Method for detecting and quantifying free light chain dimer of immunoglobulin

Abstract

Some pathological conditions are accompanied by an increase in free light chain (FLC) kappa and / or lambda dimers. For example, high levels of FLC dimers have been found in AL amyloidosis, multiple myeloma, and multiple sclerosis. Accordingly, there is a need for a sensitive method capable of specifically and quantitatively measuring the amount of FLC dimer in a sample. The present application relates to a method of detecting and quantifying immunoglobulin FLC dimers in a sample comprising proteolytic digestion of the sample under non-reducing conditions and mass spectrometry of the digested sample to detect immunoglobulin free light chain dimer peptides. The methods may help diagnose and monitor diseases characterized by abnormal production of FLC, such as plasma cytoplasm, light chain deposition disease (LCDD), autoimmune diseases, chronic kidney disease (CKD), and inflammatory conditions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 367,941, filed on July 8, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention generally relates to the field of immunoglobulin detection, and in particular to the detection and quantification of immunoglobulin free light chain dimers in samples. Background Art

[0004] Human immunoglobulins are composed of two heavy chains and two light chains that are bound together by covalent and noncovalent interactions. Light chains are produced by plasma cells in excess of heavy chains, resulting in the release of free light chains (FLCs) (1). Light chains are divided into two subtypes based on the amino acid sequence of the constant region: κ (kappa) and λ (lambda). Normal serum concentrations of κ and λ FLCs range as follows: κ, 3.3-19.4 mg / L; λ, 5.7-26.3 mg / L; and κ / λ ratio, 0.26-1.65 (2). These concentrations are maintained by a balance between continued production by plasma cells and clearance by the kidneys. The half-life of FLCs in serum is 2 to 6 hours (1). FLCs are also present in urine, cerebrospinal fluid (CSF), synovial fluid, tears, and saliva.

[0005] Several pathological conditions are associated with an increase in κ, λ, or both FLCs. Polyclonal FLC overproduction typically results from general activation of the immune system, leading to changes in κ and / or λ levels with or without changes in the κ / λ ratio, whereas monoclonal FLC overproduction is characterized by the presence of only one type of FLC and a change in the κ / λ ratio (3). Polyclonal serum FLCs have been found to be elevated in autoimmune diseases such as systemic lupus erythematosus (SLE) (4) and inflammatory conditions such as asthma (5). Infection with certain viruses (i.e., HIV) also results in an increase in circulating FLCs (6). Overall, polyclonal FLC overproduction is associated with an increased risk of death: when combined FLC concentrations exceed 47.2 mg / L, the hazard ratio for death is 2.07 (7).

[0006] Of particular concern is that increases in monoclonal FLC are seen in plasma cell dyscrasias (PCD), such as monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM), amyloid light chain (AL) amyloidosis, and non-amyloid light chain deposition disease (NALCDD). In MM, large amounts of κ or λ FLC are produced by malignant plasma cells that proliferate in the bone marrow and cause extensive bone lesions (8). In AL amyloidosis, the special properties of FLC make it easy for them to aggregate and deposit in various tissues, leading to organ damage and failure (9). MGUS and SMM are considered benign conditions, but the risk of developing MM or AL amyloidosis is increased. Serum FLC levels are often used to monitor PCD (10).

[0007] Free light chains exist as monomers, dimers, and oligomers. λ FLC is particularly prone to forming dimers and oligomers. FLC dimers are stabilized by covalent and noncovalent interactions between the two light chains (11). In κ LC, the dimers are held together by a disulfide bond between the C-terminal cysteine ​​at position 107 (C107), while in λ LC dimers, the disulfide bond is formed between the cysteine ​​at position 105 (C105) of the constant region. Conserved residues in the framework regions of the variable domains are involved in the noncovalent dimerization of FLC (12). Under certain pathological conditions, FLC dimerization can change in the direction of increased dimer formation. For example, high levels of FLC dimers have been found in AL amyloidosis, MM, and multiple sclerosis (11). FLC dimerization is independent of the total FLC concentration and should therefore be considered an independent parameter (13). The pathophysiological role of dimerization is not fully understood, but some studies suggest that FLCλ dimers may act as autoantibodies in severe autoimmune diseases (14).

