magnetic particles may be utilized with a variety of chemistries that allow for binding to the antibody. Enrichment with antibodies attached to particles allows for parallel processing of samples. Magnetic particle processing has been automated in 96-well plates for the SISCAPA enrichment step with elution in the plate for analysis by mass spectrometry. Other specific embodiments use a novel bead trap device developed to perform the bead processing step along with the nanoflow chromatography system. Anderson et al. Mol Cell Proteomics 2009;8(5):995-1005. This minimizes peptide loss to the vessel between the elution and analysis steps. Peptide enrichment can also be performed by immobilizing anti-peptide antibodies on the pipette tip. Nelson et al. Anal Chem. 1995;67(7):1153-1158. After separating the antibody-bound peptides from the free peptides, the bound peptides can be eluted. Any elution means may be used. One elution means that has been found to be efficient is 5% acetic acid / 3% acetonitrile. Other elution means, including other acids, and other concentrations of acetic acid may be used as efficient for the particular peptide.

[0076] (VI) Liquid Chromatography (LC). In certain embodiments, one or more LC purification steps are performed prior to SRM-MS. A mixture of enriched peptides (mobile phase) may be passed through a column packed with material (stationary phase) to separate the peptides based on their weight and affinity for the mobile and stationary phases of the column. Traditional LC analysis relies on chemical interactions between the sample components and the column packing material, and laminar flow of the sample through the column is the basis for separating the analytes of interest from the test sample. Those skilled in the art will appreciate that separation in such columns is a diffusion process. A variety of column packing materials are available for chromatographic separation of samples, and the selection of an appropriate separation protocol is an empirical process that depends on the characteristics of the sample, the analytes of interest, the interfering substances present and their properties, etc. A variety of packing chemistries may be used as needed (e.g., structure, polarity, solubility of the compound to be purified). In certain embodiments, the columns are polar, ion exchange (both cation and anion), hydrophobic interaction, phenyl, C-2, C-8, C-18 columns, polar coatings on porous polymers, or other commercially available. During chromatography, separation of materials is influenced by variables such as the choice of eluent (also known as the "mobile phase"), the choice of gradient elution, and gradient conditions, temperature, etc. In certain embodiments, analytes may be purified by applying a sample to a column under conditions in which the analyte of interest is reversibly retained by the column packing material and one or more other materials are not retained. In these embodiments, first mobile phase conditions may be used in which the analyte of interest is retained by the column, and then second mobile phase conditions may be used to remove the retained material from the column once the unretained material has been washed away. Alternatively, analytes may be purified by applying a sample to a column under mobile phase conditions in which the analyte of interest elutes at a different rate compared to one or more other materials. As noted above, such procedures may enrich the amount of one or more analytes of interest relative to one or more other components of the sample. In certain embodiments, the LC is nanoflow LC (nanoLC).In nanoflow LC (nanoLC), chromatographic separations are performed using flow rates in the low nanoliters per minute range, resulting in high analytical sensitivity due to the high enrichment efficiency achieved by this type of chromatography. Cutillas, Current Nanoscience, 2005;1:65-71.

[0077] (VII) Mass Spectrometry (MS). Mass spectrometers are equipped with gas phase ion spectrometers that measure parameters that can be converted to mass-to-charge (m / z) ratios of gas phase ions. Mass spectrometry refers to the detection of gas phase ions using a mass spectrometer. Mass spectrometers typically include an ion source and a mass analyzer. Examples of mass spectrometers are time-of-flight (TOF), magnetic sector, quadrupole filter, ion trap, ion cyclotron resonance, electrostatic sector analyzer, and hybrids thereof. Laser desorption mass spectrometers include mass analyzers that use laser energy as a means to desorb, volatilize, and ionize analytes. Tandem mass spectrometers include any mass spectrometer that can perform two successive stages of m / z-based discrimination or measurement of ions, including ions in an ion mixture. The phrase includes mass spectrometers equipped with two mass analyzers that can perform two successive stages of m / z-based discrimination or spatial tandem measurement of ions. The phrase further includes mass spectrometers having a single mass analyzer capable of performing two successive stages of m / z-based discrimination or tandem measurement in time of ions. Thus, the phrase expressly includes Qq-TOF mass spectrometers, ion trap mass spectrometers, ion trap-TOF mass spectrometers, TOF-TOF mass spectrometers, Fourier transform ion cyclotron resonance mass spectrometers, electrostatic sector-magnetic sector mass spectrometers, and combinations thereof.

[0078] Ionization in mass spectrometry involves a process in which analytes in a sample are ionized. Such analytes may become charged molecules that are used for further analysis. For example, ionization of a sample can be performed by electrospray ionization (ESI), laser spray ionization (LSI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB) / liquid secondary ionization (LSIMS), matrix-assisted laser desorption ionization (MALDI), field ionization, field desorption, thermospray / plasma spray ionization, and particle beam ionization. Those skilled in the art will understand that the selection of the ionization method can be determined based on the analyte to be measured, the type of sample, the type of detector, the selection of positive mode vs. negative mode, etc.

[0079] A mass analyzer comprises a mass analyzer component that takes the ionized masses, separates them based on their m / z ratio, and outputs them to a detector where they are detected and later converted to a digital output. Suitable mass analyzers for determining m / z ratio include quadrupole mass analyzers, time-of-flight (TOF) mass analyzers, magnetic or electrostatic sector mass analyzers, and ion trap (e.g., ion cyclotron resonance) mass analyzers.

[0080] Selected reaction monitoring (SRM)-MS assays target a set of predefined peptides for a given protein of interest. SRM is a tandem mass spectrometry mode that selects ions of a specific mass (parent or precursor ions) in the first stage of tandem mass spectrometry and selects the ion products of the fragmentation reaction of the precursor ions in the second mass spectrometry stage for detection. The specific pairs of m / z values ​​associated with the selected precursor ions and fragment ions are called transitions. For each signature peptide, a fragment ion is identified that provides optimal signal intensity and distinguishes the targeted peptide from other species present in the sample. Optimized transitions contribute to an effective SRM assay. Several such transitions (precursor / fragment ion pairs) are monitored over time, and a series of chromatographic traces are generated that have as coordinates the retention times and signal intensities of the specific transitions. SRM-MS analysis of signature peptides is typically performed on a triple quadrupole mass spectrometer (QQQ-MS), an instrument with the capability to selectively isolate precursor ions corresponding to the m / z of the signature peptide and selectively monitor peptide-specific fragment ions. In SRM analysis, specificity relies on multiple mass analyzers (mass filters). The first quadrupole is for selecting the desired parent or precursor ion. The third quadrupole is for monitoring the (one or more) fragment ion(s). The fragment ion(s) are generated by collision-induced dissociation in the second quadrupole. The two levels of mass selection allow high selectivity, since co-eluting background ions are filtered very effectively. Unlike conventional tandem mass spectrometry (MS / MS) experiments that interrogate all analytes in a sample, SRM analysis selectively targets (filters) specific analytes, which means that sensitivity is improved by one or two orders of magnitude compared to conventional "full scan" techniques. Moreover, SRM provides a linear response over a wide dynamic range of up to five orders of magnitude, enabling the detection of low-abundance proteins in highly complex mixtures.Therefore, SRM is a highly specific detection / monitoring method with low background interference. When multiple parent ions are monitored in one MS run, this type of analysis is known as multiple reaction monitoring (MRM). Using MRM analysis, multiple proteins and multiple regions of proteins (signature peptides) can be monitored in one mass spectrometry run. Selected reaction monitoring / multiple reaction monitoring mass spectrometry (SRM / MRM-MS) is described, for example, in U.S. Pat. No. 8,383,417, WO2013 / 106603, and U.S. Patent Application Publication No. 2013 / 105684.

[0081] In certain embodiments, the following parameters may be used to specify an LC-SRM-MS assay of a protein under a particular LC-SRM-MS system: (1) the concentration of tryptic peptides of a given protein; (2) the retention time (RT) of the peptide on the LC column; (3) the m / z value of the peptide precursor ion; (4) the declustering potential used to ionize the precursor ion; (5) the m / z value of the fragment ions generated from the peptide precursor ion; and (6) the collision energy (CE) used to fragment the peptide precursor ion optimized for a particular peptide. The RT includes the time elapsed from injection to elution of the analyte. The declustering potential (DP) includes the potential to dissolve and dissociate the ion clusters. It is also known as the "fragment voltage" or "ion transfer capillary offset voltage" depending on the manufacturer. The collision energy (CE) includes the amount of energy that the precursor ion receives when it is accelerated into the collision cell.

[0082] To facilitate accurate quantification of peptides by the methods disclosed herein, a set of isotopically labeled synthetic versions of the peptides of interest may be added to the sample in known amounts for use as internal standards. Since the isotopically labeled peptides have identical physical and chemical properties to their corresponding surrogate peptides, they will co-elute from the chromatographic column and be easily distinguishable in the resulting mass spectrum. Gerber et al. Proc. Natl. Assos. Sci. 2003; 100:6940-6945; Kirkpatrick et al. Methods 2005; 35:265-273. Isotopes that can label amino acids of a given peptide include: 13 C. 2 H, 15 N, 17 O. 18 O, and 34 In certain embodiments, the peptide is 13 C and / or 15 The peptide is labeled with a heavy isotope of N. The addition of the labeled standard may be performed before or after the proteolytic digestion. In certain embodiments, the labeled internal standard peptide is added before the LC-MRM-MS assay. Methods for synthesizing isotopically labeled peptides will be known to those skilled in the art. Thus, in certain embodiments, the experimental sample comprises an internal standard peptide. In certain embodiments, the internal standard peptide comprises a reference signature peptide. In certain embodiments, the signature peptide concentration may be determined by combining: (i) a ratio calculated from comparing the peak area of ​​the signature peptide with the peak area of ​​its corresponding reference signature peptide obtained from the LC-MRM-MS assay, and (ii) a known concentration of the reference signature peptide. The peptides selected as reference standards and suitable for quantification are sometimes called quantum peptides (Q-peptides). Q-peptides contain all the characteristics of proteotypic peptides, but also impose restrictions on the residues that may constitute the reference peptide to eradicate artificial modifications and / or incomplete cleavage. Holman et al.Bioanalysis 2012;4(14):1763-1786.

[0083] The absolute quantitative level of a given protein(s) can be determined by SRM / MRM methodology, whereby the SRM / MRM signature peak area of ​​individual peptides from a given protein in one biological sample is compared to the SRM / MRM signature peak area of ​​a known amount of "spiked" internal standard. In certain embodiments, the internal standard is a synthetic version of the same exact peptide containing one or more amino acid residues labeled with one or more heavy isotopes. Such isotopically labeled internal standards are synthesized such that mass spectrometry produces predictable and consistent SRM / MRM signature peaks that are different and distinct from the native peptide signature peaks and can be used as comparator peaks. Thus, when the internal standard is spiked in a known amount into a protein preparation from a biological sample and analyzed by mass spectrometry, the signature peak area of ​​the native peptide is compared to the signature peak area of ​​the internal standard peptide, and this numerical comparison indicates either the absolute molar concentration and / or absolute weight of the native peptide present in the original protein preparation from the biological sample. Absolute quantification data of fragment peptides is displayed according to the amount of protein analyzed per sample. Absolute quantification may be performed across many peptides, and therefore proteins, simultaneously in a single sample and / or across many samples to provide insight into the absolute amounts of proteins in individual biological samples and across cohorts of individual samples.

[0084] Another strategy for absolute quantification of peptides is equimolar with equalizer peptides. This methodology involves chemically synthesizing isotopically labeled Q-peptides of interest as dipeptides. A common amino acid sequence is located at the N-terminus of the Q-peptide and is called the equalizer peptide. After solubilization and proteolytic digestion, the amount of Q-peptide can be accurately determined by reference to a single photolabeled peptide. An appropriate amount of each standard peptide is then added to the sample of interest (either predigested or preproteolytic) to facilitate absolute quantification. Holzmann et al. Anal. Chem. 2009;81:10254-10261. Quantitative concatemer (QconCAT) proteins may be used for absolute quantification. Beynon et al. Nat. Methods 2005;2:587-589;Johnson et al. J. Am. Soc. Mass Spectrom. 2009;20:2211-2220;Ding et al. J. Proteome Res. 2011;10:3652-3659;Caroll et al. Molecular & Cellular Proteomics 2011;Sep 19:mcp-M111. In this strategy, a recombinant artificial protein that is a concatenation of affinity-tagged standard peptides from several proteins of interest is heterologously produced in Escherichia coli grown in stable isotope-enriched medium. The QconCAT protein is then affinity purified and co-digested with the sample to generate a stoichiometric mixture of all its constituent "heavy" Q peptides, which are then analyzed by proteolytic peptides from the native protein and the internal standards. A variation of the QconCAT approach, called peptide-concatenated standards (PCS), uses flanking regions between Q-peptides within an artificial protein sequence that reflects the endogenous environment. Kito et al. J. Proteome Res. 2007;6:792-800. Other specific embodiments use protein standards for absolute quantification (PSAQ). Brun et al. Mol. Cell. Proteomics 2007;6:2139-2149.PSAQ uses recombinant proteins, but rather than a concatenation of peptides from several proteins, the entire protein to be quantified is expressed in a stable isotope-labeled form. One or more PSAQs may then be added to the sample pre-digestion to facilitate quantification.

[0085] Certain embodiments use label-free strategies for protein quantification, such as intensity-based measurements (America and Cordewener, Proteomics 2008;8:731-749) or spectral counting (Lundgren et al. Expert Rev. Proteomics 2010;7:39-53).

[0086] To obtain relative quantitative levels of a given peptide, the mass spectrometry-derived signature peak area (or peak height if the peaks are well resolved) of an individual peptide or peptides from a given protein in one biological sample may be compared to the signature peak area determined for the same peptide or peptides from the same protein in one or more additional and different biological samples using the same SRM / MRM approach. In this way, the amount of a particular peptide(s) from a given protein is determined in comparison to the same peptide or peptides from the same protein across two or more biological samples under the same experimental conditions. Furthermore, SRM / MRM approaches may determine relative quantification for a given peptide(s) from a single protein in a single sample by comparing the signature peak area of ​​that peptide of the given protein against the signature peak area for another and different peptide(s) from a different protein in the same protein preparation from the biological sample. In this way, the amount of a particular peptide from a given protein, and therefore the amount of the given protein, is determined in comparison to another protein in the same sample. These approaches quantify individual peptide(s) from a given protein relative to the amount of another peptide(s) from the same or different proteins between and within samples, where the amounts determined by signature peak areas are relative to each other, regardless of the absolute weight-to-volume or weight-to-weight amounts of the peptides in the protein preparation from this biological sample. Relative quantification data for individual signature peak areas between different samples may be normalized to the amount of protein analyzed per sample. Relative quantification may be performed across many peptides simultaneously in a single sample and / or across many samples to gain insight into relative protein abundance.

[0087] Signature peptide levels may be expressed in concentration units (e.g., pmol / L). In certain embodiments, the average concentration of the signature peptide in test samples from subjects being screened for SCID, WAS, XLA, cystinosis, and / or WD may be compared to the average concentration of the corresponding peptide in normal control samples. In certain embodiments, the normal control samples may be derived from one or more normal control subjects, or from a population of normal control subjects. In certain embodiments, normal control subjects include subjects that do not have or are not known to have SCID, WAS, XLA, cystinosis, or WD. In certain embodiments, normal control subjects include subjects that do not have a genetic mutation associated with SCID, WAS, XLA, cystinosis, or WD.

[0088] In certain embodiments, the mean concentration of CD3ε signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 70 pmol / L to 400 pmol / L, 80 pmol / L to 300 pmol / L, and 90 pmol / L to 200 pmol / L. In certain embodiments, the mean concentration of CD3ε signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 70 pmol / L, 80 pmol / L, 90 pmol / L, 100 pmol / L, 110 pmol / L, 120 pmol / L, 130 pmol / L, 140 pmol / L, 150 pmol / L, 160 pmol / L, 170 pmol / L, 180 pmol / L, 190 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, 280 pmol / L, 290 pmol / L, 300 pmol / L, 310 pmol / L, 320 pmol / L, 330 pmol / L, 340 pmol / L, 350 pmol / L, 360 pmol / L, 370 pmol / L, 380 pmol / L, 390 pmol / L, 400 pmol / L, 410 pmol / L, 420 pmol / L, 430 pmol / L, 440 pmol / L, 450 pmol / L, 460 pmol / L, 470 pmol / L, 480 pmol / L, 490 pmol / L, 500 pmol / L, 500 pmol / L 20pmol / L, 230pmol / L, 240pmol / L, 250pmol / L, 260pmol / L, 270pmol / L, 280pmol / L, 290pmol / L, 300pmol / L, 310pmol / L , 320pmol / L, 330pmol / L, 340pmol / L, 350pmol / L, 360pmol / L, 370pmol / L, 380pmol / L, 390pmol / L, 400pmol / L or higher.

[0089] In certain embodiments, the mean concentrations of WASp 274 signature peptide in DBS from a population of normal control subjects include concentrations in the ranges of 600 pmol / L to 5000 pmol / L, 700 pmol / L to 3000 pmol / L, and 800 pmol / L to 2500 pmol / L. The mean concentrations of the 274 signature peptides were 600 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1100 pmol / L, 1200 pmol / L, 1300 pmol / L, 1400 pmol / L, 1500 pmol / L, 1600 pmol / L, 1700 pmol / L, 1800 pmol / L, 1900 pmol / L, 2000 pmol / L, 2100 pmol / L, 2200 pmol / L, 2300 pmol / L, 2400 pmol / L, 2500 pmol / L, 2600 pmol / L, 2700 pmol / L, and 2800 pmol / L. 800pmol / L, 2900pmol / L, 3000pmol / L, 3100pmol / L, 3200pmol / L, 3300pmol / L, 3400pmol / L, 3500pmol / L, 3600pmol / L, 3700pmol / L, 3800pmol / L, 3900pmol / L , 4000pmol / L, 4100pmol / L, 4200pmol / L, 4300pmol / L, 4400pmol / L, 4500pmol / L, 4600pmol / L, 4700pmol / L, 4800pmol / L, 4900pmol / L, 5000pmol / L or higher.

[0090] In certain embodiments, the mean concentrations of WASp 289 signature peptide in DBS from a population of normal control subjects include concentrations in the ranges of 5500 pmol / L to 15000 pmol / L, 6000 pmol / L to 14000 pmol / L, and 6500 pmol / L to 13000 pmol / L. The mean concentrations of the 289 signature peptides were 5500 pmol / L, 5600 pmol / L, 5700 pmol / L, 5800 pmol / L, 5900 pmol / L, 6000 pmol / L, 6100 pmol / L, 6200 pmol / L, 6300 pmol / L, 6400 pmol / L, 6500 pmol / L, 6600 pmol / L, 6700 pmol / L, 6800 pmol / L, 6900 pmol / L, 7000 pmol / L, and 7100 pmol / L. L, 7200pmol / L, 7300pmol / L, 7400pmol / L, 7500pmol / L, 7600pmol / L, 7700pmol / L, 7800pmol / L, 7900pmol / L, 8000pmol / L, 81 00pmol / L, 8200pmol / L, 8300pmol / L, 8400pmol / L, 8500pmol / L, 8600pmol / L, 8700pmol / L, 8800pmol / L, 8900pmol / L, 9000pm ol / L, 9800pmol / L, 9900pmol / L, 10000pmol / L, 10100pmol / L, 10200pmol / L, 10300pmol / L, 10400pmol / L, 10500pmol / L, 106 00pmol / L, 10700pmol / L, 10800pmol / L, 10900pmol / L, 11000pmol / L, 11100pmol / L, 11200pmol / L, 11300pmol / L, 11400pmol / L, 11500pmol / L, 11600pmol / L, 11700pmol / L, 11800pmol / L, 11900pmol / L, 12000pmol / L, 12100pmol / L, 12200pmol / L, 12300 pmol / L, 12400pmol / L, 12500pmol / L, 12600pmol / L, 12700pmol / L, 12800pmol / L, 12900pmol / L, 13000pmol / L, 13100pmol / L,13200pmol / L, 113300pmol / L, 13400pmol / L, 13500pmol / L, 13600pmol / L, 13700pmol / L, 13800pmol / L, 13900pmol / L, 14000pmol / L, 14100pmol / L, 14200pmol / L, 14300pmol / L, 14400pmol / L, 14500pmol / L, 14600pmol / L, 14700pmol / L, 14800pmol / L, 14900pmol / L, 15000pmol / L or higher. ,

[0091] In certain embodiments, the average concentration of the BTK 407 signature peptide in DBS from a population of normal control subjects includes concentrations in the ranges of 350 pmol / L to 2500 pmol / L, 450 pmol / L to 2400 pmol / L, and 550 pmol / L to 2300 pmol / L. In certain embodiments, the average concentration of the BTK 407 signature peptide in DBS from a population of normal control subjects includes concentrations in the ranges of 350 pmol / L, 400 pmol / L, 450 pmol / L, 500 pmol / L, 550 pmol / L, 600 pmol / L, 650 pmol / L, 700 pmol / L, 750 pmol / L, 800 pmol / L, 850 pmol / L, 900 pmol / L, 1000 pmol / L, 1100 pmol / L, Includes concentrations of 1200pmol / L, 1300pmol / L, 1400pmol / L, 1500pmol / L, 1600pmol / L, 1700pmol / L, 1800pmol / L, 1900pmol / L, 2000pmol / L, 2100pmol / L, 2200pmol / L, 2300pmol / L, 2400pmol / L, 2500pmol / L or greater.