[0008] Free light chain monomer-dimer pattern analysis (FLC-MDPA) found that monoclonal FLC dimers were significantly increased in the serum of patients with AL amyloidosis. Multivariate analysis showed that FLC-MDPA could successfully distinguish AL amyloidosis from benign PCDs, such as MGUS and SMM (15). This finding is very important because there is currently no blood-based test available for the diagnosis of AL amyloidosis. Disease diagnosis relies on painful and laborious biopsies. In addition, treatment is only initiated after organ damage has occurred. Therefore, a test that can detect the conversion of benign PCD conditions to AL amyloidosis early would prevent target organ damage. In this regard, FLC dimerization represents a promising biomarker for the diagnosis and monitoring of patients with AL amyloidosis.

[0009] Similar to patients with AL amyloidosis, the extent of FLC dimerization in MM patients is significantly higher than that in healthy controls, MGUS, or SMM patients (13). The utility of FLC dimerization in diagnosing or monitoring patients with MM remains to be demonstrated, but the noninvasive nature of this test warrants further investigation.

[0010] Multiple sclerosis (MS) is an autoimmune disease characterized by demyelination and neuronal loss. The diagnosis of this disease is challenging because other neurological diseases often have similar clinical manifestations (16). Analysis of CSF for the presence of oligoclonal IgG bands and FLC is now commonly used in the diagnostic workup of MS (17). Studies have found that FLC dimers are elevated in the CSF of MS patients (18). In 40% of MS patients, λ LC dimers are significantly increased. In the other 60% of cases, both κ monomers and dimers are increased. Overall, analysis of the FLC monomer-dimer pattern in CSF can distinguish MS from other neurological diseases with a specificity of 90% and a sensitivity of 96% (18). In order to develop a noninvasive test for MS patients, Kaplan et al. analyzed the FLC monomer-dimer pattern in saliva from MS patients and healthy individuals. They found that the level of FLC in saliva could distinguish healthy individuals from MS patients (19). Furthermore, salivary FLC correlates with relapse / remitting status and can therefore be used as a tool to monitor MS disease progression and response to treatment (20).

[0011] Currently, FLC is measured using the turbidimetric assays Flerite (The Binding Site Group Ltd., Birmingham, UK) and N Latex FLC (Siemens Healthcare Diagnostics Gmbh, Marburg, Germany) (21). Both assays use specific antibodies to detect monomeric and dimeric forms of FLC (22). This assay has become standard practice in disease screening for MM, AL amyloidosis, and, increasingly, MS (23, 24). For example, the Freelite assay is currently being used to diagnose MM and monitor response to treatment by distinguishing between complete response and strict complete response (21). Although turbidimetric analysis has been widely used in clinical practice, it is still criticized for poor accuracy, inability to detect excess antigen, differences between antibody reagent batches, and lack of consistency between different assays (22, 25).

[0012] Given the widespread application of FLC measurement, mass spectrometry-based techniques have been developed (27). The initial report by Bergen et al. focused on the characterization of intact FLC monomers and dimers from sera of patients with AL amyloidosis (27). Subsequently, the monoclonal immunoglobulin rapid accurate mass measurement (miRAMM) method was able to detect and quantify intact monoclonal FLC in the presence of a polyclonal background (28). In addition, this method was able to analyze FLC monomers and dimers and their glycosylated forms separately in sera from patients with AL amyloidosis (28). More recently, serum FLC immunoenrichment coupled with detection by MALDI-TOF mass spectrometry showed superior sensitivity compared to the Freelite assay (29). However, 24% of IFE-negative samples with abnormal κ / λ FLC ratios were not detected by the MALDI-TOF-based method, indicating that additional preanalytical optimization is required. Another limitation is the qualitative nature of the assay (29). An alternative mass spectrometry-based approach measured peptides from κ and λ light chains by selected reaction monitoring (SRM) after depletion of intact immunoglobulins. This assay allows accurate quantification of FLC down to 3.8 mg / L (κ) and 2.7 mg / L (λ), but cannot distinguish between monomeric and dimeric FLC forms (30).

[0013] Although FLC is considered a biomarker of plasma cell activity, they lack specificity and may not be sufficient for disease monitoring (3). Increases in FLC due to infection are indistinguishable from increases in FLC due to relapses of autoimmune disease. For example, FLC levels in subjects with active infection are comparable to those found in SLE patients with disease flare (31). In the case of AL amyloidosis, FLC measurements by turbidimetry may be inaccurate due to the structural abnormalities of the LC that are unique to this disorder. In addition, FLC assays are unable to distinguish between malignant conditions that require treatment, such as MM and AL amyloidosis, and benign conditions, such as MGUS and SMM. On the other hand, measurement of FLC dimers in patients with AL amyloidosis is considered a specific and sensitive tool in the diagnostic workup of this disease (15). Importantly, FLC dimerization is increased even when total FLC measurements or the κ / λ ratio are normal (13). Therefore, sensitive methods that can specifically measure the amount of FLC dimers would potentially aid in the diagnosis and monitoring of conditions such as AL amyloidosis, MM, and MS. Furthermore, quantitative assays would be more suitable for monitoring therapeutic efficacy and could better predict prognosis.

[0014] Therefore, there is a need for a sensitive method that can specifically and quantitatively measure the amount of FLC dimer in a sample, which would be very useful for diagnosis, prognosis and / or monitoring of disorders associated with overproduction of FLC, such as AL amyloidosis, MM and MS.

[0015] This specification refers to a number of documents, the contents of which are incorporated herein by reference in their entirety. Summary of the invention

[0016] In various aspects and embodiments, the present disclosure provides the following items 1 to 27:

[0017] 1. A method for detecting immunoglobulin free light chain (FLC) dimers in a sample, comprising:

[0018] (a) subjecting the sample to proteolytic digestion under non-reducing conditions to obtain a digested sample; and

[0019] (b) performing mass spectrometry analysis on the digested sample to detect immunoglobulin free light chain dimer peptides, wherein the detection of immunoglobulin free light chain dimer peptides indicates the presence of immunoglobulin free light chain dimers in the sample.

[0020] 2. The method according to item 1, wherein the sample is a serum sample, a cerebrospinal fluid sample or a saliva sample.

[0021] 3. The method according to item 1 or 2, wherein the sample is suspected to contain immunoglobulin free light chain dimers.

[0022] 4. The method according to any one of items 1 to 3, wherein the immunoglobulin free light chain dimers include dimers of κ light chains.

[0023] 5. The method according to any one of items 1 to 3, wherein the immunoglobulin free light chain dimers include dimers of λ light chains.

[0024] 6. The method according to any one of items 1 to 5, wherein proteolytic digestion of the sample comprises contacting the sample with at least one endoprotease.

[0025] 7. The method according to item 6, wherein the at least one endoprotease comprises trypsin, chymotrypsin, LysC, LysargiNase or any combination thereof.

[0026] 8. The method according to any one of items 1 to 7, wherein the method further comprises a denaturation step of the sample before the proteolytic digestion.

[0027] 9. The method according to item 8, wherein the denaturation step comprises contacting the sample with urea.

[0028] 10. The method of claim 9, wherein the denaturation step comprises contacting the sample with urea at a concentration of at least 4 M for at least 30 minutes.

[0029] 11. The method according to any one of items 1 to 10, wherein the method further comprises contacting the sample with a cysteine ​​modifying agent prior to the proteolytic digestion.

[0030] 12. The method according to item 11, wherein the cysteine ​​modifying agent comprises N-ethylmaleimide (NEM).

[0031] 13. The method according to any one of items 1 to 12, wherein the mass spectrometry is liquid chromatography coupled with parallel reaction monitoring.

[0032] 14. The method according to item 13, wherein the mass spectrometry analysis is performed on a quadrupole orbital trap mass spectrometer.

[0033] 15. The method according to any one of items 1 to 14, wherein the immunoglobulin FLC dimer is measured by spiking a known amount of a synthetic peptide containing a specific label.

[0034] 16. The method according to item 15, wherein the specific label comprises amino acids labeled with heavy isotopes.

[0035] 17. The method according to any one of items 1 to 16 further comprises enriching the kappa and / or lambda light chains in the sample before subjecting the sample to proteolytic digestion.

[0036] 18. The method according to item 17, wherein the enrichment comprises contacting the sample with one or more matrices capable of binding the κ and / or λ light chains; and eluting the κ and / or λ light chains bound to the one or more matrices.

[0037] 19. The method according to any one of items 1 to 18, wherein the immunoglobulin free light chain dimer peptide is and / or

[0038] 20. The method according to any one of items 1 to 19, wherein the sample is a biological sample from a subject suffering from a disease or disorder associated with FLC production.

[0039] 21. The method according to item 20, wherein the disease or disorder is an autoimmune disease, an inflammatory disorder, a viral infection or plasma cell dyscrasia (PCD).