[0092] In certain embodiments, the mean concentrations of the BTK 545 signature peptide in DBS from a population of normal control subjects include concentrations in the ranges of 550 pmol / L to 1600 pmol / L, 650 pmol / L to 1500 pmol / L, and 750 pmol / L to 1400 pmol / L. In certain embodiments, the average concentration of the BTK 545 signature peptide in DBS from a population of normal control subjects includes concentrations of 550 pmol / L, 600 pmol / L, 650 pmol / L, 700 pmol / L, 750 pmol / L, 800 pmol / L, 850 pmol / L, 900 pmol / L, 950 pmol / L, 1000 pmol / L, 1050 pmol / L, 1100 pmol / L, 1150 pmol / L, 1200 pmol / L, 1250 pmol / L, 1300 pmol / L, 1350 pmol / L, 1400 pmol / L, 1450 pmol / L, 1500 pmol / L, 1550 pmol / L, 1600 pmol / L or greater.

[0093] In certain embodiments, the average concentration of the CTNS 115 signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 40 pmol / L to 250 pmol / L, the range of 50 pmol / L to 200 pmol / L, and the range of 60 pmol / L to 150 pmol / L. In certain embodiments, the average concentration of the CTNS 115 signature peptide in DBS from a population of normal control subjects includes concentrations of 40 pmol / L, 45 pmol / L, 50 pmol / L, 55 pmol / L, 60 pmol / L, 65 pmol / L, 70 pmol / L, 75 pmol / L, 80 pmol / L, 85 pmol / L, 90 pmol / L, 95 pmol / L, 100 pmol / L, 110 pmol / L, 120 pmol / L, 130 pmol / L, 140 pmol / L, 150 pmol / L, 160 pmol / L, 170 pmol / L, 180 pmol / L, 190 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L or greater.

[0094] In certain embodiments, the average concentration of SHPK 363 signature peptide in DBS from a population of normal control subjects includes concentrations in the ranges of 100 pmol / L to 8000 pmol / L, 300 pmol / L to 7000 pmol / L, and 500 pmol / L to 6000 pmol / L. In certain embodiments, the average concentration of SHPK 363 signature peptide in DBS from a population of normal control subjects includes concentrations of 100 pmol / L, 200 pmol / L, 300 pmol / L, 400 pmol / L, 500 pmol / L, 600 pmol / L, 700 pmol / L, 800 pmol / L, 900 pmol / L, 1000 pmol / L, 1500 pmol / L, 2000 pmol / L, 2500 pmol / L, 3000 pmol / L, 3500 pmol / L, 4000 pmol / L, 4500 pmol / L, 5000 pmol / L, 5500 pmol / L, 6000 pmol / L, 6500 pmol / L, 7000 pmol / L, 7500 pmol / L, 8000 pmol / L or greater.

[0095] In certain embodiments, the average concentration of the ATP7B 214 signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 30 pmol / L to 100 pmol / L, the range of 40 pmol / L to 90 pmol / L, and the range of 50 pmol / L to 80 pmol / L. In certain embodiments, the average concentration of the ATP7B 214 signature peptide in DBS from a population of normal control subjects includes concentrations of 30 pmol / L, 35 pmol / L, 40 pmol / L, 45 pmol / L, 50 pmol / L, 55 pmol / L, 60 pmol / L, 65 pmol / L, 70 pmol / L, 75 pmol / L, 80 pmol / L, 85 pmol / L, 90 pmol / L, 95 pmol / L, 100 pmol / L or more.

[0096] In certain embodiments, the average concentration of the ATP7B 887 signature peptide in DBS from a population of normal control subjects includes concentrations in the ranges of 200 pmol / L to 500 pmol / L, 250 pmol / L to 450 pmol / L, and 300 pmol / L to 400 pmol / L. In certain embodiments, the average concentration of the ATP7B 887 signature peptide in DBS from a population of normal control subjects includes concentrations in the ranges of 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, 250 pmol / L, 260 pmol / L, 270 pmol / L, 280 pmol / L, 290 pmol / L, 300 pmol / L, 310 pmol / L, 320 pmol / L, 330 pmol / L, 340 pmol / L, , 350pmol / L, 360pmol / L, 370pmol / L, 380pmol / L, 390pmol / L, 400pmol / L, 410pmol / L, 420pmol / L, 430 Concentrations include pmol / L, 440pmol / L, 450pmol / L, 460pmol / L, 470pmol / L, 480pmol / L, 490pmol / L, 500pmol / L or higher.

[0097] In certain embodiments, the average concentration of the ATP7B 1056 signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 90 pmol / L to 400 pmol / L, 100 pmol / L to 300 pmol / L, and 150 pmol / L to 250 pmol / L. In certain embodiments, the average concentration of the ATP7B 1056 signature peptide in DBS from a population of normal control subjects includes concentrations in the range of 90 pmol / L, 100 pmol / L, 110 pmol / L, 120 pmol / L, 130 pmol / L, 140 pmol / L, 150 pmol / L, 160 pmol / L, 170 pmol / L, 180 pmol / L, 190 pmol / L, 200 pmol / L, 210 pmol / L, 220 pmol / L, 230 pmol / L, 240 pmol / L, and 250 pmol / L. mol / L, 250pmol / L, 260pmol / L, 270pmol / L, 280pmol / L, 290pmol / L, 300pmol / L, 310pmol / L, 320pmol / L, 3 Concentrations include 30pmol / L, 340pmol / L, 350pmol / L, 360pmol / L, 370pmol / L, 380pmol / L, 390pmol / L, 400pmol / L or higher.

[0098] The one or more standard peptides may be synthesized by any method known in the art. Such synthetic peptides may further include amino acids with one or more natural modifications. Such natural modifications may include deamination, amination, oxidation, and hydroxylation of glutamine and asparagine.

[0099] (VIII) Methods of Use. The methods of the present disclosure include identifying an individual having one or more of SCID, WAS, XLA, cystinosis, and / or WD. In certain embodiments, diagnosis of an individual having SCID, WAS, XLA, cystinosis, and / or WD is performed early, for example, as part of NBS or before symptoms of the disorder are evident in the individual.

[0100] The method of the present disclosure includes obtaining a DBS sample. In certain embodiments, the DBS is obtained according to the above method. In certain embodiments, the DBS is obtained from a DBS repository or laboratory that stores DBS for future testing.

[0101] The method of the present disclosure includes digesting the proteins in DBS with digestive enzymes. In certain embodiments, one or more punches of DBS or the whole DBS can be solubilized in a suitable buffer, and the above-mentioned suitable digestive enzymes can be added to digest the proteins present in DBS into peptide fragments. In certain embodiments, DBS can be solubilized with 0.1% ProteaseMax™ in 50 mM ammonium bicarbonate (pH 8) and digested with trypsin.

[0102] The methods of the disclosure include enrichment of signature peptides used for screening for SCID, WAS, XLA, cystinosis, and / or WD. Signature peptides include CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 120 for cystinosis; CTNS 194 for cystinosis; CTNS 360 for cystinosis; SHPK 44 for cystinosis; SHPK 363 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 325 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; ATP7B 1056 for WD; and ATP7B 1061 for WD. In certain embodiments, signature peptides include those peptides disclosed in FIG. 1: CD3ε 197 for SCID; WASp 274 for WAS; WASp 289 for WAS; BTK 407 for XLA; BTK 545 for XLA; CTNS 115 for cystinosis; CTNS 360 for cystinosis; SHPK 363 for cystinosis; ATP7B 325 for WD; and ATP7B 1056 for WD. In certain embodiments, the signature peptides include peptides in Table 1: CTNS 120 for cystinosis; CTNS 194 for cystinosis; SHPK 44 for cystinosis; SHPK 388 for cystinosis; ATP7B 214 for WD; ATP7B 466 for WD; ATP7B 589 for WD; ATP7B 621 for WD; ATP7B 887 for WD; and ATP7B 1061 for WD. In certain embodiments, enriching for signature peptides includes contacting the mixture of peptide fragments from digested DBS with one or more binding entities that recognize the signature peptides. In certain embodiments, the binding entity is an antibody or an antigen-binding fragment thereof. In certain embodiments, the antibody includes those disclosed in Tables 2 and 3 above.In certain embodiments, the amino acid sequences of the antibodies of the disclosure include SEQ ID NOs: 79, 81, 83, 85, 86, 88, 90, 91, 93, 95, 96, 98, 100, 101, 103, 105, 106, 108, 110, 111, 113, 115, 116, 118, 120, 121, 123, 125, 126, 128, 130, 131, 133, 135, 136, 138, 140, and 141. In certain embodiments, the coding sequences of the antibodies of the disclosure include SEQ ID NOs: 78, 80, 82, 84, 87, 89, 92, 94, 97, 99, 102, 104, 107, 109, 112, 114, 117, 119, 122, 124, 127, 129, 132, 134, 137, and 139. In particular embodiments, the antibodies include those that bind to CD3ε 197, WASp 274, WASp 289, BTK 407, BTK 545, CTNS 115, CTNS 120, CTNS 194, SHPK 44, SHPK 363, SHPK 388, ATP7B 214, ATP7B 325, ATP7B 466, ATP7B 589, ATP7B 621, ATP7B 887, ATP7B 1056, and ATP7B 1061.

[0103] In a particular embodiment, antibodies comprising SEQ ID NOs: 22-27, 63, and 64 are used to enrich for CD3ε peptides comprising SEQ ID NO:1.

[0104] In certain embodiments, antibodies including SEQ ID NOs: 28-33, 65, and 66 are used to enrich for WASp peptides including SEQ ID NO: 2. In certain embodiments, antibodies are used to enrich for WASp peptides including SEQ ID NO: 3.

[0105] In certain embodiments, antibodies including SEQ ID NOs:34-38, 67, and 68 are used to enrich for BTK peptides including SEQ ID NO:4. In certain embodiments, antibodies are used to enrich for BTK peptides including SEQ ID NO:5.

[0106] In certain embodiments, the following antibody combinations may be used to screen for SCID, WAS, and XLA: antibodies that bind to CD3ε peptide, including SEQ ID NO:1, including SEQ ID NO:22-27, 63, and 64; antibodies that bind to WASp peptide, including SEQ ID NO:2, including SEQ ID NO:28-33, 65, and 66; antibodies that bind to WASp peptide, including SEQ ID NO:3; antibodies that bind to BTK peptide, including SEQ ID NO:4, including SEQ ID NO:34-38, 67, and 68; and antibodies that bind to BTK peptide, including SEQ ID NO:5.

[0107] In certain embodiments, the following antibody combinations may be used to screen for SCID, WAS, and XLA: antibodies that bind to CD3ε peptides, including SEQ ID NO:1, including SEQ ID NO:22-27, 63, and 64; antibodies that bind to WASp peptides, including SEQ ID NO:2, including SEQ ID NO:28-33, 65, and 66; antibodies that bind to WASp peptides, including SEQ ID NO:3; and antibodies that bind to BTK peptides, including SEQ ID NO:4, including SEQ ID NO:34-38, 67, and 68.

[0108] In certain embodiments, the following antibody combinations may be used to screen for SCID, WAS, and XLA: antibodies that bind to CD3ε peptides, including SEQ ID NO:1, including SEQ ID NO:22-27, 63, and 64; antibodies that bind to WASp peptides, including SEQ ID NO:2, including SEQ ID NO:28-33, 65, and 66; antibodies that bind to BTK peptides, including SEQ ID NO:4, including SEQ ID NO:34-38, 67, and 68; and antibodies that bind to BTK peptides, including SEQ ID NO:5.

[0109] In certain embodiments, the following antibody combinations may be used to screen for SCID, WAS, and XLA: antibodies that bind to CD3ε peptide, including SEQ ID NO:1, including SEQ ID NO:22-27, 63, and 64; antibodies that bind to WASp peptide, including SEQ ID NO:2, including SEQ ID NO:28-33, 65, and 66; and antibodies that bind to BTK peptide, including SEQ ID NO:4, including SEQ ID NO:34-38, 67, and 68.

[0110] In certain embodiments, antibodies including SEQ ID NOs: 39-44, 69, and 70 are used to enrich for CTNS peptides including SEQ ID NO: 6. In certain embodiments, antibodies including SEQ ID NOs: 45-50, 71, and 72 are used to enrich for CTNS peptides including SEQ ID NOs: 7 and 8. In certain embodiments, antibodies are used to enrich for CTNS peptides of SEQ ID NO: 12. In certain embodiments, antibodies are used to enrich for CTNS peptides of SEQ ID NO: 13.

[0111] In certain embodiments, antibodies including SEQ ID NOs:51-56, 73, and 74 are used to enrich for SHPK peptides including SEQ ID NO:9. In certain embodiments, antibodies are used to enrich for SHPK peptides of SEQ ID NO:14. In certain embodiments, antibodies are used to enrich for SHPK peptides of SEQ ID NO:15.

[0112] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0113] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 9, including SEQ ID NO: 51-56, 73, and 74; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0114] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies that bind to CTNS peptides, including SEQ ID NO: 6, including SEQ ID NO: 39-44, 69, and 70; antibodies that bind to CTNS peptides, including SEQ ID NO: 7 and 8, including SEQ ID NO: 45-50, 71, and 72; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 9, including SEQ ID NO: 51-56, 73, and 74; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0115] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0116] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0117] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0118] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0119] In certain embodiments, the following combinations of antibodies may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NO: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0120] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies including SEQ ID NO: 12; antibodies including SEQ ID NO: 13 that bind to CTNS peptides; and antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9.

[0121] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0122] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0123] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0124] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0125] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NO: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 14.

[0126] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: an antibody that binds to a CTNS peptide, including SEQ ID NO: 12; an antibody that binds to a CTNS peptide, including SEQ ID NO: 13; an antibody that binds to an SHPK peptide, including SEQ ID NO: 9, including SEQ ID NO: 51-56, 73, and 74; an antibody that binds to an SHPK peptide, including SEQ ID NO: 14; and an antibody that binds to an SHPK peptide, including SEQ ID NO: 15.

[0127] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NO: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0128] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NO: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0129] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0130] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0131] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies including SEQ ID NO: 13 that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0132] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies including SEQ ID NO: 12 that bind to CTNS peptides, including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0133] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0134] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0135] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0136] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0137] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0138] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0139] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0140] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0141] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; and antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9.

[0142] In certain embodiments, the following antibody combinations may be used to screen for cystinosis: antibodies comprising SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NO: 6; and antibodies comprising SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9.

[0143] One skilled in the art will recognize that other combinations of antibodies selected from the following may be used to screen for cystinosis: antibodies including SEQ ID NOs: 39-44, 69, and 70 that bind to CTNS peptides, including SEQ ID NOs: 6; antibodies including SEQ ID NOs: 45-50, 71, and 72 that bind to CTNS peptides, including SEQ ID NOs: 7 and 8; antibodies that bind to CTNS peptides, including SEQ ID NO: 12; antibodies that bind to CTNS peptides, including SEQ ID NO: 13; antibodies including SEQ ID NOs: 51-56, 73, and 74 that bind to SHPK peptides, including SEQ ID NO: 9; antibodies that bind to SHPK peptides, including SEQ ID NO: 14; and antibodies that bind to SHPK peptides, including SEQ ID NO: 15.

[0144] In certain embodiments, antibodies are used to enrich for ATP7B peptides comprising SEQ ID NO: 10. In certain embodiments, antibodies comprising SEQ ID NOs: 57-62, 75, and 76 are used to enrich for ATP7B peptides comprising SEQ ID NO: 11 or 21. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 16. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 17. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 18. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 19. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 20. In certain embodiments, antibodies are used to enrich for ATP7B peptides of SEQ ID NO: 21.

[0145] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0146] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0147] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0148] In certain embodiments, the following combinations of antibodies may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0149] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0150] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...8; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0151] In certain embodiments, the following combinations of antibodies may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0152] In certain embodiments, the following combinations of antibodies may be used to screen for WD: an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 10; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 16; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 17; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 18; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 19; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 20; and an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 21.

[0153] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0154] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 19.

[0155] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0156] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0157] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...8; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0158] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0159] In certain embodiments, the following combinations of antibodies may be used to screen for WD: an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 10; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 16; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 17; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 18; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 19; and an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 20.

[0160] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0161] In certain embodiments, the following combinations of antibodies may be used to screen for WD: an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 16; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 17; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 18; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 19; an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 20; and an antibody that binds to an ATP7B peptide comprising SEQ ID NO: 21.

[0162] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0163] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0164] In certain embodiments, the following combinations of antibodies may be used to screen for WD: antibodies comprising SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0165] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0166] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies including SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0167] In certain embodiments, the following combinations of antibodies may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...9; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0168] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...8; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0169] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...8; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0170] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0171] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0172] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 1 ...7; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0173] In certain embodiments, the following antibody combinations may be used to screen for WD: antibodies comprising SEQ ID NOs: 57-62, 75, and 76 that bind to ATP7B peptides, including SEQ ID NO: 11 or 21; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 20.

[0174] One skilled in the art will recognize that other combinations of antibodies selected from the following may be used to screen for WD: antibodies that bind to ATP7B peptides, including SEQ ID NO: 10; antibodies that bind to ATP7B peptides, including SEQ ID NO: 11 or 21, including SEQ ID NO: 57-62, 75, and 76; antibodies that bind to ATP7B peptides, including SEQ ID NO: 16; antibodies that bind to ATP7B peptides, including SEQ ID NO: 17; antibodies that bind to ATP7B peptides, including SEQ ID NO: 18; antibodies that bind to ATP7B peptides, including SEQ ID NO: 19; antibodies that bind to ATP7B peptides, including SEQ ID NO: 20; and antibodies that bind to ATP7B peptides, including SEQ ID NO: 21.

[0175] The disclosed methods optionally include performing liquid chromatography on the immunoaffinity-enriched peptides to separate the peptides prior to MS analysis, which may separate the peptides based on their weight and affinity for the mobile and stationary phases of the column.

[0176] The method of the present disclosure comprises performing SRM-MS or MRM-MS on the immunoaffinity enriched peptides to quantify the amount of a given signature peptide. In certain embodiments, SRM-MS or MRM-MS is performed as described above. In certain embodiments, quantifying the signature peptide comprises using a reference peptide that is introduced into the assay in a known amount. In certain embodiments, the reference peptide can be identical to the signature peptide in all respects, except that it is differentially labeled, for example, with one or more heavy isotopes, to distinguish the reference peptide from the signature peptide.

[0177] In certain embodiments, SRM-MS or MRM-MS detects the reduction or absence of CD3ε peptide. In certain embodiments, the CD3ε peptide comprises SEQ ID NO:1.

[0178] In certain embodiments, SRM-MS or MRM-MS detects a reduction or absence of the WASp peptide. In certain embodiments, the WASp peptide comprises SEQ ID NO: 2. In certain embodiments, the WASp peptide comprises SEQ ID NO: 3.

[0179] In certain embodiments, SRM-MS or MRM-MS detects a reduction or absence of the BTK peptide. In certain embodiments, the BTK peptide comprises SEQ ID NO: 4. In certain embodiments, the BTK peptide comprises SEQ ID NO: 5.

[0180] In certain embodiments, SRM-MS or MRM-MS detects a reduction or absence of the CTNS peptide. In certain embodiments, the CTNS peptide comprises SEQ ID NO: 6. In certain embodiments, the CTNS peptide comprises SEQ ID NO: 7 and 8. In certain embodiments, the CTNS peptide comprises SEQ ID NO: 12. In certain embodiments, the CTNS peptide comprises SEQ ID NO: 13.

[0181] In certain embodiments, SRM-MS or MRM-MS detects a decrease or absence of SHPK peptide. In certain embodiments, the SHPK peptide comprises SEQ ID NO: 9. In certain embodiments, the SHPK peptide comprises SEQ ID NO: 14. In certain embodiments, the SHPK peptide comprises SEQ ID NO: 15.

[0182] In certain embodiments, SRM-MS or MRM-MS detects a decrease or absence of ATP7B peptide. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 10. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 11 or 21. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 16. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 17. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 18. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 19. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 20. In certain embodiments, the ATP7B peptide comprises SEQ ID NO: 21.

[0183] In certain embodiments, a pre-determined cut-off value is used as the threshold value for the pre-determined signature peptide. A concentration of the pre-determined signature peptide that exceeds the threshold value indicates that the assayed DBS is from an individual that is not affected by SCID, WAS, XLA, cystinosis, or WD. A concentration of the pre-determined signature peptide that is below the threshold value or is absent indicates that the assayed DBS is from an individual that is affected by SCID, WAS, XLA, cystinosis, or WD. In certain embodiments, this threshold value can be determined by analyzing a population of normal controls and calculating the standard deviation (SD) of the concentration of the pre-determined signature peptide in this population. The threshold value can be set at a certain SD from the mean concentration of the pre-determined signature peptide. In certain embodiments, the threshold value is -1 SD, -1.1 SD, -1.2 SD, -1.3 SD, -1.4 SD, -1.5 SD, -1.6 SD, -1.7 SD, -1.8 SD, -1.9 SD, -2.0 SD, -2.1 SD, -2.2 SD, -2.3 SD, -2.4 SD, -2.5 SD, -2.6 SD, -2.7 SD, -2.8 SD, -2.9 SD, -3.0 SD, or more SD from the mean concentration of the predetermined signature peptide. In certain embodiments, for the diagnosis or screening of SCID, WAS, XLA, or cystinosis, the threshold value may be determined by analysis of a population of normal controls and calculation of the standard deviation (SD) of the ratio of the concentration of the predetermined signature peptide to the endogenous concentration of ATP7B in this population. The peptide concentration cutoff for each PIDD can be set to a particular SD derived from the mean concentration of each signature peptide or the ratio of the concentration of a particular signature peptide to the endogenous concentration of ATP7B.

[0184] In a particular embodiment, the threshold concentration of CD3ε 197 peptide is determined based on the CD3ε 197 peptide concentration in a population of normal controls. 197 mean concentrations included -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of CD3ε197 peptide include 60 pmol / L or less, 55 pmol / L or less, 50 pmol / L or less, 45 pmol / L or less, 40 pmol / L or less, 39 pmol / L or less, 38 pmol / L or less, 37 pmol / L or less, 36 pmol / L or less, 35 pmol / L or less, 34 pmol / L or less, 33 pmol / L or less, 32 pmol / L or less, 31 pmol / L or less, 30 pmol / L or less.

[0185] In a particular embodiment, the threshold concentration of the WASp 274 peptide is 274 mean concentrations including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of WASp 274 peptide include 220 pmol / L or less, 215 pmol / L or less, 210 pmol / L or less, 205 pmol / L or less, 200 pmol / L or less, 195 pmol / L or less, 190 pmol / L or less, 185 pmol / L or less, 180 pmol / L or less, 175 pmol / L or less, 170 pmol / L or less, 165 pmol / L or less, 160 pmol / L or less, 155 pmol / L or less, or 150 pmol / L or less.

[0186] In certain embodiments, the threshold concentration of the WASp 289 peptide is determined based on the level of WASp 289 in a normal control population. 289 mean concentrations including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of WASp 289 peptide include 2500 pmol / L or less, 2490 pmol / L or less, 2480 pmol / L or less, 2470 pmol / L or less, 2460 pmol / L or less, 2450 pmol / L or less, 2440 pmol / L or less, 2430 pmol / L or less, 2420 pmol / L or less, 2410 pmol / L or less, or 2400 pmol / L or less.

[0187] In certain embodiments, the threshold concentration of the BTK 407 peptide is determined based on the BTK concentration in a normal control population. 407 mean concentrations included -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of BTK 407 peptide include 80 pmol / L or less, 75 pmol / L or less, 70 pmol / L or less, 65 pmol / L or less, 60 pmol / L or less, 55 pmol / L or less, 50 pmol / L or less, 49 pmol / L or less, 48 ​​pmol / L or less, 47 pmol / L or less, 46 pmol / L or less, 45 pmol / L or less, 44 pmol / L or less, 43 pmol / L or less, 42 pmol / L or less, 41 pmol / L or less, 40 pmol / L or less, 35 pmol / L or less, or 30 pmol / L or less.

[0188] In certain embodiments, the threshold concentration of the BTK 545 peptide is determined based on the BTK concentration in a normal control population. including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more from the mean concentration of 545. In certain embodiments, threshold concentrations of BTK 545 peptide include 110 pmol / L or less, 109 pmol / L or less, 108 pmol / L or less, 107 pmol / L or less, 106 pmol / L or less, 105 pmol / L or less, 104 pmol / L or less, 103 pmol / L or less, 102 pmol / L or less, 101 pmol / L or less, or 100 pmol / L or less.

[0189] In a particular embodiment, the threshold concentration of the CTNS 115 peptide is determined based on the CTNS 115 concentration in a normal control population. 115 mean concentrations included -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of CTNS 115 peptide include 60 pmol / L or less, 59 pmol / L or less, 58 pmol / L or less, 57 pmol / L or less, 56 pmol / L or less, 55 pmol / L or less, 54 pmol / L or less, 53 pmol / L or less, 52 pmol / L or less, 51 pmol / L or less, or 50 pmol / L or less.

[0190] In a particular embodiment, the threshold concentration of SHPK 363 peptide is determined based on the SHPK 363 concentration in a normal control population. from 363 mean concentrations including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of SHPK 363 peptide include 2000 pmol / L or less, 1950 pmol / L or less, 1900 pmol / L or less, 1850 pmol / L or less, 1800 pmol / L or less, 1750 pmol / L or less, 1700 pmol / L or less, 1650 pmol / L or less, 1600 pmol / L or less, 1550 pmol / L or less, and 1500 pmol / L or less.

[0191] In a particular embodiment, the threshold concentration of the ATP7B 1056 peptide is determined based on the ATP7B from the mean concentrations of 1056, including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of ATP7B 1056 peptide include 90 pmol / L or less, 85 pmol / L or less, 80 pmol / L or less, 75 pmol / L or less, 70 pmol / L or less, 65 pmol / L or less, 60 pmol / L or less, 55 pmol / L or less, 50 pmol / L or less, 45 pmol / L or less, 40 pmol / L or less, 35 pmol / L or less, and 30 pmol / L or less.

[0192] In a particular embodiment, the threshold concentration of the ATP7B 214 peptide is determined based on the ATP7B 214 concentration in a normal control population. 214 mean concentrations included -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more. In certain embodiments, threshold concentrations of ATP7B 214 peptide include 30 pmol / L or less, 29 pmol / L or less, 28 pmol / L or less, 27 pmol / L or less, 26 pmol / L or less, 25 pmol / L or less, 24 pmol / L or less, 23 pmol / L or less, 22 pmol / L or less, 21 pmol / L or less, or 20 pmol / L or less.

[0193] In a particular embodiment, the threshold concentration of the ATP7B 887 peptide is determined based on the ATP7B including -1.0SD, -1.25SD, -1.3SD, -1.35SD, -1.4SD, -1.45SD, -1.5SD, -1.55SD, -1.6SD, -1.65SD, -1.7SD, -1.75SD, -1.8SD, -1.85SD, -1.9SD, -1.95SD, -2.0SD, -2.25SD, -2.3SD, -2.35SD, -2.4SD, -2.45SD, -2.5SD, -2.55SD, -2.6SD, -2.65SD, -2.7SD, -2.75SD, -2.8SD, -2.85SD, -2.9SD, -2.95SD, -3.0SD, or more from the mean concentration of 887. In certain embodiments, threshold concentrations of ATP7B 887 peptide include 190 pmol / L or less, 185 pmol / L or less, 180 pmol / L or less, 175 pmol / L or less, 170 pmol / L or less, 165 pmol / L or less, 160 pmol / L or less, 155 pmol / L or less, 150 pmol / L or less, 145 pmol / L or less, and 140 pmol / L or less.

[0194] In certain embodiments, the signature peptide may be considered as a primary biomarker for the diagnosis or screening of a given disease. The primary signature peptide may include a peptide that is first used to diagnose or screen for a given disease. In certain embodiments, the primary marker can be reproducibly obtained from digestion of the corresponding protein, has high affinity antibodies for immunoaffinity enrichment, and / or is reproducible across independent liquid chromatography columns and / or mass spectrometry instruments. In certain embodiments, the signature peptide may be considered as a secondary marker for the diagnosis or screening of a given disease. The secondary signature peptide may include a peptide that is secondly used to confirm the diagnosis or screening of a given disease with the primary marker. In certain embodiments, BTK 545 may be a secondary marker to BTK 407 in diagnosing XLA. In certain embodiments, WASp 289 may be a secondary marker to WASp 274 in diagnosing WAS.

[0195] In certain embodiments, the antibodies of the present disclosure may also be used in complementary clinical trials for the diagnosis of primary immunodeficiencies, cystinosis and WD for patients with equivocal biochemical results and for patients with variants of unknown significance by genetic testing.

[0196] The methods disclosed herein include treating a subject (e.g., a human) based on the results of screening for SCID, WAS, XLA, cystinosis, and / or WD with the compositions and methods disclosed herein. Treating a subject includes delivery of a therapeutically effective amount. A therapeutically effective amount includes an amount that provides an effective amount, prophylactic treatment, and / or therapeutic treatment.

[0197] "Effective amount" is the amount of composition required to produce a desired physiological change in a subject. For example, an effective amount can provide symptom relief, symptom elimination, or cure of SCID, WAS, XLA, cystinosis, and / or WD. Effective amounts are often administered for research purposes. Effective amounts disclosed herein can cause statistically significant effects in animal models or in vitro assays related to the evaluation of disease development, progression, and / or resolution.

[0198] Certain embodiments may include administering the composition as a "prophylactic treatment." Prophylactic treatment includes treatment administered to subjects who do not exhibit signs or symptoms of SCID, WAS, XLA, cystinosis, and / or WD, or who exhibit only early signs or symptoms of SCID, WAS, XLA, cystinosis, and / or WD, where the treatment is administered with the intent of reducing or decreasing the risk of developing the disorder. Thus, the prophylactic treatment serves as a preventative treatment against SCID, WAS, XLA, cystinosis, and / or WD.

[0199] In certain embodiments, prophylactic treatment may prevent, delay, or reduce the onset of SCID, WAS, XLA, cystinosis, and / or WD. In certain embodiments, prophylactic treatment may be administered before, at the same time, or after other preventive measures, such as the use of antibiotics for WAS and XLA. In certain embodiments, prophylactic treatment may prevent or reduce the severity of symptoms or complications associated with SCID, WAS, XLA, cystinosis, and / or WD. Symptoms and complications of SCID may include: failure to thrive; rashes that look like eczema; chronic diarrhea; recurrent oral candidiasis; and Pneumocystis pneumonia. Symptoms and complications of WAS may include: bleeding; eczema; bloody diarrhea; and recurrent infections. Symptoms and complications of XLA may include: infection; diarrhea; failure to thrive; joint disease; kidney inflammation; red blood cell breakdown; and inflammation of the skin and muscles. Symptoms and complications of cystinosis may include: polyuria, polydipsia, dehydration, vomiting, metabolic acidosis, hypophosphatemic rickets, constipation, failure to thrive, recurrent bouts of fever, heat intolerance, and anorexia / loss of appetite. Symptoms and complications of WD may include: fatigue, loss of appetite or abdominal pain, jaundice, golden-brown eye discoloration (Kayser-Fleischer rings), fluid accumulation in the legs or abdomen, problems with speech, swallowing, or physical coordination, and uncontrolled movements or muscle contractures.

[0200] "Therapeutic treatment" includes treatment administered to a subject exhibiting symptoms or signs of SCID, WAS, XLA, cystinosis, and / or WD, with the purpose of reducing or eliminating the signs or symptoms of SCID, WAS, XLA, cystinosis, and / or WD. In certain embodiments, the therapeutic treatment may provide immune function to a subject diagnosed with SCID, WAS, and / or XLA. In certain embodiments, the therapeutic treatment may reduce cystine accumulation in cells of a subject suffering from cystinosis. In certain embodiments, the therapeutic treatment may reduce copper accumulation in organs of a subject suffering from WD. In certain embodiments, the therapeutic treatment may reduce, control, or eliminate symptoms and complications of SCID, WAS, XLA, cystinosis, and / or WD, as described above.

[0201] Prophylactic and therapeutic treatments need not be mutually exclusive, and in certain embodiments, an administered dosage may accomplish more than one type of treatment.

[0202] In certain embodiments, the therapeutically effective amount provides immune system function to a subject diagnosed with SCID, WAS, and / or XLA. Thus, in certain embodiments, the methods of treatment disclosed herein include stem cell transplantation, immunoglobulin infusion, antibiotic infusion, and / or gene therapy for disorders such as SCID, WAS, and XLA. In certain embodiments, the methods of treatment include enzyme therapy for SCID. Providing immune function includes reducing the frequency or number of bacterial, viral, or parasitic infections, increasing life expectancy, and / or increasing growth.

[0203] In certain embodiments, the therapeutically effective amount prevents accumulation of cystine in cells of a subject diagnosed with cystinosis. In certain embodiments, the method of treatment comprises providing cysteamine for cystinosis. In certain embodiments, providing cysteamine reduces or eliminates the symptoms of cystinosis, as described above.

[0204] In certain embodiments, the therapeutically effective amount prevents copper accumulation in organs of a subject diagnosed with WD. In certain embodiments, the method of treatment includes D-penicillamine, trientine, zinc salts, and / or liver transplantation for WD. In certain embodiments, preventing copper accumulation in organs reduces or eliminates the symptoms of WD, as described above.

[0205] In certain embodiments, administration of the therapeutic composition may be accompanied by administration of a separate adjuvant. Exemplary adjuvants include alum, bentonite, latex, and acrylic particles; incomplete Freund's adjuvant, complete Freund's adjuvant; aluminum-based salts such as aluminum hydroxide; calcium-based salts; silica or any TLR biological ligand(s); Sigma Adjuvant System (SAS); Ribi adjuvant.

[0206] For administration, a therapeutically effective amount (also referred to herein as a dose) can be initially estimated based on results from in vitro assays and / or animal model studies. Such information can be used to more accurately determine a useful dose in a subject of interest. The actual dose administered to a particular subject can be determined by a physician, veterinarian, or researcher, taking into account parameters such as the target, body weight, severity of condition, previous or concurrent therapeutic interventions, subject's idiopathic disease, and physical and physiological factors, including the route of administration.

[0207] A therapeutically effective amount of cells is 10 4 cells / kg~10 9 In certain embodiments, the therapeutically effective amount of cells may range from 10 4 cells / kg, 10 5 cells / kg, 10 6 cells / kg, 10 7 cells / kg, 10 8 cells / kg, 10 9 It may contain 100,000 cells / kg or more.

[0208] Useful doses may range from 0.1 to 5 μg / kg or 0.5 to 1 μg / kg. In certain embodiments, doses may include 1 μg / kg, 15 μg / kg, 30 μg / kg, 50 μg / kg, 55 μg / kg, 70 μg / kg, 90 μg / kg, 150 μg / kg, 350 μg / kg, 500 μg / kg, 750 μg / kg, 1000 μg / kg, 0.1 to 5 mg / kg, or 0.5 to 1 mg / kg. In certain embodiments, doses may include 1 mg / kg, 10 mg / kg, 30 mg / kg, 50 mg / kg, 70 mg / kg, 100 mg / kg, 300 mg / kg, 500 mg / kg, 700 mg / kg, 1000 mg / kg or more.

[0209] A therapeutically effective amount may be achieved by single or multiple administrations during the course of a treatment regimen (e.g., daily, every other day, every 3, 4, 5, 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or yearly).

[0210] (IX) Kits. Kits for testing for congenital disorders are also provided. The kits may include a lancet for pricking blood, a filter card for collecting blood drops, a solution for solubilizing DBS, and appropriate buffers and enzymes for digesting marker proteins in DBS. The kits may further include one or more containers containing anti-peptide binding agents (e.g., antibodies) and / or reagents or supplies for assessing the absence or reduction of CD3ε, WASp, BTK, CTNS, SHPK, and / or ATP7B. In certain embodiments, the kit comprises one or more containers containing the following anti-peptide antibodies: anti-CD3ε 197, anti-WASp 274, anti-WASp 289, anti-BTK 407, anti-BTK 545, anti-CTNS 115, anti-CTNS 120, anti-CTNS 194, anti-CTNS 360, anti-SHPK 44, anti-SHPK 363, anti-SHPK 388, anti-ATP7B 214, anti-ATP7B 325, anti-ATP7B 466, anti-ATP7B 589, anti-ATP7B 621, anti-ATP7B 887, anti-ATP7B 1056, and / or anti-ATP7B 1061. The antibodies may be immobilized on a solid support, such as a column or beads. The kit may further comprise an elution buffer for releasing the peptide from the antibody. In certain embodiments, the kits may include one or more labeled reference peptides to perform absolute quantification of the signature peptides. In certain embodiments, the kits may also include some or all of the necessary laboratory and / or medical supplies required for effective use of the kit, such as gauze, sterile adhesive strips, gloves, tubing, etc. Modifications may be made to any of the contents of the kits described herein.

[0211] The components of the kit may be prepared for storage and later use. Associated with such container(s) may be a notice in a format prescribed by a government agency regulating the manufacture, use, or sale of the kit, which notice reflects approval by the agency of manufacture, use, or sale, as appropriate.

[0212] Optionally, the kit further comprises instructions for using the kit in this method. In various embodiments, the instructions may include appropriate instructions for interpreting the results associated with the use of the kit; appropriate disposal of associated waste; and the like. The instructions may be in the form of printed instructions provided in the kit, or the instructions may be printed on a part of the kit itself. The instructions may be in the form of a sheet, pamphlet, booklet, CD-ROM, or computer readable device, or may provide instructions to instructions at a remote location, such as a website.

[0213] Variants of the sequences disclosed and referred to herein are also included. Guidance on determining which amino acid residues can be substituted, inserted, or deleted without impairing biological activity can be found using computer programs well known in the art, such as DNASTAR™ (Madison, Wisconsin) software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the family of amino acids that are related in their side chains.

[0214] In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and may generally be made without changing the biological activity of the resulting molecule.Those skilled in the art generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, for example, Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, Benjamin / Cummings Pub.Co., p.224). Naturally occurring amino acids are generally classified into conservative substitution families as follows: Group 1: alanine (Ala), glycine (Gly), serine (Ser), and threonine (Thr); Group 2: (acidic): aspartic acid (Asp) and glutamic acid (Glu); Group 3: (acidic; also classified as polar negatively charged residues and their amides): asparagine (Asn), glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar positively charged residues): arginine (Arg), lysine (Lys), and histidine (His); Group 6 (large aliphatic, non-polar residues): isoleucine (Il) e), leucine (Leu), methionine (Met), valine (Val), and cysteine ​​(Cys); Group 7 (uncharged polar): tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr; Group 8 (large aromatic residues): phenylalanine (Phe), tryptophan (Trp), and Tyr; Group 9 (non-polar): proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu, and Ile; Group 10 (small aliphatic, non-polar, or slightly polar residues): Ala, Ser, Thr, Pro, and Gly; and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

[0215] In making such changes, the hydropathic index of amino acids may be taken into consideration. The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32). Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: Ile(+4.5); Val(+4.2); Leu(+3.8); Phe(+2.8); Cys(+2.5); Met(+1.9); Ala(+1.8); Gly(-0.4); Thr(-0.7); Ser(-0.8); Trp(-0.9); Tyr(-1.3); Pro(-1.6); His(-3.2); Glutamate(-3.5); Gln(-3.5); Aspartate(-3.5); Asn(-3.5); Lys(-3.9); and Arg(-4.5).

[0216] It is known in the art that certain amino acids can be substituted with other amino acids having similar hydropathic index or score and still result in proteins with similar biological activity, i.e., still obtain biologically functional equivalent proteins. When making such changes, substitution of amino acids with hydropathic index within ±2 is preferred, substitution of amino acids within ±1 is particularly preferred, and substitution of amino acids within ±0.5 is even more particularly preferred. It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity.

[0217] As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Aspartate (+3.0±1); Glutamate (+3.0±1); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Thr (-0.4); Pro (-0.5±1); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); Trp (-3.4). It is understood that an amino acid may be substituted with another amino acid having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular an immunologically equivalent, protein. In such changes, substitutions of amino acids whose hydrophilicity values ​​are within ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

[0218] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.

[0219] As noted elsewhere, variants of a gene sequence may include codon-optimized variants, sequence polymorphisms, splice variants, and / or mutations that do not affect the function of the encoded product to a statistically significant extent.

[0220] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include sequences having at least 70% sequence identity, 80% sequence identity, 85% sequence identity, 90% sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98% sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequences disclosed herein.

[0221] "Percent sequence identity" refers to the relationship between two or more sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein, nucleic acid, or gene sequences, as determined by the match between strings of such sequences. "Identity" (often referred to as "similarity") can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M., and Devereux, J., eds.) Oxford University Press, NY (1992). Preferred methods for determining identity are designed to obtain the best match between the sequences tested. Methods for determining identity and similarity are codified in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiple alignment of sequences can also be performed using the Clustal alignment method (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10).Related programs also include the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP, BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc., Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher. Plenum, New York, 1999). York, NY. Within the context of this disclosure, when sequence analysis software is used for the analysis, it will be understood that the results of the analysis will be based on the "default values" of the referenced program. As used herein, "default values" refers to any set values ​​or parameters that originally load the software when first initialized.

[0222] Variants also include nucleic acid molecules that hybridize to sequences disclosed herein under stringent hybridization conditions and provide the same function as the reference sequences. Exemplary stringent hybridization conditions include overnight incubation at 42°C in a solution containing 50% formamide, 5X SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 μg / ml denatured, sheared salmon sperm DNA, followed by washing the filter with 0.1X SSC at 50°C. Changes in stringency of hybridization and signal detection are primarily achieved by manipulating formamide concentration (lower percentages of formamide result in lower stringency); salt conditions, or temperature. For example, moderately high stringency conditions include overnight incubation at 37° C. in a solution containing 6XSSPE (20XSSPE=3M NaCl; 0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg / ml salmon sperm block DNA; followed by washing with 1XSSPE, 0.1% SDS at 50° C. Additionally, to achieve lower stringency, washes performed following stringent hybridization may be performed at higher salt concentrations (e.g., 5X SSC). Variations in the above conditions may be achieved by inclusion and / or substitution of alternative blocking reagents used to suppress background in hybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Inclusion of certain blocking reagents may require modification of the above hybridization conditions due to compatibility issues.