[0040] 22. The method according to item 21, wherein the PCD is monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM), amyloid light chain (AL) amyloidosis or non-amyloid light chain deposition disease (NALCDD).

[0041] 23. The method according to item 20, wherein the sample is a serum sample from a human subject suffering from multiple myeloma.

[0042] 24. The method according to item 23, wherein the method further comprises quantifying the amount of immunoglobulin FLC dimer in the sample, and wherein the amount of immunoglobulin FLC dimer is correlated with tumor burden.

[0043] 25. The method according to item 20, wherein the sample is from a human subject suffering from or suspected of suffering from multiple sclerosis.

[0044] 26. The method according to item 25, wherein the sample is a cerebrospinal fluid sample.

[0045] 27. The method according to item 25, wherein the method comprises comparing the amount of immunoglobulin FLC dimer in the sample with the amount of immunoglobulin FLC dimer in a control sample from a healthy subject.

[0046] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-limiting description of specific embodiments thereof, which is given by way of example only with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In the attached picture:

[0048] Figure 1A and 1B Depicted is the MS / MS spectrum confirming the identity of the kappa FLC dimer peptide. Figure 1A : m / z 541.22 The fragmentation pattern of the precursor identified the disulfide-linked κFLC peptide SFNRGEC (SEQ ID NO: 1). Recombinant κFLC was treated with N-ethylmaleimide (NEM), digested with LysC, and analyzed by LC-MS / MS on a Q Exactive mass spectrometer. Figure 1B : The synthetic dimer peptide SFNRGEC was analyzed by LC-MS / MS as a positive control. The fragmentation pattern of the precursor at m / z 541.22 was similar to Figure 1A The maps were annotated using the pLabel tool of pLink software (32).

[0049] Figure 2A and 2B Depicted is the MS / MS spectrum confirming the identity of the λ-FLC dimer peptide. Figure 2A: m / z 806.35 The fragmentation pattern of the precursor corresponds to the λFLC peptide TVAPTECS (SEQ ID NO: 2) linked by disulfide bonds. Recombinant λFLC was treated with NEM, digested with LysC, and analyzed by LC-MS / MS on a Q Exactive mass spectrometer. Figure 2B : Synthetic dimer peptide TVAPTECS was analyzed by LC-MS / MS as a positive control. The fragmentation pattern of the precursor at m / z 806.35 was similar to Figure 2A The maps were annotated using the pLabel tool of pLink software (32).

[0050] Figures 3A-3D The extracted ion chromatogram of FLC dimer peptide is depicted. Synthetic κFLC dimer peptide SFNRGEC ( Figure 3A ), the dimeric peptide SFNRGE ( Figure 3B ), synthetic λFLC dimer peptide TVAPTECS ( Figure 3C ), the dimeric peptide TVAPTEC ( Figure 3D ). The retention time (in minutes) and mass accuracy (in ppm) of the most intense fragment ions are shown above the peaks. These images were generated and analyzed using Skyline 20 software (33).

[0051] Figure 4A -B depicts rFLCκ spiked into normal human serum ( Figure 4A ) and λ( Figure 4B ) dilution curve. Data were generated using PRM analysis on a Q Exactive mass spectrometer. Dimeric peptides SFNRGEC and TVAPTECS generated by LysC digestion under non-reducing conditions were measured for κ and λ rFLC, respectively. These graphs show the linear correlation between peptide peak area and spiked rFLC concentration. Goodness of fit was measured using R 2 Perform the evaluation (as shown).

[0052] Figure 5 Dilution curves of λrFLC spiked into normal human serum are depicted. Data were generated using PRM analysis on a QExactive mass spectrometer. The dimeric peptide KTVAPTECS generated by LysargiNase digestion under non-reducing conditions was analyzed in the presence of a normal human serum background. The graph shows a linear correlation between the m / z 623.3 peak area and the spiked rFLCλ concentration. 2 The goodness of fit of the linear model is shown.

[0053] Figure 6 Depicted are the peak areas of the kappa FLC dimer peptide SFNRGEC (light grey bars, SEQ ID No: 1) and the lambda FLC dipeptide TVAPTECS (dark grey bars, SEQ ID NO: 2) generated by LysC digestion of control serum in 4M or 8M urea for 1, 4 or 22 hours as indicated on the X-axis. Mean ± SD are shown.