[0223] (X) Exemplary embodiments. 1. A method for screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and / or X-linked agammaglobulinemia (XLA) in a subject, the method comprising: obtaining a dried blood spot (DBS) sample from a subject; enzymatically digesting blood-derived proteins in the DBS to generate one or more peptides; Concentrating the mixture comprising: a CD3ε signature peptide of SCID using an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide; a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to said first WASp signature peptide; a second WASp signature peptide of the WAS with an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide; and / or a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; said concentrating (enriching); performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; 20. Diagnosing a subject, comprising: SCID, if the concentration of the CD3ε signature peptide is below a predetermined threshold concentration or if the CD3ε signature peptide is absent; if the concentrations of said first and / or second WASp signature peptides are below a corresponding pre-defined threshold concentration or if said first and / or second WASp signature peptides are absent, WAS; and / or if the concentrations of the first and / or second BTK signature peptides are lower than a corresponding predetermined threshold concentration or if the first and / or second BTK signature peptides are absent, XLA; and diagnosing the subject as having.

[0224] 2. The method of embodiment 1, wherein: the SCID CD3ε signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:1; the first WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:2; the second WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:3; the first BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or The method, wherein the second BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:5.

[0225] 3. The method according to embodiment 1 or 2, comprising: The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; The antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and / or The antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38. The method.

[0226] 4. The method according to any one of embodiments 1 to 3, wherein the method is performed as part of a newborn screening (NBS) procedure in which the subject is further screened for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

[0227] 5. The method of any one of embodiments 1 to 4, wherein the subject is free of clinical symptoms of SCID, WAS, and / or XLA.

[0228] 6. The method of any one of the preceding claims, wherein the enzyme is trypsin.

[0229] 7. The method of any one of embodiments 1 to 6, wherein the corresponding predetermined threshold concentration of each signature peptide is calculated from the standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

[0230] 8. The method of any one of embodiments 1 to 7, wherein the corresponding predetermined threshold concentration is -1 standard deviation (SD), -1.1 SD, -1.2 SD, -1.3 SD, -1.4 SD, -1.5 SD, -1.6 SD, -1.7 SD, -1.8 SD, -1.9 SD, -2.0 SD, -2.1 SD, -2.2 SD, -2.3 SD, -2.4 SD, -2.5 SD, -2.6 SD, -2.7 SD, -2.8 SD, -2.9 SD, -3.0 SD, or more SD from the mean concentration of each signature peptide in DBS from a population of normal control subjects.

[0231] 9. The method of any one of embodiments 1-8, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises a VH domain of SEQ ID NO:63.

[0232] 10. The method of any one of embodiments 1-9, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises the VL domain of SEQ ID NO:64.

[0233] 11. The method according to any one of the preceding embodiments, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises a VH domain of SEQ ID NO: 65.

[0234] 12. The method according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises the VL domain of SEQ ID NO: 66.

[0235] 13. The method according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises a heavy chain of SEQ ID NO: 86.

[0236] 14. The method according to any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises a light chain of SEQ ID NO: 91.

[0237] 15. The method of any one of embodiments 1 to 14, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a VH domain of SEQ ID NO: 67.

[0238] 16. The method of any one of embodiments 1 to 15, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a VL domain of SEQ ID NO: 68.

[0239] 17. The method of any one of embodiments 1 to 16, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a heavy chain of SEQ ID NO: 96.

[0240] 18. The method of any one of embodiments 1 to 17, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a light chain of SEQ ID NO: 101.

[0241] 19. The method according to any one of the preceding claims, further comprising: Concentrating the mixture, comprising: a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide; a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide; a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide; a second SHPK signature peptide of cystinosis using an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third SHPK signature peptide; screening for cystinosis by said enrichment; diagnosing the subject as having cystinosis if the concentration of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptide is lower than a corresponding predetermined threshold concentration or if the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptide is absent; The method further comprises:

[0242] 20. The method of embodiment 19, wherein: the first CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:6; the second CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 7 or 8; the third CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:12; the fourth CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 13; the first SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:9; the second SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 14; and / or The method, wherein the third SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:15.

[0243] 21. The method of embodiment 19 or 20, The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and / or The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; The method.

[0244] 22. The method of any one of embodiments 19 to 21, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 69.

[0245] 23. The method of any one of embodiments 19 to 22, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a VL domain of SEQ ID NO: 70.

[0246] 24. The method of any one of embodiments 19 to 23, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 106.

[0247] 25. The method of any one of embodiments 19 to 24, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 111.

[0248] 26. The method of any one of embodiments 19 to 25, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 71.

[0249] 27. The method of any one of embodiments 19 to 26, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 72.

[0250] 28. The method of any one of embodiments 19 to 27, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 116.

[0251] 29. The method of any one of embodiments 19 to 28, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 121.

[0252] 30. The method of any one of embodiments 19 to 29, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 73.

[0253] 31. The method of any one of embodiments 19 to 30, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 74.

[0254] 32. The method of any one of embodiments 19 to 31, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 126.

[0255] 33. The method of any one of embodiments 19 to 32, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a light chain of SEQ ID NO: 131.

[0256] 34. The method according to any one of the preceding claims, wherein the concentrating comprises: a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said first ATP7B signature peptide; a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said second ATP7B signature peptide; a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to said third ATP7B signature peptide; a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fourth ATP7B signature peptide; a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fifth ATP7B signature peptide; a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said sixth ATP7B signature peptide; and / or The seventh ATP7B signature peptide is detected using an antibody or antigen-binding fragment thereof. screening for Wilson's disease (WD) by said enrichment; diagnosing the subject as having WD if the concentration of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is lower than a corresponding predetermined threshold concentration or if the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is absent; The method further comprises:

[0257] 35. The method of embodiment 34, wherein: the first ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 10; the second ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 11 or 21; the third ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 16; the fourth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 17; the fifth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 18; the sixth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 19; and / or The seventh ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 20; The method.

[0258] 36. The method of embodiment 34 or 35, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62; The method.

[0259] 37. A method according to any one of embodiments 34 to 36, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a VH domain of SEQ ID NO: 75.

[0260] 38. A method according to any one of embodiments 34 to 37, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a VL domain of SEQ ID NO: 76.

[0261] 39. A method according to any one of embodiments 34 to 38, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a heavy chain of SEQ ID NO: 136.

[0262] 40. A method according to any one of embodiments 34 to 39, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a light chain of SEQ ID NO: 141.

[0263] 41. A method according to any one of embodiments 1 to 40, wherein the concentration of the peptide is determined from a reference signature peptide of corresponding known concentration added prior to LC-MRM-MS.

[0264] 42. The method of any one of embodiments 7 to 41, wherein the average concentration of the CD3α signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 70 pmol / L to 400 pmol / L.

[0265] 43. The method of any one of embodiments 7 to 42, wherein the average concentration of the first WASp signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 600 pmol / L to 5000 pmol / L.

[0266] 44. The method of any one of embodiments 7 to 43, wherein the average concentration of the second WASp signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 5500 pmol / L to 15000 pmol / L.

[0267] 45. The method of any one of embodiments 7 to 44, wherein the average concentration of the first BTK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 350 pmol / L to 2500 pmol / L.

[0268] 46. ​​The method of any one of embodiments 7 to 45, wherein the average concentration of the second BTK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 550 pmol / L to 1600 pmol / L.

[0269] 47. The method of any one of embodiments 7 to 46, wherein the average concentration of the first CTNS signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 40 pmol / L to 250 pmol / L.

[0270] 48. The method of any one of embodiments 7 to 47, wherein the average concentration of the first SHPK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 100 pmol / L to 8000 pmol / L.

[0271] 49. The method of any one of embodiments 7 to 48, wherein the average concentration of the second ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 90 pmol / L to 400 pmol / L.

[0272] 50. The method of any one of embodiments 7 to 49, wherein the average concentration of the third ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 30 pmol / L to 100 pmol / L.

[0273] 51. A method according to any one of embodiments 7 to 50, wherein the average concentration of the seventh ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 200 pmol / L to 500 pmol / L.

[0274] 52. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and / or X-linked agammaglobulinemia (XLA) in a subject; obtaining a dried blood spot (DBS) sample from the subject; enzymatically digesting blood-derived proteins of said DBS to generate one or more peptides; Concentrating the mixture comprising: a CD3ε signature peptide of SCID using an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide; a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to said first WASp signature peptide; a second WASp signature peptide of the WAS with an antibody or antigen-binding fragment thereof that binds to the second WASp signature peptide; a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide; and / or a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; said concentrating (enriching); enriching the endogenous ATP7B peptide with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; calculating the ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; The subject matter is as follows: SCID, if the ratio of CD3ε signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding pre-defined threshold ratio or if the CD3ε signature peptide is absent; if the ratio of said first and / or second WASp signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding pre-defined threshold ratio or if said first and / or second WASp signature peptide is absent, WAS; and / or if the ratio of the first and / or second BTK signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding pre-defined threshold ratio or if the first and / or second BTK signature peptide is absent, XLA; diagnosing the patient as having The method comprising:

[0275] 53. The method according to embodiment 52, wherein the CD3ε signature peptide of SCID is encoded by the amino acid sequence set forth in SEQ ID NO:1; the first WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:2; the second WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:3; and / or the first BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or The method, wherein the second BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:5.

[0276] 54. The method of embodiment 52 or 53, wherein: The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and / or The method, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38.

[0277] 55. The method of any one of embodiments 42 to 44, wherein the endogenous ATP7B peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21.

[0278] 56. A method according to any one of embodiments 42 to 45, wherein the antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

[0279] 57. A method according to any one of embodiments 52 to 56, wherein each predetermined threshold ratio is calculated from the standard deviation of the mean ratio of each peptide concentration:endogenous ATP7B peptide concentration in a population of samples.

[0280] 58. The method according to any one of embodiments 52 to 57, wherein the method is performed as part of a newborn screening (NBS) procedure to further screen the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

[0281] 59. The method of any one of embodiments 52-58, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and / or XLA in the subject.

[0282] 60. The method of any one of embodiments 52-59, wherein the enzyme is trypsin.

[0283] 61. The method of any one of embodiments 52-60, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises a VH domain of SEQ ID NO: 63.

[0284] 62. The method of any one of embodiments 52 to 61, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises the VL domain of SEQ ID NO: 64.

[0285] 63. A method according to any one of embodiments 52 to 62, wherein the antibody or antigen-binding fragment thereof used for enriching the first WASp signature peptide of WAS comprises a VH domain of SEQ ID NO: 65.

[0286] 64. A method according to any one of embodiments 52 to 63, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises the VL domain of SEQ ID NO: 66.

[0287] 65. The method of any one of embodiments 52 to 64, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises a heavy chain of SEQ ID NO: 86.

[0288] 66. The method of any one of embodiments 52 to 65, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first WASp signature peptide of WAS comprises a light chain of SEQ ID NO: 91.

[0289] The method of any one of embodiments 52 to 66, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of 67.XLA comprises a VH domain of SEQ ID NO:67.

[0290] 68. The method of any one of embodiments 52 to 67, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a VL domain of SEQ ID NO: 68.

[0291] 69. The method of any one of embodiments 52 to 68, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a heavy chain of SEQ ID NO: 96.

[0292] 70. The method of any one of embodiments 52 to 69, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a light chain of SEQ ID NO: 101.

[0293] 71. The method according to any one of embodiments 52 to 70, Concentrating the mixture, comprising: a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide; a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide; a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide; a second SHPK signature peptide of cystinosis using an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or detecting a third SHPK signature peptide of cystinosis using an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide; screening for cystinosis by said enrichment; Calculating the ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; diagnosing the subject as having cystinosis if a ratio of the first CTNS peptide concentration to endogenous ATP7B peptide concentration, the second CTNS peptide concentration to endogenous ATP7B peptide concentration, the third CTNS peptide concentration to endogenous ATP7B peptide concentration, the fourth CTNS peptide concentration to endogenous ATP7B peptide concentration, the first SHPK peptide concentration to endogenous ATP7B peptide concentration, the second SHPK peptide concentration to endogenous ATP7B peptide concentration, and / or the third SHPK signature peptide concentration to endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio or if the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptide is absent; The method further comprises:

[0294] 72. The method according to embodiment 71, wherein: the first CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:6; the second CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 7 or 8; the third CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:12; the fourth CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 13; the first SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:9; the second SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 14; and / or The method, wherein the third SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:15.

[0295] 73. The method according to embodiment 71 or 72, wherein The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and / or The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; The method.

[0296] 74. The method of any one of embodiments 71 to 73, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

[0297] 75. The method of any one of embodiments 71 to 74, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 70.

[0298] 76. The method of any one of embodiments 71 to 75, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 106.

[0299] 77. The method of any one of embodiments 71 to 76, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 111.

[0300] 78. The method of any one of embodiments 71 to 77, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 71.

[0301] 79. The method of any one of embodiments 71 to 78, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 72.

[0302] 80. The method of any one of embodiments 71 to 79, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 116.

[0303] 81. The method of any one of embodiments 71 to 80, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 121.

[0304] 82. The method of any one of embodiments 71 to 81, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 73.

[0305] 83. The method of any one of embodiments 71 to 82, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 74.

[0306] 84. The method of any one of embodiments 71 to 83, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 126.

[0307] 85. The method of any one of embodiments 71 to 84, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a light chain of SEQ ID NO: 131.

[0308] 86. The method of any one of embodiments 52 to 85, wherein the antibody or antigen-binding fragment thereof used for enriching the endogenous ATP7B peptide comprises a VH domain of SEQ ID NO: 75.

[0309] 87. The method of any one of embodiments 52 to 86, wherein the antibody or antigen-binding fragment thereof used for enriching the endogenous ATP7B signature peptide comprises the VL domain of SEQ ID NO: 76.

[0310] 88. The method of any one of embodiments 52 to 87, wherein the antibody or antigen-binding fragment thereof used for enriching the endogenous ATP7B signature peptide comprises a heavy chain of SEQ ID NO: 136.

[0311] 89. The method of any one of embodiments 52 to 88, wherein the antibody or antigen-binding fragment thereof used for enriching the endogenous ATP7B signature peptide comprises a light chain of SEQ ID NO: 141.

[0312] 90. The method of any one of embodiments 52 to 89, wherein the concentration of the peptide is determined from a corresponding known concentration of a reference signature peptide added prior to LC-MRM-MS.

[0313] 91. A method for detecting one or more signature peptides of severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and / or X-linked agammaglobulinemia (XLA) in one or more dried blood spot (DBS) samples, comprising: obtaining the one or more dried blood spot (DBS) samples; enzymatically digesting blood-derived proteins of each DBS to generate one or more peptides; Concentrating the mixture, comprising: a CD3ε signature peptide of SCID using an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide; a first WASp signature peptide of WAS with an antibody or antigen-binding fragment thereof that binds to said first WASp signature peptide; a second WASp signature peptide of the WAS with an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; and / or a first BTK signature peptide of XLA using an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide. enriching a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to the second BTK signature peptide; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to detect one or more signature peptides of SCID, WAS, and XLA in one or more DBS samples; The method comprising:

[0314] 92. The method of embodiment 91, wherein: the CD3ε signature peptide of SCID is encoded by the amino acid sequence set forth in SEQ ID NO:1; the first WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:2; the second WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:3; and / or the first BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or The second BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:5. The method.

[0315] 93. The method of embodiment 91 or 92, wherein: The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; The antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and / or The antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38. The method.

[0316] 94. The method of any one of embodiments 91-93, wherein the enzyme is trypsin.

[0317] 95. The method of any one of embodiments 91 to 94, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises a VH domain of SEQ ID NO: 63.

[0318] 96. The method of any one of embodiments 91 to 95, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID comprises the VL domain of SEQ ID NO: 64.

[0319] 97. A method according to any one of embodiments 91 to 96, wherein the antibody or antigen-binding fragment thereof used for enriching the first WASp signature peptide of WAS comprises a VH domain of SEQ ID NO: 65.

[0320] 98. A method according to any one of embodiments 91 to 97, wherein the antibody or antigen-binding fragment thereof used for enriching the first WASp signature peptide of WAS comprises the VL domain of SEQ ID NO: 66.

[0321] 99. A method according to any one of embodiments 91 to 98, wherein the antibody or antigen-binding fragment thereof used for enriching the first WASp signature peptide of WAS comprises a heavy chain of SEQ ID NO: 86.

[0322] 100. A method according to any one of embodiments 91 to 99, wherein the antibody or its antigen-binding fragment used for enriching the first WASp signature peptide of WAS comprises a light chain of SEQ ID NO: 91.

[0323] 101. The method of any one of embodiments 91 to 100, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a VH domain of SEQ ID NO: 67.

[0324] 102. The method of any one of embodiments 91 to 101, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a VL domain of SEQ ID NO: 68.

[0325] 103. The method of any one of embodiments 91 to 102, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a heavy chain of SEQ ID NO: 96.

[0326] 104. The method of any one of embodiments 91 to 103, wherein the antibody or antigen-binding fragment thereof used for enrichment of the first BTK signature peptide of XLA comprises a light chain of SEQ ID NO: 101.

[0327] 105. The method according to any one of embodiments 91 to 104, wherein the concentrating comprises: a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide; a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide; a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide; a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or detecting a third SHPK signature peptide of cystinosis using an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide; detecting one or more signature peptides of cystinosis by said enrichment; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thereby detecting one or more signature peptides of cystinosis in one or more DBS samples; The method further comprises:

[0328] 106. The method of embodiment 105, wherein the first CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:6; the second CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 7 or 8; the third CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:12; the fourth CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 13; the first SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:9; the second SHPK signature peptide for cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 14; and / or The method, wherein the third SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:15.

[0329] 107. The method according to embodiment 105 or 106, wherein The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; and / or The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; The method.

[0330] 108. The method of any one of embodiments 105 to 107, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 69.

[0331] 109. The method of any one of embodiments 105 to 108, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a VL domain of SEQ ID NO: 70.

[0332] 110. The method of any one of embodiments 105 to 109, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 106.

[0333] 111. The method of any one of embodiments 105 to 110, wherein the antibody or antigen-binding fragment thereof used for enriching the first CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 111.

[0334] 112. The method of any one of embodiments 105 to 111, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 71.

[0335] 113. The method of any one of embodiments 105 to 112, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second CTNS signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 72.

[0336] 114. The method of any one of embodiments 105 to 113, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 116.

[0337] 115. The method of any one of embodiments 105 to 114, wherein the antibody or antigen-binding fragment thereof used for enriching the second CTNS signature peptide of cystinosis comprises a light chain of SEQ ID NO: 121.

[0338] 116. The method of any one of embodiments 105 to 115, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a VH domain of SEQ ID NO: 73.

[0339] 117. The method of any one of embodiments 105 to 116, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises the VL domain of SEQ ID NO: 74.

[0340] 118. The method of any one of embodiments 105 to 117, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a heavy chain of SEQ ID NO: 126.

[0341] 119. The method of any one of embodiments 105 to 118, wherein the antibody or antigen-binding fragment thereof used for enriching the first SHPK signature peptide of cystinosis comprises a light chain of SEQ ID NO: 131.

[0342] 120. The method according to any one of embodiments 91 to 119, further comprising: enriching the endogenous ATP7B peptide with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide; and calculating the ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide.

[0343] 121. The method of embodiment 120, wherein the endogenous ATP7B peptide is encoded by the amino acid sequence set forth in SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, or SEQ ID NO:21.

[0344] 122. The method of embodiment 120 or 121, wherein the antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

[0345] 123. The method according to any one of embodiments 91 to 122, wherein the concentrating comprises: a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said first ATP7B signature peptide; a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said second ATP7B signature peptide; a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to said third ATP7B signature peptide; a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fourth ATP7B signature peptide; a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fifth ATP7B signature peptide; a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said sixth ATP7B signature peptide; and / or a seventh ATP7B signature peptide, using the antibody or antigen-binding fragment thereof; detecting one or more signature peptides of Wilson's disease (WD) by said enrichment; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to detect one or more signature peptides of WD in one or more DBS samples; The method further comprises:

[0346] 124. The method according to embodiment 123, wherein the first ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 10; the second ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 11 or 21; the third ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 16; the fourth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 17; the fifth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 18; the sixth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 19; and / or The method, wherein the seventh ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO:20.

[0347] 125. The method of embodiment 123 or 124, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

[0348] 126. A method according to any one of embodiments 123 to 125, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a VH domain of SEQ ID NO: 75.

[0349] 127. A method according to any one of embodiments 123 to 126, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a VL domain of SEQ ID NO: 76.

[0350] 128. A method according to any one of embodiments 123 to 127, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a heavy chain of SEQ ID NO: 136.

[0351] 129. A method according to any one of embodiments 123 to 128, wherein the antibody or antigen-binding fragment thereof used for enrichment of the second ATP7B signature peptide of WD comprises a light chain of SEQ ID NO: 141.

[0352] 130. The method of any one of embodiments 91 to 129, further comprising comparing said concentration of each signature peptide with a corresponding predetermined threshold concentration.

[0353] 131. The method of embodiment 130, wherein the corresponding predetermined threshold concentration is -1 standard deviation (SD), -1.1 SD, -1.2 SD, -1.3 SD, -1.4 SD, -1.5 SD, -1.6 SD, -1.7 SD, -1.8 SD, -1.9 SD, -2.0 SD, -2.1 SD, -2.2 SD, -2.3 SD, -2.4 SD, -2.5 SD, -2.6 SD, -2.7 SD, -2.8 SD, -2.9 SD, -3.0 SD, or more SD from the mean concentration of each signature peptide in a population of normal control subjects.

[0354] 132. The method of embodiment 130 or 131, wherein the corresponding predetermined threshold concentration of each signature peptide is calculated from the standard deviation of the mean concentration of each signature peptide in a population of normal control subjects.

[0355] 133. The method of embodiment 132, wherein the average concentration of the CD3ε signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 70 pmol / L to 400 pmol / L.

[0356] 134. The method of embodiment 132 or 133, wherein the average concentration of the first WASp signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 600 pmol / L to 5000 pmol / L.

[0357] 135. The method of any one of embodiments 132 to 134, wherein the average concentration of the second WASp signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 5500 pmol / L to 15000 pmol / L.

[0358] 136. The method of any one of embodiments 132-135, wherein the average concentration of the first BTK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 350 pmol / L to 2500 pmol / L.

[0359] 137. The method of any one of embodiments 132-136, wherein the average concentration of the second BTK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 550 pmol / L to 1600 pmol / L.

[0360] 138. The method of any one of embodiments 132 to 137, wherein the average concentration of the first CTNS signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 40 pmol / L to 250 pmol / L.

[0361] 139. The method of any one of embodiments 132 to 138, wherein the average concentration of the first SHPK signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 100 pmol / L to 8000 pmol / L.

[0362] 140. The method of any one of embodiments 132 to 139, wherein the average concentration of the second ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 90 pmol / L to 400 pmol / L.

[0363] 141. The method of any one of embodiments 132 to 140, wherein the average concentration of the third ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 30 pmol / L to 100 pmol / L.

[0364] 142. A method according to any one of embodiments 132 to 141, wherein the average concentration of the seventh ATP7B signature peptide in DBS from a population of normal control subjects comprises a concentration in the range of 200 pmol / L to 500 pmol / L.

[0365] 143. The method of any one of embodiments 91 to 142, wherein the concentration of the peptide is determined from a corresponding known concentration of a reference signature peptide added prior to LC-MRM-MS.

[0366] 144. An assay for screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, or Wilson's disease (WD) in a subject, the assay comprising: (I) (i) an antibody or antigen-binding fragment thereof that binds to a CD3ε signature peptide of SCID; (ii) an antibody or antigen-binding fragment thereof that binds to a first WASp signature peptide of the WAS; (iii) an antibody or antigen-binding fragment thereof that binds to a second WASp signature peptide of the WAS; (iv) an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA; (v) an antibody or antigen-binding fragment thereof that binds to a second BTK signature peptide of XLA; (vi) an antibody or antigen-binding fragment thereof that binds to a first CTNS signature peptide of cystinosis; (vii) an antibody or antigen-binding fragment thereof that binds to a second CTNS signature peptide of cystinosis; (viii) an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide of cystinosis; (ix) an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide of cystinosis; (x) an antibody or antigen-binding fragment thereof that binds to a first SHPK signature peptide of cystinosis; (xi) an antibody or antigen-binding fragment thereof that binds to a second SHPK signature peptide of cystinosis; (xii) an antibody or antigen-binding fragment thereof that binds to a third SHPK signature peptide of cystinosis; (xiii) an antibody or antigen-binding fragment thereof that binds to a first ATP7B signature peptide of WD; (xiv) an antibody or antigen-binding fragment thereof that binds to a second ATP7B signature peptide of WD; (xv) an antibody or antigen-binding fragment thereof that binds to the third ATP7B signature peptide of WD; (xvi) an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide of WD; (xvii) an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide of WD; (xviii) an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide of WD; and / or (xix) an antibody or antigen-binding fragment thereof that binds to the seventh ATP7B signature peptide of WD; and / or (II) the corresponding reference signature peptide.

[0367] 145. The assay according to embodiment 144, wherein (i) the CD3ε signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:1; (ii) the first WASp signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:2; (iii) the second WASp signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:3; (iv) the first BTK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or (v) the second BTK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:5; (vi) the first CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:6; (vii) the second CTNS signature peptide and / or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8; (viii) the third CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 12; (ix) the fourth CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 13; (x) the first SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:9; (xi) the second SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 14; (xii) the third SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 15; (xiii) the first ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 10; (xiv) the second ATP7B signature peptide and / or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21; (xv) the third ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 16; (xvi) the fourth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 17; (xvii) the fifth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 18; (xviii) the sixth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 19; and / or (xix) the seventh ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 20; The assay.

[0368] 146. The assay according to embodiment 144 or 145, wherein The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; The antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33; The antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38; The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; and / or The assay, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

[0369] 147. The assay of any one of embodiments 144 to 146, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID comprises a VH domain of SEQ ID NO: 63.

[0370] 148. The assay of any one of embodiments 144 to 147, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID comprises a VL domain of SEQ ID NO: 64.

[0371] 149. The assay of any one of embodiments 144 to 148, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a VH domain of SEQ ID NO: 65.

[0372] 150. An assay according to any one of embodiments 144 to 149, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a VL domain of SEQ ID NO: 66.

[0373] 151. The assay of any one of embodiments 144 to 150, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a heavy chain of SEQ ID NO: 86.

[0374] 152. The assay of any one of embodiments 144 to 151, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a light chain of SEQ ID NO: 91.

[0375] 153. The assay of any one of embodiments 144 to 152, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA comprises a VH domain of SEQ ID NO: 67.

[0376] 154. The assay of any one of embodiments 144 to 153, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA comprises a VL domain of SEQ ID NO: 68.

[0377] 155. The assay of any one of embodiments 144 to 154, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA comprises a heavy chain of SEQ ID NO: 96.

[0378] 156. The assay of any one of embodiments 144 to 155, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA comprises a light chain of SEQ ID NO: 101.

[0379] 157. The assay of any one of embodiments 144 to 156, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

[0380] 158. The assay of any one of embodiments 144 to 157, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

[0381] 159. The assay of any one of embodiments 144 to 158, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

[0382] 160. The assay of any one of embodiments 144 to 159, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

[0383] 161. The assay of any one of embodiments 144 to 160, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

[0384] 162. The assay of any one of embodiments 144 to 161, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

[0385] 163. The assay of any one of embodiments 144 to 162, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

[0386] 164. The assay of any one of embodiments 144 to 163, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

[0387] 165. The assay of any one of embodiments 144 to 164, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

[0388] 166. The assay of any one of embodiments 144 to 165, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

[0389] 167. The assay of any one of embodiments 144 to 166, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

[0390] 168. The assay of any one of embodiments 144 to 167, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

[0391] 169. The assay of any one of embodiments 144 to 168, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

[0392] 170. The assay of any one of embodiments 144 to 169, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

[0393] 171. The assay of any one of embodiments 144 to 170, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

[0394] 172. The assay of any one of embodiments 144 to 171, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

[0395] 173. The assay of any one of embodiments 144 to 172, wherein the reference signature peptide is isotopically labeled.

[0396] 174. The assay of any one of embodiments 144 to 173, wherein the antibody or antigen-binding fragment thereof is attached to a magnetic bead.

[0397] 175. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO: 27.

[0398] 176. The isolated antibody or antigen-binding fragment of embodiment 175, wherein the VH domain comprises SEQ ID NO: 63.

[0399] 177. The isolated antibody or antigen-binding fragment of embodiment 175 or 176, wherein the VL domain comprises SEQ ID NO:64.

[0400] 178. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 28, CDR2 of SEQ ID NO: 29, and CDR3 of SEQ ID NO: 30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 31, CDR2 of SEQ ID NO: 32, and CDR3 of SEQ ID NO: 33.

[0401] 179. The isolated antibody or antigen-binding fragment of embodiment 178, wherein the VH domain comprises SEQ ID NO: 65.

[0402] 180. The isolated antibody or antigen-binding fragment of embodiment 178 or 179, wherein the VL domain comprises SEQ ID NO:66.

[0403] 181. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 178 to 180, wherein the heavy chain comprises SEQ ID NO: 86.

[0404] 182. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 178 to 181, wherein the light chain comprises SEQ ID NO: 91.

[0405] 183. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38.

[0406] 184. The isolated antibody or antigen-binding fragment of embodiment 183, wherein the VH domain comprises SEQ ID NO: 67.

[0407] 185. The isolated antibody or antigen-binding fragment thereof according to embodiment 183 or 184, wherein the VL domain comprises SEQ ID NO: 68.

[0408] 186. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 183 to 185, wherein the heavy chain comprises SEQ ID NO: 96.

[0409] 187. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 183 to 186, wherein the light chain comprises SEQ ID NO: 101.

[0410] 188. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44.

[0411] 189. The isolated antibody or antigen-binding fragment thereof of embodiment 188, wherein the VH domain comprises SEQ ID NO: 69.

[0412] 190. The isolated antibody or antigen-binding fragment thereof of embodiment 188 or 189, wherein the VL domain comprises SEQ ID NO: 70.

[0413] 191. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 188 to 190, wherein the heavy chain comprises SEQ ID NO: 106.

[0414] 192. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 188 to 191, wherein the light chain comprises SEQ ID NO: 111.

[0415] 193. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50.

[0416] 194. The isolated antibody or antigen-binding fragment thereof of embodiment 193, wherein the VH domain comprises SEQ ID NO:71.

[0417] 195. The isolated antibody or antigen-binding fragment thereof according to embodiment 193 or 194, wherein the VL domain comprises SEQ ID NO: 72.

[0418] 196. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 193 to 195, wherein the heavy chain comprises SEQ ID NO: 116.

[0419] 197. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 193 to 196, wherein the light chain comprises SEQ ID NO: 121.

[0420] 198. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56.

[0421] 199. The isolated antibody or antigen-binding fragment thereof of embodiment 198, wherein the VH domain comprises SEQ ID NO:73.

[0422] 200. The isolated antibody or antigen-binding fragment thereof of embodiment 198 or 199, wherein the VL domain comprises SEQ ID NO: 74.

[0423] 201. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 198 to 200, wherein the heavy chain comprises SEQ ID NO: 126.

[0424] 202. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 198-201, wherein the light chain comprises SEQ ID NO: 131.

[0425] 203. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62.

[0426] 204. The isolated antibody or antigen-binding fragment thereof of embodiment 203, wherein the VH domain comprises SEQ ID NO: 75.

[0427] 205. The isolated antibody or antigen-binding fragment thereof according to embodiment 203 or 204, wherein the VL domain comprises SEQ ID NO: 76.

[0428] 206. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 203 to 205, wherein the heavy chain comprises SEQ ID NO: 136.

[0429] 207. The isolated antibody or antigen-binding fragment thereof according to any one of embodiments 203 to 206, wherein the light chain comprises SEQ ID NO: 141.

[0430] 208. A kit comprising: (I) (i) an antibody or antigen-binding fragment thereof that binds to a CD3ε signature peptide of SCID; (ii) an antibody or antigen-binding fragment thereof that binds to a first WASp signature peptide of the WAS; (iii) an antibody or antigen-binding fragment thereof that binds to a second WASp signature peptide of the WAS; (iv) an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA; (v) an antibody or antigen-binding fragment thereof that binds to a second BTK signature peptide of XLA; (vi) an antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis; (vii) an antibody or antigen-binding fragment thereof that binds to a second CTNS signature peptide of cystinosis; (viii) an antibody or antigen-binding fragment thereof that binds to the third CTNS signature peptide of cystinosis; (ix) an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide of cystinosis; (x) an antibody or antigen-binding fragment thereof that binds to a first SHPK signature peptide of cystinosis; (xi) an antibody or antigen-binding fragment thereof that binds to a second SHPK signature peptide of cystinosis; (xii) an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide of cystinosis; (xiii) an antibody or antigen-binding fragment thereof that binds to a first ATP7B signature peptide of WD; (xiv) an antibody or antigen-binding fragment thereof that binds to a second ATP7B signature peptide of WD; (xv) an antibody or antigen-binding fragment thereof that binds to the third ATP7B signature peptide of WD; (xvi) an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide of WD; (xvii) an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide of WD; (xviii) an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide of WD; and / or (xix) an antibody or antigen-binding fragment thereof that binds to the seventh ATP7B signature peptide of WD and / or (II) the corresponding reference signature peptide; and (III) instructions for use of the contents of the kit; The kit.

[0431] 209. The kit of embodiment 208, wherein: (i) the CD3ε signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:1; (ii) the first WASp signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:2; (iii) the second WASp signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:3; (iv) the first BTK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or (v) the second BTK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:5; (vi) the first CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:6; (vii) the second CTNS signature peptide and / or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 7 or 8; (viii) the third CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 12; (ix) the fourth CTNS signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 13; (x) the first SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO:9; (xi) the second SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 14; (xii) the third SHPK signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 15; (xiii) the first ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 10; (xiv) the second ATP7B signature peptide and / or its reference signature peptide is encoded by an amino acid sequence set forth in SEQ ID NO: 11 or 21; (xv) the third ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 16; (xvi) the fourth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 17; (xvii) the fifth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 18; (xviii) the sixth ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 19; and / or (xix) The above kit, wherein the seventh ATP7B signature peptide and / or its reference signature peptide is encoded by the amino acid sequence set forth in SEQ ID NO: 20.

[0432] 210. The kit of embodiment 208 or 209, wherein The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; The antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; The antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38; The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 51, CDR2 of SEQ ID NO: 52, and CDR3 of SEQ ID NO: 53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 54, CDR2 of SEQ ID NO: 55, and CDR3 of SEQ ID NO: 56; and / or The antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62; The kit.

[0433] 211. The kit according to any one of embodiments 208 to 210, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID comprises a VH domain of SEQ ID NO: 63.

[0434] 212. The kit according to any one of embodiments 208 to 211, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID comprises a VL domain of SEQ ID NO: 64.

[0435] 213. A kit according to any one of embodiments 208 to 212, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a VH domain of SEQ ID NO: 65.

[0436] 214. A kit according to any one of embodiments 208 to 213, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a VL domain of SEQ ID NO: 66.

[0437] 215. A kit according to any one of embodiments 208 to 214, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a heavy chain of SEQ ID NO: 86.

[0438] 216. A kit according to any one of embodiments 208 to 215, wherein the antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide of WAS comprises a light chain of SEQ ID NO: 91.

[0439] The kit of any one of embodiments 208 to 216, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of 217.XLA comprises a VH domain of SEQ ID NO:67.

[0440] The kit of any one of embodiments 208 to 217, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of 218.XLA comprises a VL domain of SEQ ID NO:68.

[0441] 219. A kit described in any one of embodiments 208 to 218, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of XLA comprises a heavy chain of SEQ ID NO: 96.

[0442] The kit of any one of embodiments 208 to 219, wherein the antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide of 220.XLA comprises a light chain of SEQ ID NO: 101.

[0443] 221. The kit according to any one of embodiments 208 to 220, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO: 69.

[0444] 222. The kit according to any one of embodiments 208 to 221, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO: 70.

[0445] 223. The kit according to any one of embodiments 208 to 222, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO: 106.

[0446] 224. The kit of any one of embodiments 208 to 223, wherein the antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO: 111.

[0447] 225. The kit of any one of embodiments 208 to 224, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO: 71.

[0448] 226. The kit of any one of embodiments 208 to 225, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO: 72.

[0449] 227. The kit of any one of embodiments 208 to 226, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO: 116.

[0450] 228. The kit of any one of embodiments 208 to 227, wherein the antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO: 121.

[0451] 229. The kit of any one of embodiments 208 to 228, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VH domain of SEQ ID NO: 73.

[0452] 230. The kit of any one of embodiments 208 to 229, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a VL domain of SEQ ID NO: 74.

[0453] 231. The kit according to any one of embodiments 208 to 230, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO: 126.

[0454] 232. The kit of any one of embodiments 208 to 231, wherein the antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO: 131.

[0455] 233. The kit of any one of embodiments 208 to 323, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO: 75.

[0456] 234. The kit of any one of embodiments 208 to 233, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO: 76.

[0457] 235. The kit according to any one of embodiments 208 to 234, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO: 136.

[0458] 236. The kit according to any one of embodiments 208 to 235, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO: 141.

[0459] 237. The kit according to any one of embodiments 208 to 236, further comprising one or more of a filter paper card, a punch tool, a digestive enzyme, a digestion buffer, a solid support of an antibody or an antigen-binding fragment thereof; and an elution buffer.

[0460] 238. The kit according to any one of embodiments 208 to 237, wherein the reference signature peptide is isotopically labeled.

[0461] 239. The kit according to any one of embodiments 208 to 238, wherein the antibody or antigen-binding fragment thereof is attached to magnetic beads. EXAMPLES

[0462] (XI) Experimental Example

[0463] Example 1. Summary. A study was conducted to evaluate whether a multiplex assay based on peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) could reliably and accurately distinguish affected patients with CD3ε-associated severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and X-linked agammaglobulinemia (XLA) from each other and from unaffected normal control dried blood spot (DBS) samples. A blinded multiplex analysis of proteolytically generated peptides from CD3ε, WASp, and BTK (SCID, WAS, and XLA, respectively) in DBS samples from 42 patients with primary immunodeficiency disorders (PIDD), 40 normal adult controls, and 62 normal newborns was performed. The immuno-SRM assay reliably quantified target peptides in DBS, including intra- and inter-assay precision (11–22% and 11–43%), linearity (1.39–2000 fmol peptide), and stability (difference ≤0.09% at 72 h). Analysis of signature peptides showed a statistically significant reduction (or absence) of peptide levels in affected patients compared to controls (SCID: p=0.05, WAS and BTK: p=0.0001). Immuno-SRM-based quantification of proteolytic peptides from CD3ε, WASp, and BTK in DBS distinguishes associated PIDD cases from controls. This approach may be used to perform large-scale multiplexed newborn screening for selective PIDD.

[0464] Materials and Methods. Patient samples. PIDD and normal control blood samples were obtained from Seattle Children's Immunology Diagnostic Laboratory. Neonatal DBS were retrieved from Washington State Newborn Screening Laboratory (Shoreline, WA) after Institutional Review Board approval. XLA DBS were collected from 20 suspected Vietnamese patients and shipped to Seattle Children's Hospital by regular mail. Genotypes of these patients by Sanger sequencing were previously reported in Segundo et al. Front Immunol. Frontiers;2018;9:289. In total, DBS samples were obtained from 42 PIDD patients and 40 normal controls. Normal control and PIDD patient DBS were prepared by pipetting 70 μL of blood / 12 mm spots onto filter paper cards (Protein Saver 903 Cards, Whatman, Piscataway, NJ), dried overnight at room temperature, and stored in sealed plastic bags at −80°C until use. Affected patient samples were shipped from the collection site and stored at −80°C until use.

[0465] Surrogate peptide selection and antibody production. Surrogate peptides for CD3ε, WASp, and BTK were selected in silico by trypsin digestion and NCBI BLAST tool. Final peptide selection followed accepted key criteria for immuno-SRM development, including peptide length, lack of post-transcriptional modifications, and uniqueness to the human genome by BLAST search as previously described. Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402; Abbatiello et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2015;14:2357-2374; Hoofnagle et al. Clin. Chem. 2016;62:48-69. Peptide selection and monoclonal antibody production for ATP7B signature peptides have been previously reported. Jung et al. 2017, supra. The crude peptides were then empirically screened to determine their suitability for detection and quantification by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0466] Affinity purified rabbit polyclonal antibodies (pAb) or monoclonal antibodies (mAb) were successfully generated against five peptides by Pacific Immunology (Ramona, CA) or Fred Hutchinson Cancer Research Center Immunology lab (Seattle WA). Briefly, signature peptides were synthesized with C-terminal extension (GSGC, SEQ ID NO: 77) or N-terminal cysteine ​​extension and conjugated to keyhole limpet hemocyanin (KHL) for immunization. Two New Zealand white rabbits or five mice were injected per peptide. pAb or mAb of all selected peptides were successfully affinity purified from 25 mL of antiserum.

[0467] Immuno-SRM assay reagents. ProteaseMAX™ detergent (no. V2072) and proteomics grade trypsin (no. V5113) were purchased from Promega (Madison, WI). Bovine serum albumin standard (200 mg / mL) and (3-[3-cholamidopropyl)=dimethylammonio]-1-propanesulfonate) (Pierce™ CHAPS, no. PI28300) detergent were obtained from Thermo Fisher Scientific (Waltham, MA). Ammonium bicarbonate (40867-50G-F) was purchased from Fluka Analytical (Munich, Germany). Acetonitrile (no. A955), water (no. W6, LCMS optima grade), formic acid (no. PI28905), and phosphate buffered saline (PBS, no. 10010-023) were obtained from Thermo Fisher Scientific (Waltham, Mass.).

[0468] Heavy stable isotope-labeled peptides were obtained from Anaspec (Fremont, Calif.). Stable isotope-labeled peptides were purified by HPLC to >95% and modified with either C-terminal arginine or lysine. 13 C and 15 Labeling with the N atom resulted in a mass shift of +8 or +10 Da, respectively. Aliquots were stored in 5% acetonitrile / 0.1% formic acid at -20°C until use.

[0469] Antibodies were immobilized on 2.8 μm Dynabeads Protein G magnetic beads (no. 10004D, Invitrogen, Carlsbad, CA) at a ratio of 1 μg antibody to 2.5 μL beads. Briefly, 250 μL of beads were added to a 1.5 mL Eppendorf tube (022363204 Eppendorf) and washed twice with 250 μL of 1× PBS, followed by addition of 100 μg of antibody and 1× PBS + 0.03% CHAPS (no. 28300, Thermo Scientific, Waltham, MA) to obtain a total volume of 250 μL. The antibody was allowed to bind to the beads overnight with tumbling at 4°C. The next day, the antibody was immobilized to the beads by chemical cross-linking. Briefly, antibody beads were collected using magnetic pull-down, excess PBS was discarded, and 300 μL of freshly prepared 20 mM DMP (dimethyl pimelimidate dihydrochloride, no. D8388, Sigma Aldrich, St. Louis, MO) in 200 mM triethanolamine, pH 8.5 (no. T58300, Sigma Aldrich, St. Louis, MO) was added. The sample was tumbled at room temperature for 30 minutes, and the DMP in triethanolamine was discarded. 250 μL of 150 mM monoethanolamine (no. 411000, Sigma Aldrich, St. Louis, MO) was added, and the beads were tumbled at room temperature for 30 minutes twice. Antibody beads were washed twice with 250 μL of 5% acetic acid + 0.03% CHAPS (tumbling at room temperature for 5 minutes each time) and once more with 250 μL of 1× PBS + 0.03% CHAPS. CD3ε, WASp, and BTK antibody-bound beads were then washed, incubated with 5% acetic acid + 3% acetonitrile (ACN), washed with 250 μL 1×PBS + 0.03% CHAPS, and the latter two steps were repeated once. All antibody-bound beads were washed with 250 μL 1×PBS + 0.03% CHAPS until a neutral pH (7.0) was reached. The washed antibody-bound beads were then resuspended in 250 μL 1×PBS + 0.03% CHAPS and 2.5 μL NaN3 (52002-5G Sigma Aldrich) for antifungal properties and stored at 4°C until use.

[0470] DBS protein extraction and trypsin digestion. For each sample (blinded normal control or patient), 17 punches of 3 mm diameter were punched across one DBS spot (13 mm) containing 70 μL of blood using a standard leather punch tool. The final sample representation was SCID: n=3, WAS: n=11, XLA: n=26, and normal control (n=40). The punches were placed in 1.5 mL Eppendorf tubes and 490 μL of 0.1% ProteaseMax™ in 50 mM ammonium bicarbonate (pH 8) was added to each tube. The tubes were vortexed for 1 h in an Eppendorf MixMate (Eppendorf, Hamburg, Germany), after which 10 μL of each sample was aliquoted and diluted 200-fold for the Bradford assay to measure protein concentration. Reduction of disulfide bonds was performed with 5 mM 2M DTT, and 490 μL of 0.1% ProteaseMax™ in 50 mM ammonium bicarbonate (pH 8) was added to each tube before incubating in a 37° C. water bath for 30 minutes. Trypsin was then added at an enzyme-to-protein ratio (w / w) of 1:50, and acetonitrile was added to a final concentration of 15%. The mixture was incubated overnight in a 37° C. water bath for digestion, followed by centrifugation at 13,000 RPM for 10 minutes, after which each supernatant was transferred to a new tube and dried in a Savant™ SpeedVac™ High Capacity Concentrator (Thermo Fisher Scientific, Waltham, MA). All tryptic DBS digests were stored at −80° C. until use.

[0471] For samples analyzed from the NBS lab in Washington, five or six 3 mm punches were used for protein extraction and digestion (n=62). The procedure was the same as for the previous sample, but with reduced volumes as follows: 150 μL of 0.1% ProteaseMax™ and 0.78 μL of DTT for each addition.

[0472] Peptide immunoaffinity enrichment. DBS digests were resuspended in 1x PBS + 0.03% CHAPS to give a nominal protein digest concentration of 1 μg / μL. Cross-linked antibody-coated beads were added to a total mass of 2 μg pAb for each target. 20 μL of 1M Tris pH 8.0 (15568-025 UltraPure, Invitrogen, Carlsbad, CA) was then added. Isotopically labeled peptides were added as internal standards (IS). The suspension was incubated overnight with tumbling at 4°C to achieve peptide capture. The next day, antibody bead:peptide complexes were washed twice with 100 μL PBS + 0.01% CHAPS and once with 100 μL 0.01% PBS + 0.01% CHAPS. Finally, peptides were eluted by incubation with 30 μL 5% acetic acid / 3% ACN. Released peptides were stored at -80°C until analysis. The procedure for samples analyzed from the Washington State NBS laboratory was the same as for previous samples, except the volumes were reduced to: 58.1 μL 1× PBS + 0.03% CHAPS, 0.59 μg pAb of each peptide, 3.13 μL internal standard (IS), and 12.5 μL TRIS.

[0473] Liquid chromatography-tandem mass spectrometry. Enriched samples were analyzed at two laboratory sites utilizing two separate LC-MS / MS systems and instrument configurations (described below) to examine inter-laboratory variability in data collection. Measured peptide concentrations were then compared for method validation. Peptide parent and daughter ion spectra have been reported previously. Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402.

[0474] Laboratory Site 1: The equipment included a Waters Xevo TQ-XS MS equipped with ion key source technology connected to a Waters M-Class gradient and loading pump (Waters, Milford, MA). Chromatographic solvents were A: HO + 0.1% formic acid (FA) and B: ACN + 0.1% FA. First, the peptide mixture was loaded onto an M-Class Trap Symmetry 300 μm × 50 mm C18 column (100 Å, 5 μm) using a constant flow rate of 98:2 A:B at 20 μL / min for 3 min. The flow was then reversed and the peptides were separated using gradient flow across a 150 μm × 100 mm BEH C18 ikey (130 Å, 1.7 μm). The gradient programming method is shown in Table 4. Peptides monitored at this location were CD3ε 197, WASp 274, WASp 289, BTK 407, and ATP7B 1056.

[0475] [Table 4]

[0476] Transition and collision energy (CE) parameters were derived from linear regression of previously optimized values ​​in Skyline and values ​​generated using Waters IntelliStart technology to identify the most intense fragments upon ionization. SRM transitions were acquired at unit / unit resolution in both Q1 and Q3 quadrupoles with dwell times of 5 ms and pause times of 3 ms between mass ranges resulting in a cycle time of 1.5 s. All samples were run in a blinded fashion.

[0477] Laboratory site 2: LC-MS was performed on a SCIEX 5500 QTRAP mass spectrometer interfaced with an Eksigent 425 LC and Nanoflex Chip system. Chromatographic solvents were A: H2O+0.1% FA and B: 90% ACN+0.1% FA. Peptides were loaded on a 0.2 x 0.5 mm trap column (Reprosil-Pur AQ C18, 3 μm, 120A) at 2% B for 4 min using a flow rate of 4 μL / min. Peptides were eluted on a 0.075 x 150 mm column (Reprosil-Pur AQ C18, 3 μm, 120A) at 300 nL / min. The gradient program is shown in Table 4. Collision energy settings were obtained from Skyline. MacLean et al. Bioinformatics. 2010;26:966-968. Transitions were acquired with unit / unit resolution, dwell times of 10 ms, and pauses of 5 ms between mass ranges, resulting in a cycle time of 0.75 s. All data was acquired in a blinded fashion.

[0478] Method performance evaluation. Response curves were performed to determine the linearity and sensitivity of the assay in the background matrix of DBS. Punches from normal control DBS (4 punches per sample) were extracted in triplicate using extraction buffer (ProteaseMax™, ammonium bicarbonate). Trypsin digestion was performed on the extracted proteins and the digests were pooled to create a common background matrix. Heavy stable isotope standards were spiked into the digests and serially diluted to create samples with different peptide amounts (2000, 200, 12.5, 4.17, 1.39, 0.69 fmol). Two micrograms of each antibody covalently coupled to magnetic protein G beads were added to the background matrix and incubated overnight. The antibody beads were washed with PBS and the eluates were analyzed by SRM.

[0479] Reproducibility and intra- and inter-assay precision were characterized by performing measurements of endogenous (light) peptide signals over five separate days. Each sample was analyzed in five complete process replicates (including punch, extraction, digestion, enrichment, and mass spectrometry) per day.

[0480] Finally, stability was assessed by comparing endogenous (light) peptides detected in DBS stored at room temperature for 1 and 3 days with those detected in DBS stored in sealed containers at -80°C. Each sample was processed in triplicate as described above. The percentage difference was calculated for each time point.

[0481] Interlaboratory validation of the analytical assay. DBS extraction, trypsin digestion, and peptide capture of patient samples were all performed at laboratory site 1. The peptide solution eluted from the antibody beads was split into two 15 μl aliquots for analysis at laboratory site 1 and laboratory site 2 for interlaboratory validation of the analytical performance of the assay.

[0482] Data analysis. All SRM data were analyzed and plotted using Skyline (MacCoss Lab Software, open source, Seattle, WA). MacLean et al. Bioinformatics. 2010;26:966-968. Endogenous target peptide concentrations were quantified by comparing the ratio of the peak area of ​​the signature peptide to its IS added at a known concentration (100 fmol). Statistics were generated using Graphpad Prism (San Diego, CA). Receiver operating characteristic (ROC) curves were constructed using Graphpad Prism and 95% confidence intervals.

[0483] Results. Peptide Selection and Antibody Development. Selected peptide sequences, molecular weights, parent ions, and daughter ions are listed in Figure 1. Fragmentation patterns of peptides of interest have been reported previously. Kerfoot et al. Proteomics Clin Appl. 2012;6:394-402. Affinity-purified polyclonal antibodies (Pacific Immunology, Ramona, CA) were generated against all five peptides and pursued for use with human samples due to their ability to successfully capture the target sequences and the lack of background signal contributed by copurified peptide contaminants. Kuhn et al. Clin. Chem. 2009;55:1108-1117; Hoofnagle et al. Clin. Chem. 2008;54:1796-1804; Whiteaker et al. Mol. Cell Proteomics. American Society for Biochemistry and Molecular Biology; 2011;10:M110.005645.

[0484] Method performance evaluation. Analytical figures of merit are reported in Table 5. Overall, linear responses ranged from 1.39 to 2000 fmol of peptide (Figure 2A-E). The median coefficient of variation (CV) of all points on the response curve was 11%. The lower limit of quantification (LLOQ) was defined by the lowest point yielding a CV < 20%. LLOQs ranged from 0.69 to 12.5 fmol. Five peptides were detected above the LLOQ in DBS samples. Across all peptides, the average intra-assay (i.e., intra-day) variability ranged from 11 to 22%, and the inter-assay (i.e., inter-day) variability ranged from 11 to 43%. Of note, a single peptide (BTK 545-558) showed variability above 20% CV.

[0485] [Table 5]

[0486] Finally, stability was assessed by comparing endogenous (light) peptides detected in DBS stored at room temperature for 1 and 3 days to those detected in DBS stored at -80 °C in a sealed container. The results are shown in Table 5. All five peptides had endogenous signal above the LLOQ with little variation over time. Representative multiple reaction monitoring (MRM) chromatograms for each peptide are shown in Figures 3A-E.

[0487] Overall, there was a high level of agreement between the concentrations determined by the two separate instrumental analyses. Correlation plots comparing the two measurements, Figures 4A-D, show R 2 The linearity of the measured concentration is shown by a value of 0.97 or more. The measured values ​​of WASp 289 are 2 = 0.85 and may therefore be useful as a secondary marker for WASp 274 (Figure 5).

[0488] Peptide concentrations. After analysis, normal controls were unblinded to define normal ranges for comparison of affected patients. The mean peptide concentrations from normal controls were as follows (mean ± SD): CD3ε = 228.68 ± 150.98 pmol / L, WASp 274 = 1176.96 ± 456.68 pmol / L, WASp 289 = 10326.98 ± 4513.13 pmol / L, BTK 407 = 635.09 ± 260.40 pmol / L, and BTK 545 = 1038.44 ± 465.77 pmol / L. Analysis of signature peptides found in each case a statistically significant (p < 0.05 - 0.0001) decrease in peptide levels in patients compared to the control group (Figures 6A - 6E). Peptide levels in the majority of affected patients were significantly reduced or absent. For each patient, the concentration of ATP7B 1056 was also determined using the immuno-SRM methodology previously developed in Jung et al. 2017 (supra). These protein concentrations served as quality control (QC) measurements and were used to assess the reproducibility of the digestion and process, with sample-to-sample consistency (Figure 3A-E).

[0489] Peptide concentration cutoffs for each PIDD diagnosis were arbitrarily set at -1.25SD (CD3ε), -2.15SD (WASp274), -1.75SD (WASp289), -2SD (BTK545), and -2.25SD (BTK407). Using these ranges resulted in two false positive indications in normal controls. NC4 and NC20 were indicative of WAS and SCID patients, respectively. NC signature peptide values ​​are shown in Figure 8. Cutoffs to reliably identify PIDD are shown in Table 6.

[0490] [Table 6]

[0491] Using these cutoffs, a specific PIDD diagnosis was predicted for each patient. The predicted diagnoses showed excellent concordance with the clinical or genetic diagnosis, as shown in Figure 9. All molecularly confirmed cases of WAS and BTK were also diagnosed by immuno-SRM analysis. Two patients clinically diagnosed with agammaglobulinemia, patients 10 and 13, had normal levels of BTK protein by immuno-SRM. Molecularly, no BTK mutations were identified in these patients. Segundo et al. Front Immunol. Frontiers;2018;9:289. Interestingly, patient 12, who had agammaglobulinemia, had low levels of BTK protein but no mutations were found in the coding region of BTK. Furthermore, patient 41, a case of X-linked hypomorphic SCID, was identified as normal by immuno-SRM. For each signature peptide utilized, area under the curve (AUC) analysis of the ROC plot revealed areas between 0.925 and 0.999 with p-values ​​ranging from 0.015 to 0.0001 (Figure 10A, 10B). Overall, there was agreement between clinical diagnosis and immuno-SRM assay results in 97.6% of cases. Interesting outliers and discordant cases are discussed further below.

[0492] Signature peptide concentrations for the NBS laboratory samples are shown in Figure 11. Each DBS sample showed significant measured peptide concentrations above the previously established diagnostic cutoff for PIDD, indicating an unaffected status.

[0493] Immuno-SRM is demonstrated as a sensitive and specific proteomic screening method for the multiplex detection of patients with three life-threatening PIDDs (i.e., SCID, WAS, and XLA) from DBS. The results clearly distinguish patients with PIDD from normal controls, with endogenous peptides of the transmembrane protein CD3ε as well as low levels of the intracellular proteins WASp and BTK correlating with the target diseases (SCID, WAS, and XLA, respectively). These diagnoses can be performed in a single run, with a total run time of 20 min or 6.67 min per disease target. The disclosed results also show the stability of the peptides in DBS, with minimal fluctuations in concentration after 72 h of storage at room temperature (Table 5).

[0494] The immuno-SRM platform reliably detected endogenous peptides from normal control DBS in this highly multiplexed method. Normal control DBS (N=40) were unblinded and utilized to define the normal range and potential positive screening cutoffs (Figure 8). In clinical laboratories, reference ranges for diagnostic tests are determined by normal distributions in the general population. Initial cutoffs for screening tests are usually conservative, aiming to detect all true positives without generating an excessively high screening positivity rate compared to the incidence of the disease (Table 6). However, these cutoffs are continually validated and adjusted according to population-based studies. Considering these parameters, the definition of a positive screening result ranged from 1.25 to 2.25 standard deviations (SD) below the mean of the peptides in this example. The cutoffs selected yielded two false positive normal controls, one WAS (NC4) and one SCID (NC20) (Figure 8). In the case of NC4, rescreening showed WASp levels in the normal range. These preliminary cutoffs are not static and will become more clearly defined as more normal control and patient samples are screened.

[0495] Using these cutoffs, all molecularly confirmed WAS and BTK patients covering a wide range of mutations were clearly identified (Figure 9). As a hypothesis, peptide concentrations are decreased in the majority of BTK and WAS cases, regardless of genotype. Qasim et al. Br. J. Haematol. 2001;113:861-865; Jin et al. Blood. American Society of Hematology; 2004;104:4010-4019; Futatani et al. British Journal of Haematology. 2001;114(1):141-9. These peptides therefore provide biomarkers for diagnosis and screening. Of the three SCID patients available for testing, two were positively identified by CD3ε analysis. The third patient had low CD3ε levels compared to the majority of normal controls, but within the defined cutoffs, and had a "hypomorphic" mutation in IL2RG known to generate a partially functional protein. This is reflected in the patient's slightly lower total CD3+ T cell count (800 cells / μL), but not their absence as in classical SCID. Because CD3ε is expressed only by CD3+ T cells in peripheral blood, the amount of CD3ε protein present reflects the total number of CD3+ T cells. Thus, patients with hypomorphic SCID, patients with "leaky" SCID who have expanded oligoclonal T cell populations, or patients who have expanded maternally derived T cells may be missed by the Immuno-SRM approach.

[0496] ROC curves were constructed to evaluate the diagnostic ability of the immuno-SRM analysis. These plots relate the true positive rate to the false positive rate, with increasingly stringent cut-off values. The lower the diagnostic cut-off, the more capable the test is of scoring true positives, but this process is also more likely to lead to false positives. Thus, screening tests that maintain a high true positive rate and low false positive rate will be graphed close to the y-axis, resulting in a large AUC (Figure 10A, Figure 10B). These values ​​indicate a high diagnostic accuracy of the immuno-SRM analysis of the signature peptides of PIDD.

[0497] QC monitoring of digestion and process performance is included in the current immuno-SRM multiplex in the form of ATP7B signature peptide measurements. As not all detected metabolites are useful NBS targets, calculation of metabolite ratios and secondary metabolite analysis are used to improve the sensitivity and specificity of NBS for specific diseases, such as C3:C2 ratios and 2-methylcitric acid analysis in methylmalonic aciduria. Lindner et al. J. Inherit. Metab. Dis. 2nd ed. 2008;31:379-385. Furthermore, target ratios may account for sample-to-sample variability introduced by a number of factors, such as sample collection quality, storage, efficiency of extraction and digestion, as well as blood characteristics. Razavi et al. Bioanalysis. Future Science Ltd London,UK;2016;8:1597-1609. Here, ATP7B concentrations were found to be largely consistent across screened samples (Figure 7). The absence of ATP7B may help flag improperly processed or handled specimens. As a first experiment, each PIDD peptide was compared in ratio to the endogenous concentration of ATP7B in the same sample. The predictions obtained based on peptide concentrations showed perfect agreement with the clinical diagnosis, demonstrating that immuno-SRM and ATP7B ratios are effective and complementary tools for PIDD diagnosis (Figure 12 and Table 7). These types of ratios are useful in clinical immuno-SRM screening when the selected peptides prove to be ubiquitous and remarkably invariant signals across a large cohort of samples.

[0498] [Table 7]

[0499] One case demonstrated the importance of having both primary and secondary signature peptides for a protein of interest. Patient 18 was found to have a positive BTK diagnosis after analysis of BTK545 instead of the primary marker BTK407. BTK407 levels were significantly decreased compared to the mean, 167.86 vs. 642.16 pmol / L, but not low enough to trigger a positive screen. In contrast, BTK545 levels were almost absent, as the patient harbors a p.Y551N mutation located within the amino acid sequence 545-558 contained within the signature peptide itself (Figure 9). In this case, the multiplexed peptides allowed confirmation of a positive diagnosis that was initially borderline.

[0500] It is noteworthy that normal levels of BTK were found in two clinically defined agammaglobulinemia patients (samples #10 and #13) who lacked BTK mutations by Sanger sequencing (Figure 9). Thus, these patients likely do not have XLA, but may have other autosomal forms of agammaglobulinemia, although more extensive genetic testing was not performed. Another patient (sample #12) had reduced levels of BTK protein but no identifiable mutation in BTK. This suggests that a mutation was missed during sequencing of the coding region and intron-exon junctions, or that this patient may harbor a BTK mutation affecting either regulatory elements, polyadenylation signals, or intronic regions. These cases highlight the clinical utility of immuno-SRM.

[0501] In addition, two samples were obtained from the same WAS patient before and after bone marrow transplantation (BMT) (samples no. 29 and 30 in FIG. 9, respectively). Immuno-SRM analysis before BMT confirmed that the patient had WAS. After BMT, the patient was identified as normal. This case highlights the ability of immuno-SRM to track the therapeutic course of BMT to confirm successful immune system reconstitution. Similar principles may be applied to patients with monogenic disorders undergoing gene therapy.

[0502] Overall, the analysis demonstrates that the disclosed assay has a wide linear range and acceptable precision for determining the concentrations of target peptides in DBS (Table 5). Correlation plots show significant agreement of sample analysis by different MS instruments at two separate laboratories (Figures 4A-D and 5). Four of the five peptides, CD3ε 197, WASp 274, BTK 407, and ATP7B 1056, showed low linearity and low chromatin concentration during analysis. 2 The R value was >0.97, which was almost identical. 2 = 0.85 overlap, showing the ability to vary slightly and therefore may be a secondary marker for WASp 274 when performing clinical analysis. Furthermore, BTK 545 showed a variability of over 20%CV, making it suitable as a secondary marker for BTK 407. These results indicate that immuno-SRM analysis has clinical applicability and transferability. In clinical practice, the use of reference standards and / or calibrators will aid in inter-laboratory validation of findings.

[0503] The feasibility of utilizing immuno-SRM analysis in the NBS context was tested using randomly selected samples provided by the NBS laboratory in Washington state. Due to limited sample availability and to test the utility of signature peptide analysis from smaller samples, the amount of DBS used was reduced from one whole spot to five or six 3 mm punches. Peptides of interest were readily enriched and analyzed with minimal modifications to sample processing. The concentrations of signature peptides were all greater than predefined cutoffs obtained from the analysis of known normal controls (Figure 11). Thus, these patients are designated as normal. The ability to reliably perform this analysis with a greatly reduced sample input makes the immuno-SRM analysis even more suitable for translation to NBS. This high-throughput multiplexing method could effectively reduce run times per disease and is suitable for NBS, where current automated methods typically have run times of less than 3 minutes. Rashed et al. Clin. Chem. 1997;43:1129-1141; Khalid et al. J Med Screen. SAGE Publications, Sage UK: London, England; 2008;15:112-117. The successful prediction of BTK patients using DBS shipped at ambient temperature by conventional mail from Vietnam also highlights the potential utility of a diagnostic test in resource-poor settings where collecting and shipping DBS is economical.

[0504] NBS, one of the most successful public health initiatives in modern times, relies on the detection of accumulated metabolites resulting from downstream enzymatic deficiencies. However, many genetic disorders, including PIDD, are characterized by the absence or reduction of proteins, limiting the scope of current NBS methods. Qasim et al. Br. J. Haematol. 2001;113:861-865; Jin et al. Blood. American Society of Hematology; 2004;104:4010-4019. By being able to detect PIDD-associated peptides from DBS, immuno-SRM fills this gap in current coverage and allows the expansion of NBS to treatable diseases for which there are no current metabolic biomarkers. Immuno-SRM provides rapid quantified evidence of protein deficiency and can be performed simultaneously with initial screening and molecular analysis from DBS without further invasive procedures. Quantification of these signature peptides lays the foundation for immuno-SRM as a highly multiplexable screening and diagnostic tool for a variety of congenital diseases.

[0505] Example 2. Development of a quantitative assay for NBS of cystinosis using DBS. The immuno-SRM assay was performed as described in Example 1, but using the signature peptide of cystinosis. An antibody recognizing the CTNS 115 peptide was developed and used to screen patient samples. This peptide biomarker provides robust and specific detection of CTNS up to 16.9 pmol / L in DBS (Table 8). Measured concentrations of CTNS range from 5.5 to 79.8 pmol / L. Levels of SHPK range from 922.3 to 5787.6 pmol / L. Samples CTNS00001-CTNS 00013 were blinded and predicted mutation status was based on protein levels evidenced by immuno-SRM. The most common form of cystinosis is due to a 57 kb deletion mutation that results in the loss of the CTNS protein and the adjacent protein SHPK. Samples in bold are confirmed or predicted to contain a homozygous 57 kb deletion mutation. ND: Not detected (Table 8).

[0506] [Table 8]

[0507] Example 3. Multiplex immuno-SRM assay of CTNS and SHPK peptides for diagnosis of cystinosis. Multiplex quantification of CTNS and SHPK peptide biomarkers from diseased and normal control DBS has been achieved in a single experiment, with results consistent with the underlying patient genotype. CTNS 115 peptide was combined with SHPK 363, a signature peptide biomarker of sedoheptulokinase (SHPK), for multiplex analysis of cystinosis patient samples. These assays involved the combined use of both CTNS115 and SHPK363 antibodies in an immuno-SRM workflow. In this analysis, 4 micrograms of CTNS 115 mAb and 0.25 micrograms of SHPK363 mAb were used. Multiplex analysis was performed on 4 normal controls, 6 confirmed cystinosis patients, and 13 blinded samples (Table 8). CTNS115 is completely absent in known patient samples. Furthermore, patient samples confirmed to be homozygous for the 57 kb deletion lack SHPK363, while heterozygous patient samples have varying levels of SHPK protein. Interestingly, sample CTNS 209 (compound heterozygous for the 57 kb deletion and c.838A>G; p.K280R) has reduced levels of SHPK compared to samples CTNS 2...

Claims

1. 1. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and / or X-linked agammaglobulinemia (XLA) in a subject, comprising: obtaining a dried blood spot (DBS) sample from the subject; enzymatically digesting proteins from the DBS blood to produce one or more peptides; Concentrating the mixture, the concentration comprising: a CD3ε signature peptide of SCID of SEQ ID NO:1 with an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDRs of SEQ ID NO:27; a first WASp signature peptide of WAS of SEQ ID NO:2 with an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; a second WASp signature peptide of the WAS of SEQ ID NO:3 using an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; a first BTK signature peptide of XLA of SEQ ID NO:4 with an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide and comprises a heavy chain variable (VH) domain that comprises a CDR1 of SEQ ID NO:34, a CDR2 of SEQ ID NO:35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain that comprises a CDR1 of SEQ ID NO:36, a CDR2 of SEQ ID NO:37, and a CDR3 of SEQ ID NO:38; and / or a second BTK signature peptide of XLA of SEQ ID NO:5 using an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; said concentrating; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; The subject is a SCID if the concentration of the CD3ε signature peptide is below a predetermined threshold concentration or if the CD3ε signature peptide is absent; if the concentration of the first and / or second WASp signature peptide is below a corresponding predetermined threshold concentration or if the first and / or second WASp signature peptide is absent, WAS; and XLA if the concentrations of the first and / or second BTK signature peptides are below a corresponding predetermined threshold concentration or if the first and / or second BTK signature peptides are absent; To diagnose a patient as having: The method comprising:

2. 13. The method of claim 1, wherein the method is performed as part of a newborn screening (NBS) procedure that further screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

3. 2. The method of claim 1, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and / or XLA in the subject.

4. The method of claim 1 , wherein the enzyme is trypsin.

5. 2. The method of claim 1, wherein the corresponding predetermined threshold concentration for each signature peptide is calculated from the standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

6. 6. The method of claim 5, wherein the average concentration of the CD3ε signature peptide of SCID of SEQ ID NO:1 in DBS from a population of normal control subjects comprises a concentration ranging from 70 pmol / L to 400 pmol / L.

7. 6. The method of claim 5, wherein the average concentration of the WASp signature peptide of WAS of SEQ ID NO: 2 in DBS from a population of normal control subjects comprises a concentration in the range of 600 pmol / L to 5000 pmol / L.

8. 6. The method of claim 5, wherein the average concentration of the second WASp signature peptide of WAS of SEQ ID NO: 3 in DBS from a population of normal control subjects comprises a concentration in the range of 5500 pmol / L to 15000 pmol / L.

9. 6. The method of claim 5, wherein the average concentration of the first BTK signature peptide of XLA of SEQ ID NO: 4 in DBS from a population of normal control subjects comprises a concentration in the range of 350 pmol / L to 2500 pmol / L.

10. 6. The method of claim 5, wherein the average concentration of the second BTK signature peptide of XLA of SEQ ID NO:5 in DBS from a population of normal control subjects comprises a concentration ranging from 550 pmol / L to 1600 pmol / L.

11. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of the SCID of SEQ ID NO:1 comprises a VH domain of SEQ ID NO:

63.

12. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of SCID of SEQ ID NO:1 comprises a VL domain of SEQ ID NO:

64.

13. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a VH domain of SEQ ID NO:

65.

14. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a VL domain of SEQ ID NO:

66.

15. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a heavy chain of SEQ ID NO:

86.

16. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a light chain of SEQ ID NO:

91.

17. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO:

67.

18. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a VL domain of SEQ ID NO:

68.

19. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a heavy chain of SEQ ID NO:

96.

20. 2. The method of claim 1, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a light chain of SEQ ID NO:

101.

21. 2. The method of claim 1 , Concentrating the mixture, the concentration comprising: a first CTNS signature peptide of cystinosis of SEQ ID NO: 6 using an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide and that comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO: 44; a second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 using an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide and that comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; a third CTNS signature peptide of cystinosis of SEQ ID NO: 12 using an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis of SEQ ID NO: 13 using an antibody or antigen-binding fragment thereof that binds to the fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis of SEQ ID NO:9 using an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide and that comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56; a second SHPK signature peptide of cystinosis of SEQ ID NO: 14 using an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; The third SHPK signature peptide of cystinosis of SEQ ID NO: 15 is detected by an antibody or antigen-binding fragment thereof that binds to the third SHPK signature peptide, screening for cystinosis by said enrichment; diagnosing the subject as having cystinosis if the concentrations of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptides are lower than corresponding pre-defined threshold concentrations or if the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptides are absent; The method further comprises:

22. 22. The method of claim 21, wherein the corresponding predetermined threshold concentration of each signature peptide is calculated from the standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

23. 23. The method of claim 22, wherein the average concentration of the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 in DBS from a population of normal control subjects comprises a concentration in the range of 40 pmol / L to 250 pmol / L.

24. 23. The method of claim 22, wherein the average concentration of the cystinosis first SHPK signature peptide of SEQ ID NO: 9 in DBS from a population of normal control subjects comprises a concentration in the range of 100 pmol / L to 8000 pmol / L.

25. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a VH domain of SEQ ID NO:

69.

26. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a VL domain of SEQ ID NO:

70.

27. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a heavy chain of SEQ ID NO:

106.

28. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a light chain of SEQ ID NO:

111.

29. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO:

71.

30. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO:

72.

31. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO:

116.

32. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO:

121.

33. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VH domain of SEQ ID NO:

73.

34. 22. The method of claim 21, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VL domain of SEQ ID NO:

74.

35. 22. The method of claim 21 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a heavy chain of SEQ ID NO:

126.

36. 22. The method of claim 21 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO:

131.

37. 10. The method of claim 1, wherein the concentrating comprises: and / or using an antibody or antigen-binding fragment thereof that binds to the first ATP7B signature peptide, said first ATP7B signature peptide of the WD of SEQ ID NO: 10; and / or said second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 using an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) domain that binds to said second ATP7B signature peptide and that comprises CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62; using an antibody or antigen-binding fragment thereof that binds to the third ATP7B signature peptide, said third ATP7B signature peptide of the WD of SEQ ID NO: 16; Using an antibody or antigen-binding fragment thereof that binds to the fourth ATP7B signature peptide, said fourth ATP7B signature peptide of the WD of SEQ ID NO: 17; Using an antibody or antigen-binding fragment thereof that binds to the fifth ATP7B signature peptide, said fifth ATP7B signature peptide of the WD of SEQ ID NO: 18; Using an antibody or antigen-binding fragment thereof that binds to the sixth ATP7B signature peptide, said sixth ATP7B signature peptide of the WD of SEQ ID NO: 19; screening for Wilson's Disease (WD) by enriching the seventh ATP7B signature peptide of WD of SEQ ID NO: 20 with an antibody or antigen-binding fragment thereof that binds to the seventh ATP7B signature peptide; diagnosing the subject as having WD if the concentration of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is lower than a corresponding pre-defined threshold concentration or if the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is absent; The method further comprises:

38. 38. The method of claim 37, wherein the corresponding predetermined threshold concentration of each signature peptide is calculated from the standard deviation of the mean concentration of each signature peptide in DBS from a population of normal control subjects.

39. 39. The method of claim 38, wherein the average concentration of the second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 in DBS from a population of normal control subjects comprises a concentration in the range of 90 pmol / L to 400 pmol / L.

40. 39. The method of claim 38, wherein the average concentration of the third ATP7B signature peptide of WD of SEQ ID NO: 16 in DBS from a population of normal control subjects comprises a concentration in the range of 30 pmol / L to 100 pmol / L.

41. 39. The method of claim 38, wherein the average concentration of the seventh ATP7B signature peptide of WD of SEQ ID NO: 20 in DBS from a population of normal control subjects comprises a concentration in the range of 200 pmol / L to 500 pmol / L.

42. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO:

75.

43. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO:

76.

44. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO:

136.

45. The method of claim 37, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO:

141.

46. 1. A method for screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), and / or X-linked agammaglobulinemia (XLA) in a subject, comprising: obtaining a dried blood spot (DBS) sample from the subject; enzymatically digesting proteins from the DBS blood to produce one or more peptides; Concentrating the mixture, the concentration comprising: a CD3ε signature peptide of SCID of SEQ ID NO:1 with an antibody or antigen-binding fragment thereof, which binds to said CD3ε signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; a first WASp signature peptide of WAS of SEQ ID NO:2 with an antibody or antigen-binding fragment thereof, said antibody or antigen-binding fragment comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; a second WASp signature peptide of WAS of SEQ ID NO:3 with an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; a first BTK signature peptide of XLA of SEQ ID NO: 4 using an antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide and comprises a heavy chain variable (VH) domain that comprises a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain that comprises a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38; a second BTK signature peptide of XLA of SEQ ID NO:5 using an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; said concentrating; and enriching for the following endogenous ATP7B peptides: said SEQ ID NO:10 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:10; SEQ ID NO: 11 or 21 with an antibody or antigen-binding fragment thereof that binds to said endogenous ATP7B peptide of SEQ ID NO: 11 or 21 and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62; SEQ ID NO:16 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:16; SEQ ID NO:17 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:17; SEQ ID NO:18 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:18; SEQ ID NO:19 using an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:19; SEQ ID NO:20 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:20; or SEQ ID NO:21 with an antibody or antigen-binding fragment thereof that binds to the endogenous ATP7B peptide of SEQ ID NO:21; said concentrating; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; calculating the ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; 2. Diagnosing a subject, comprising: SCID if the ratio of CD3ε signature peptide concentration:endogenous ATP7B peptide concentration is lower than a corresponding predefined threshold ratio or if the CD3ε signature peptide is absent; if the ratio of the first and / or second WASp signature peptide concentration to endogenous ATP7B peptide concentration is lower than a corresponding pre-defined threshold ratio or if the first and / or second WASp signature peptide is absent, WAS; and If the ratio of the first and / or second BTK signature peptide concentration to the endogenous ATP7B peptide concentration is lower than a corresponding predetermined threshold ratio, or if the first and / or second BTK signature peptide is absent, XLA is detected. diagnosing the subject as having The method comprising:

47. 47. The method of claim 46, wherein each predetermined threshold ratio is calculated from the standard deviation of the mean ratio of each peptide concentration:endogenous ATP7B peptide concentration in a population of samples.

48. 47. The method of claim 46, wherein the method is performed as part of a newborn screening (NBS) procedure that further screens the subject for one or more of phenylketonuria, primary congenital hypothyroidism, cystic fibrosis, and sickle cell disease.

49. 47. The method of claim 46, wherein the method is performed in the absence of clinical symptoms of SCID, WAS, and / or XLA in the subject.

50. 47. The method of claim 46, wherein the enzyme is trypsin.

51. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of the SCID of SEQ ID NO:1 comprises a VH domain of SEQ ID NO:

63.

52. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of the SCID of SEQ ID NO:1 comprises a VL domain of SEQ ID NO:

64.

53. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a VH domain of SEQ ID NO:

65.

54. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a VL domain of SEQ ID NO:

66.

55. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a heavy chain of SEQ ID NO:

86.

56. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a light chain of SEQ ID NO:

91.

57. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO:

67.

58. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO:

68.

59. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a heavy chain of SEQ ID NO:

96.

60. 47. The method of claim 46, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a light chain of SEQ ID NO:

101.

61. 47. The method of claim 46, wherein concentrating comprises: a first CTNS signature peptide of cystinosis of SEQ ID NO:6 with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide and comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:40, CDR3 of SEQ ID NO:41, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO:42, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:44; a second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 with an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 48; a third CTNS signature peptide of cystinosis of SEQ ID NO: 12, using an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis of SEQ ID NO: 13, using an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis of SEQ ID NO:9 with an antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide and comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56; a second SHPK signature peptide of cystinosis of SEQ ID NO: 14, using an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or a third SHPK signature peptide of cystinosis of SEQ ID NO: 15, using an antibody or antigen-binding fragment thereof that binds to said third SHPK signature peptide; screening for cystinosis by said enrichment; calculating the ratio of signature peptide concentration:endogenous ATP7B peptide concentration for each signature peptide; diagnosing the subject as having cystinosis if a ratio of the first CTNS peptide concentration to endogenous ATP7B peptide concentration, the second CTNS peptide concentration to endogenous ATP7B peptide concentration, the third CTNS peptide concentration to endogenous ATP7B peptide concentration, the fourth CTNS peptide concentration to endogenous ATP7B peptide concentration, the first SHPK peptide concentration to endogenous ATP7B peptide concentration, the second SHPK peptide concentration to endogenous ATP7B peptide concentration, and / or the third SHPK signature peptide concentration to endogenous ATP7B peptide concentration is lower than a corresponding pre-defined threshold ratio or if the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptide is absent; The method further comprises:

62. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VH domain of SEQ ID NO:

69.

63. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO:

70.

64. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO:

106.

65. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO:

111.

66. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO:

71.

67. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises the VL domain of SEQ ID NO:

72.

68. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO:

116.

69. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO:

121.

70. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VH domain of SEQ ID NO:

73.

71. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VL domain of SEQ ID NO:

74.

72. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a heavy chain of SEQ ID NO:

126.

73. 62. The method of claim 61 , wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a light chain of SEQ ID NO:

131.

74. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used to enrich for the endogenous ATP7B peptide of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO:

75.

75. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used to enrich for the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO:

76.

76. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used to enrich for the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO:

136.

77. The method of claim 61, wherein the antibody or antigen-binding fragment thereof used to enrich for the endogenous ATP7B signature peptide of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO:

141.

78. 1. A method for detecting one or more signature peptides of Severe Combined Immunodeficiency (SCID), Wiskott-Aldrich Syndrome (WAS), and / or X-linked Agammaglobulinemia (XLA) in one or more dried blood spot (DBS) samples, comprising: obtaining one or more dried blood spot (DBS) samples; enzymatically digesting proteins from the blood of each DBS to produce one or more peptides; Concentrating the mixture, the concentration comprising: a CD3ε signature peptide of the SCID of SEQ ID NO:1 with an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; a first WASp signature peptide of WAS of SEQ ID NO:2 with an antibody or antigen-binding fragment thereof that binds to said first WASp signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; a second WASp signature peptide of WAS of SEQ ID NO:3 with an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; and / or a first BTK signature peptide of XLA of SEQ ID NO: 4 with an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide and comprises a heavy chain variable (VH) domain that comprises a CDR1 of SEQ ID NO: 34, a CDR2 of SEQ ID NO: 35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain that comprises a CDR1 of SEQ ID NO: 36, a CDR2 of SEQ ID NO: 37, and a CDR3 of SEQ ID NO: 38; and / or a second BTK signature peptide of XLA of SEQ ID NO:5 with an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; said concentrating; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to detect one or more signature peptides of SCID, WAS, and XLA in one or more DBS samples; The method comprising:

79. 79. The method of claim 78, wherein the enzyme is trypsin.

80. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of the SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO:

63.

81. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used for enrichment of the CD3ε signature peptide of the SCID of SEQ ID NO: 1 comprises the VL domain of SEQ ID NO:

64.

82. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO:

65.

83. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO: 2 comprises the VL domain of SEQ ID NO:

66.

84. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a heavy chain of SEQ ID NO:

86.

85. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first WASp signature peptide of WAS of SEQ ID NO:2 comprises a light chain of SEQ ID NO:

91.

86. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VH domain of SEQ ID NO:

67.

87. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO: 4 comprises a VL domain of SEQ ID NO:

68.

88. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a heavy chain of SEQ ID NO:

96.

89. 79. The method of claim 78, wherein the antibody or antigen-binding fragment thereof used to enrich for the first BTK signature peptide of XLA of SEQ ID NO:4 comprises a light chain of SEQ ID NO:

101.

90. 79. The method of claim 78, wherein concentrating comprises: a first CTNS signature peptide of cystinosis of SEQ ID NO:6 with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide and comprises a heavy chain variable (VH) domain that comprises CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:40, and CDR3 of SEQ ID NO:41, and a light chain variable (VL) domain that comprises CDR1 of SEQ ID NO:42, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:44; a second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8, using an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 48; a third CTNS signature peptide of cystinosis of SEQ ID NO: 12, using an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis of SEQ ID NO: 13, using an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis of SEQ ID NO:9 with an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56; a second SHPK signature peptide of cystinosis of SEQ ID NO: 14, using an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or a third SHPK signature peptide of cystinosis of SEQ ID NO: 15, using an antibody or antigen-binding fragment thereof that binds to said third SHPK signature peptide; detecting one or more signature peptides of cystinosis by said enrichment; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thereby detecting one or more signature peptides of cystinosis in one or more DBS samples; The method further comprises:

91. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a VH domain of SEQ ID NO:

69.

92. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a VL domain of SEQ ID NO:

70.

93. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a heavy chain of SEQ ID NO:

106.

94. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a light chain of SEQ ID NO:

111.

95. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO:

71.

96. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises the VL domain of SEQ ID NO:

72.

97. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO:

116.

98. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the second CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO:

121.

99. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VH domain of SEQ ID NO:

73.

100. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VL domain of SEQ ID NO:

74.

101. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a heavy chain of SEQ ID NO:

126.

102. 91. The method of claim 90, wherein the antibody or antigen-binding fragment thereof used to enrich for the first SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a light chain of SEQ ID NO:

131.

103. 79. The method of claim 78, wherein concentrating comprises: a first ATP7B signature peptide of WD of SEQ ID NO: 10 using an antibody or antigen-binding fragment thereof that binds to said first ATP7B signature peptide; and / or a second ATP7B signature peptide of WD of SEQ ID NO: 11 or 21 with an antibody or antigen-binding fragment thereof that binds to said second ATP7B signature peptide and comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 57, CDR2 of SEQ ID NO: 58, and CDR3 of SEQ ID NO: 59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 60, CDR2 of SEQ ID NO: 61, and CDR3 of SEQ ID NO: 62; a third ATP7B signature peptide of WD of SEQ ID NO: 16 using an antibody or antigen-binding fragment thereof that binds to said third ATP7B signature peptide; a fourth ATP7B signature peptide of the WD of SEQ ID NO: 17 using an antibody or antigen-binding fragment thereof that binds to said fourth ATP7B signature peptide; a fifth ATP7B signature peptide of the WD of SEQ ID NO: 18 using an antibody or antigen-binding fragment thereof that binds to said fifth ATP7B signature peptide; a sixth ATP7B signature peptide of the WD of SEQ ID NO: 19 using an antibody or antigen-binding fragment thereof that binds to said sixth ATP7B signature peptide; and / or a seventh ATP7B signature peptide of the WD of SEQ ID NO: 20 using an antibody or antigen-binding fragment thereof that binds to said seventh ATP7B signature peptide; detecting one or more signature peptides of Wilson's disease (WD) by said enrichment; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides, thereby detecting one or more signature peptides of WD in one or more DBS samples; The method further comprises:

104. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO:

75.

105. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO:

76.

106. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO:

136.

107. The method of claim 103, wherein the antibody or antigen-binding fragment thereof used to enrich for the second ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO:

141.

108. 1. An assay for screening for Severe Combined Immunodeficiency Disorder (SCID), Wiskott-Aldrich Syndrome (WAS), X-linked Agammaglobulinemia (XLA) in a subject, and / or Wilson's Disease (WD), comprising: (i) an antibody or antigen-binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27 (binding to the CD3ε signature peptide of the SCID of SEQ ID NO:1); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33 (binding to the WASp signature peptide of the WAS of SEQ ID NO:2); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO: 38 (which binds to the BTK signature peptide of XLA of SEQ ID NO: 4); and / or a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:57, CDR2 of SEQ ID NO:58, and CDR3 of SEQ ID NO:59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62 (binding to the ATP7B peptide of SEQ ID NO:11 or 21); and / or (ii) an antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO:3; An antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:5; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 10; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 16; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 17; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 18; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 19; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 20; and / or An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO:21; and / or (iii) a reference signature peptide comprising: CD3ε peptide of SCID of SEQ ID NO:1; WASp peptide of WAS of SEQ ID NO:2; WASp peptide of WAS of SEQ ID NO:3; BTK peptide of XLA of SEQ ID NO:4; BTK peptide of XLA of SEQ ID NO:5; ATP7B peptide of WD of SEQ ID NO: 10; ATP7B peptide of WD of SEQ ID NO: 11; ATP7B peptide of WD of SEQ ID NO: 16; ATP7B peptide of WD of SEQ ID NO: 17; ATP7B peptide of WD of SEQ ID NO: 18; ATP7B peptide of WD of SEQ ID NO: 19; ATP7B peptide of SEQ ID NO: 20 WD; and / or ATP7B peptide of WD of SEQ ID NO: 21, The assay comprising:

109. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VH domain of SEQ ID NO:

63.

110. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide of SCID of SEQ ID NO: 1 comprises a VL domain of SEQ ID NO:

64.

111. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VH domain of SEQ ID NO:

65.

112. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a VL domain of SEQ ID NO:

66.

113. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO:2 comprises a heavy chain of SEQ ID NO:

86.

114. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO: 2 comprises a light chain of SEQ ID NO:

91.

115. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:4 comprises a VH domain of SEQ ID NO:

67.

116. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:4 comprises a VL domain of SEQ ID NO:

68.

117. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:4 comprises a heavy chain of SEQ ID NO:

96.

118. The assay of claim 108, wherein the antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:4 comprises a light chain of SEQ ID NO:

101.

119. 109. The assay of claim 108 for further screening for cystinosis, comprising: (iii) an antibody or antigen-binding fragment thereof comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:40, and CDR3 of SEQ ID NO:41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:42, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:44 (binding to the cystinosis CTNS signature peptide of SEQ ID NO:6); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:45, CDR2 of SEQ ID NO:46, and CDR3 of SEQ ID NO:47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:49, and CDR3 of SEQ ID NO:50 (binding to the cystinosis CTNS signature peptide of SEQ ID NO:7 or 8); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56 (binding to the cystinosis SHPK signature peptide of SEQ ID NO:9); and / or (iv) an antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of SEQ ID NO: 12; An antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of SEQ ID NO: 13; An antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of SEQ ID NO: 14; and / or An antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of SEQ ID NO: 15; and / or (v) a reference signature peptide comprising: Cystinosis CTNS peptide of SEQ ID NO:6; Cystinosis CTNS peptide of SEQ ID NO: 7 or 8; Cystinosis CTNS peptide of SEQ ID NO: 12; Cystinosis CTNS peptide of SEQ ID NO: 13; Cystinosis SHPK peptide of SEQ ID NO:9; Cystinosis SHPK peptide of SEQ ID NO: 14; and / or Cystinosis SHPK peptide of SEQ ID NO: 15, The assay comprising:

120. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO:6 comprises a VH domain of SEQ ID NO:

69.

121. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a VL domain of SEQ ID NO:

70.

122. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a heavy chain of SEQ ID NO:

106.

123. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 6 comprises a light chain of SEQ ID NO:

111.

124. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VH domain of SEQ ID NO:

71.

125. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a VL domain of SEQ ID NO:

72.

126. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a heavy chain of SEQ ID NO:

116.

127. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of cystinosis of SEQ ID NO: 7 or 8 comprises a light chain of SEQ ID NO:

121.

128. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VH domain of SEQ ID NO:

73.

129. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a VL domain of SEQ ID NO:

74.

130. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO:9 comprises a heavy chain of SEQ ID NO:

126.

131. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of cystinosis of SEQ ID NO: 9 comprises a light chain of SEQ ID NO:

131.

132. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VH domain of SEQ ID NO:

75.

133. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a VL domain of SEQ ID NO:

76.

134. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a heavy chain of SEQ ID NO:

136.

135. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of the WD of SEQ ID NO: 11 or 21 comprises a light chain of SEQ ID NO:

141.

136. The assay of claim 119, wherein the reference signature peptide is isotopically labeled.

137. The assay of claim 119, wherein the antibody or antigen-binding fragment thereof is attached to a magnetic bead.

138. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 22, CDR2 of SEQ ID NO: 23, and CDR3 of SEQ ID NO: 24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 25, CDR2 of SEQ ID NO: 26, and CDR3 of SEQ ID NO:

27.

139. The isolated antibody or antigen-binding fragment thereof of claim 138, wherein the VH domain comprises SEQ ID NO:

63.

140. The isolated antibody or antigen-binding fragment thereof of claim 138, wherein the VL domain comprises SEQ ID NO:

64.

141. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:

33.

142. The isolated antibody or antigen-binding fragment thereof of claim 141, wherein the VH domain comprises SEQ ID NO:

65.

143. The isolated antibody or antigen-binding fragment thereof of claim 141, wherein the VL domain comprises SEQ ID NO:

66.

144. 142. The isolated antibody or antigen-binding fragment thereof of claim 141, wherein the heavy chain comprises SEQ ID NO:

86.

145. 142. The isolated antibody or antigen-binding fragment thereof of claim 141, wherein the light chain comprises SEQ ID NO:

91.

146. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 34, CDR2 of SEQ ID NO: 35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 36, CDR2 of SEQ ID NO: 37, and CDR3 of SEQ ID NO:

38.

147. The isolated antibody or antigen-binding fragment thereof of claim 146, wherein the VH domain comprises SEQ ID NO:

67.

148. The isolated antibody or antigen-binding fragment thereof of claim 146, wherein the VL domain comprises SEQ ID NO:

68.

149. The isolated antibody or antigen-binding fragment thereof of claim 146, wherein the heavy chain comprises SEQ ID NO:

96.

150. The isolated antibody or antigen-binding fragment thereof of claim 146, wherein the light chain comprises SEQ ID NO:

101.

151. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 39, CDR2 of SEQ ID NO: 40, and CDR3 of SEQ ID NO: 41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 42, CDR2 of SEQ ID NO: 43, and CDR3 of SEQ ID NO:

44.

152. 152. The isolated antibody or antigen-binding fragment thereof of claim 151, wherein the VH domain comprises SEQ ID NO:

69.

153. 152. The isolated antibody or antigen-binding fragment thereof of claim 151, wherein the VL domain comprises SEQ ID NO:

70.

154. 152. The isolated antibody or antigen-binding fragment thereof of claim 151, wherein the heavy chain comprises SEQ ID NO:

106.

155. 152. The isolated antibody or antigen-binding fragment thereof of claim 151, wherein the light chain comprises SEQ ID NO:

111.

156. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO:

50.

157. The isolated antibody or antigen-binding fragment thereof of claim 156, wherein the VH domain comprises SEQ ID NO:

71.

158. 157. The isolated antibody or antigen-binding fragment thereof of claim 156, wherein the VL domain comprises SEQ ID NO:

72.

159. 157. The isolated antibody or antigen-binding fragment thereof of claim 156, wherein the heavy chain comprises SEQ ID NO:

116.

160. 157. The isolated antibody or antigen-binding fragment thereof of claim 156, wherein the light chain comprises SEQ ID NO:

121.

161. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:

56.

162. 162. The isolated antibody or antigen-binding fragment thereof of claim 161, wherein the VH domain comprises SEQ ID NO:

73.

163. 162. The isolated antibody or antigen-binding fragment thereof of claim 161, wherein the VL domain comprises SEQ ID NO:

74.

164. 162. The isolated antibody or antigen-binding fragment thereof of claim 161, wherein the heavy chain comprises SEQ ID NO:

126.

165. 162. The isolated antibody or antigen-binding fragment thereof of claim 161, wherein the light chain comprises SEQ ID NO:

131.

166. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:57, CDR2 of SEQ ID NO:58, and CDR3 of SEQ ID NO:59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:

62.

167. 167. The isolated antibody or antigen-binding fragment thereof of claim 166, wherein the VH domain comprises SEQ ID NO:

75.

168. 167. The isolated antibody or antigen-binding fragment thereof of claim 166, wherein the VL domain comprises SEQ ID NO:

76.

169. 167. The isolated antibody or antigen-binding fragment thereof of claim 166, wherein the heavy chain comprises SEQ ID NO:

136.

170. 167. The isolated antibody or antigen-binding fragment thereof of claim 166, wherein the light chain comprises SEQ ID NO:

141.

171. 1. A kit comprising: (i) an antibody or antigen-binding fragment thereof, comprising: a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27 (binding to the CD3ε signature peptide of the SCID of SEQ ID NO:1); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33 (binding to the WASp signature peptide of the WAS of SEQ ID NO:2); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:34, CDR2 of SEQ ID NO:35, and CDR3 of sequence GDI, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:36, CDR2 of SEQ ID NO:37, and CDR3 of SEQ ID NO:38 (binding to the BTK signature peptide of XLA of SEQ ID NO:4); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:40, and CDR3 of SEQ ID NO:41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:42, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:44 (binding to the cystinosis CTNS signature peptide of SEQ ID NO:6); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:45, CDR2 of SEQ ID NO:46, and CDR3 of SEQ ID NO:47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:48, CDR2 of SEQ ID NO:49, and CDR3 of SEQ ID NO:50 (binding to the cystinosis CTNS signature peptide of SEQ ID NO:7 or 8); a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:51, CDR2 of SEQ ID NO:52, and CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:54, CDR2 of SEQ ID NO:55, and CDR3 of SEQ ID NO:56 (which binds to the cystinosis SHPK signature peptide of SEQ ID NO:9); and / or a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:57, CDR2 of SEQ ID NO:58, and CDR3 of SEQ ID NO:59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62 (binding to the ATP7B peptide of SEQ ID NO:11 or 21); the antibody or antigen-binding fragment thereof comprising and / or (ii) an antibody or antigen-binding fragment thereof that binds to the WASp signature peptide of WAS of SEQ ID NO:3; An antibody or antigen-binding fragment thereof that binds to the BTK signature peptide of XLA of SEQ ID NO:5; An antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of SEQ ID NO: 12; An antibody or antigen-binding fragment thereof that binds to the CTNS signature peptide of SEQ ID NO: 13; An antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of SEQ ID NO: 14; An antibody or antigen-binding fragment thereof that binds to the SHPK signature peptide of SEQ ID NO: 15; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 10; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 16; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 17; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 18; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 19; An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO: 20; and / or An antibody or antigen-binding fragment thereof that binds to the ATP7B signature peptide of SEQ ID NO:21; and / or (iii) a reference signature peptide, comprising: CD3ε peptide of SCID of SEQ ID NO:1; WASp peptide of WAS of SEQ ID NO:2; WASp peptide of WAS of SEQ ID NO:3; BTK peptide of XLA of SEQ ID NO:4; BTK peptide of XLA of SEQ ID NO:5; Cystinosis CTNS peptide of SEQ ID NO:6; Cystinosis CTNS peptide of SEQ ID NO: 7 or 8; Cystinosis CTNS peptide of SEQ ID NO: 12; Cystinosis CTNS peptide of SEQ ID NO: 13; Cystinosis SHPK peptide of SEQ ID NO:9; Cystinosis SHPK peptide of SEQ ID NO: 14; Cystinosis SHPK peptide of SEQ ID NO: 15; ATP7B peptide of WD of SEQ ID NO: 10; ATP7B peptide of WD of SEQ ID NO: 11; ATP7B peptide of WD of SEQ ID NO: 16; ATP7B peptide of WD of SEQ ID NO: 17; ATP7B peptide of WD of SEQ ID NO: 18; ATP7B peptide of WD of SEQ ID NO: 19; ATP7B peptide of SEQ ID NO: 20 WD; and / or ATP7B peptide of WD of SEQ ID NO: 21, The reference signature peptide comprising: The kit comprising:

172. 172. The kit of claim 171, further comprising one or more of a filter paper card, a punch tool, digestive enzymes, a digestion buffer, a solid support for an antibody or antigen-binding fragment thereof, and an elution buffer.

173. The kit of claim 171, wherein the reference signature peptide is isotopically labeled.

174. The kit of claim 171, wherein the antibody or antigen-binding fragment thereof is attached to a magnetic bead.

175. 1. A method of screening for severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), cystinosis, and / or Wilson's disease (WD) in a subject, comprising: obtaining a dried blood spot (DBS) sample from the subject; enzymatically digesting proteins from DBS blood to produce one or more peptides; Concentrating the mixture, the concentration comprising: a CD3ε signature peptide of SCID using an antibody or antigen-binding fragment thereof that binds to said CD3ε signature peptide; a first WASp signature peptide of a WAS with an antibody or antigen-binding fragment thereof that binds to said first WASp signature peptide; a second WASp signature peptide of the WAS with an antibody or antigen-binding fragment thereof that binds to said second WASp signature peptide; a first BTK signature peptide of XLA with an antibody or antigen-binding fragment thereof that binds to said first BTK signature peptide; a second BTK signature peptide with an antibody or antigen-binding fragment thereof that binds to said second BTK signature peptide; a first CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first CTNS signature peptide; a second CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second CTNS signature peptide; a third CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third CTNS signature peptide; a fourth CTNS signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said fourth CTNS signature peptide; a first SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said first SHPK signature peptide; a second SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said second SHPK signature peptide; and / or a third SHPK signature peptide of cystinosis with an antibody or antigen-binding fragment thereof that binds to said third SHPK signature peptide; a first ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said first ATP7B signature peptide; a second ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said second ATP7B signature peptide; a third ATP7B signature peptide with an antibody or antigen-binding fragment that binds to said third ATP7B signature peptide; a fourth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fourth ATP7B signature peptide; a fifth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said fifth ATP7B signature peptide; a sixth ATP7B signature peptide with an antibody or antigen-binding fragment thereof that binds to said sixth ATP7B signature peptide; and / or a seventh ATP7B signature peptide using an antibody or antigen-binding fragment thereof; said concentrating; performing liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) on the enriched peptides to determine the concentration of the peptides; 2. Diagnosing a subject, comprising: SCID if the concentration of the CD3ε signature peptide is below a predetermined threshold concentration or if the CD3ε signature peptide is absent; if the concentrations of the first and / or second WASp signature peptides are lower than corresponding pre-defined threshold concentrations or if the first and / or second WASp signature peptides are absent, WAS; diagnosing the subject as having XLA if the concentrations of the first and / or second BTK signature peptides are lower than corresponding pre-defined threshold concentrations or if the first and / or second BTK signature peptides are absent; cystinosis if the concentrations of the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptides are lower than corresponding pre-defined threshold concentrations or if the first CTNS, the second CTNS, the third CTNS, the fourth CTNS, the first SHPK, the second SHPK, and / or the third SHPK signature peptides are absent; and / or If the concentration of the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is lower than a corresponding predetermined threshold concentration, or if the first ATP7B, the second ATP7B, the third ATP7B, the fourth ATP7B, the fifth ATP7B, the sixth ATP7B, and / or the seventh ATP7B signature peptide is absent, a WD is detected. diagnosing the patient as having The method comprising:

176. 176. The method of claim 175, wherein: the CD3ε signature peptide of SCID is encoded by the amino acid sequence set forth in SEQ ID NO:1; The first WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:2; The second WASp signature peptide of WAS is encoded by the amino acid sequence set forth in SEQ ID NO:3; The first BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:4; and / or the second BTK signature peptide of XLA is encoded by the amino acid sequence set forth in SEQ ID NO:5; the first CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:6; the second CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 7 or 8; the third CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 12; the fourth CTNS signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 13; the first SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO:9; the second SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 14; the third SHPK signature peptide of cystinosis is encoded by the amino acid sequence set forth in SEQ ID NO: 15; the first ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 10; the second ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 11 or 21; the third ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 16; the fourth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 17; the fifth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 18; the sixth ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO: 19; and / or The seventh ATP7B signature peptide of WD is encoded by the amino acid sequence set forth in SEQ ID NO:20; The method.

177. 176. The method of claim 175, wherein: The antibody or antigen-binding fragment thereof that binds to the CD3ε signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:22, CDR2 of SEQ ID NO:23, and CDR3 of SEQ ID NO:24, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:25, CDR2 of SEQ ID NO:26, and CDR3 of SEQ ID NO:27; The antibody or antigen-binding fragment thereof that binds to the first WASp signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:28, CDR2 of SEQ ID NO:29, and CDR3 of SEQ ID NO:30, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:31, CDR2 of SEQ ID NO:32, and CDR3 of SEQ ID NO:33; and / or The antibody or antigen-binding fragment thereof that binds to the first BTK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO:34, a CDR2 of SEQ ID NO:35, and a CDR3 of sequence GDI, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO:36, a CDR2 of SEQ ID NO:37, and a CDR3 of SEQ ID NO:38; The antibody or antigen-binding fragment thereof that binds to the first CTNS signature peptide of cystinosis comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:39, CDR2 of SEQ ID NO:40, and CDR3 of SEQ ID NO:41, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:42, CDR2 of SEQ ID NO:43, and CDR3 of SEQ ID NO:44; The antibody or antigen-binding fragment thereof that binds to the second CTNS signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO: 45, CDR2 of SEQ ID NO: 46, and CDR3 of SEQ ID NO: 47, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO: 48, CDR2 of SEQ ID NO: 49, and CDR3 of SEQ ID NO: 50; The antibody or antigen-binding fragment thereof that binds to the first SHPK signature peptide comprises a heavy chain variable (VH) domain comprising a CDR1 of SEQ ID NO:51, a CDR2 of SEQ ID NO:52, and a CDR3 of SEQ ID NO:53, and a light chain variable (VL) domain comprising a CDR1 of SEQ ID NO:54, a CDR2 of SEQ ID NO:55, and a CDR3 of SEQ ID NO:56, and / or The method, wherein the antibody or antigen-binding fragment thereof that binds to the second ATP7B signature peptide comprises a heavy chain variable (VH) domain comprising CDR1 of SEQ ID NO:57, CDR2 of SEQ ID NO:58, and CDR3 of SEQ ID NO:59, and a light chain variable (VL) domain comprising CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62.

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