[0054] Figure 7 Depict the normalized peak areas of the κFLC dimer peptide SFNRGEC (light grey bar) and the λFLC dimer peptide TVAPTECS (dark grey bar) produced by LysC digestion of control serum and serum from MM patients 1916 (IgG-κM protein) and 2666 (IgA-λM protein). The two peptides from human serum albumin were used as internal standards for target peptide signal normalization. Mean values ​​± SD are shown.

[0055] Figure 8 Depicted are the normalized peak areas of the κFLC dimer peptide SFNRGEC (light grey bars) and the λFLC dimer peptide TVAPTECS (dark grey bars) generated by LysC digestion of control serum, control serum enriched with κ affinity and protein L resin for κLC, and control serum enriched with λ affinity resin for λLC. The fold increase in the average peak area under each enrichment condition compared to the control value is shown.

[0056] Fig. 9 Dilution curves of κ and λ SIL peptides spiked into normal human serum are depicted. The SIL dimer peptide SFN with heavy arginine was detected at m / z 547.89 and 812.36, respectively. R GEC (circle) and T with heavy valine V APTECS (squares) was used for quantification. The graph shows the linear correlation between the SIL peptide peak area and the spike concentration. The goodness of fit was evaluated by R 2 (Each curve is shown) for evaluation.

[0057] Fig.10 Describe the λFLC dimer monitoring curve of MM patient 2666.Use the λ dimer SIL peptide of adding mark to calculate the absolute concentration (in mg / L) of λFLC (Y axis).The time (in months) since diagnosis is shown on the X axis.The table under the icon lists the λFLC dimer concentration, the response to treatment and the M protein amount measured by serum protein electrophoresis (SPEP).CR represents the complete response to dexamethasone / bortezomib / doxorubicin treatment.PR represents the partial response to treatment, and R represents relapse.NQ represents the data that cannot be quantified.

[0058] Fig.11Depicted are the amounts of kappa FLC dimer (light grey bars) and lambda FLC dimer (dark grey bars) measured by absolute quantification in control serum, control saliva and control CSF with spiked SIL dimer peptide. The logarithmic scale on the Y axis shows the mean concentration values ​​(mg / L).

[0059] Fig.12 Describe the amount of kappa FLC dimer (light grey bar) and lambda FLC dimer (dark grey bar) measured in control CSF, CSF from multiple sclerosis patients and CSF from patients with other neurological disorders (non-MS, as shown on the X-axis) by absolute quantification. The asterisk * indicates that the value is at least 1.85 times higher than the corresponding value in control #1. The patient ID shown on the X-axis corresponds to the patient ID in the first column of Table 2. The logarithmic scale on the Y-axis shows the average concentration value (mg / L). DETAILED DESCRIPTION

[0060] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0061] The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (ie, meaning "including, but not limited to,") unless otherwise noted.

[0062] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0063] The use of any and all examples, or exemplary language ("for example," "such as") provided herein, is intended merely to better illustrate implementations of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0064] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0065] In this document, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent error variation of the device or method used to determine the value, or contains a value close to the recited value, such as within 10% of the recited value (or range of values).

[0066] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values ​​within a range are also incorporated into the specification as if they were individually recited herein.

[0067] Where features or aspects of the disclosure are described in terms of Markush groups or alternative lists, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group or alternative list.

[0068] Unless specifically defined otherwise, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0069] Unless otherwise indicated, the molecular biology, recombinant protein, cell culture, and immunological techniques used in the present disclosure are standard procedures well known to those skilled in the art. This technique is described and explained in, for example, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Humes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates to date), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and JE Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates to date).

[0070] The method reported herein relies on the unique fragments of FLC dimers in the detection and quantification samples such as human biological fluids. The detection method involves enzymatic digestion of the sample under non-reducing conditions before performing sample analysis. The small FLC dimer fragments monitored in the detection method can be detected and quantified with high sensitivity and high accuracy.

[0071] The present disclosure provides a method for detecting and quantifying free light chain dimers of immunoglobulins in a sample, comprising: (a) performing proteolytic digestion on the sample under non-reducing conditions to obtain a digested sample; and (b) performing mass spectrometry analysis on the digested sample to detect and quantify free light chain dimer peptides of immunoglobulins, wherein the detection of free light chain dimer peptides of immunoglobulins indicates the presence of free light chain dimers of immunoglobulins in the sample.

[0072] The present disclosure provides a method for identifying and quantifying disulfide-bonded free light chain dimers in samples such as biological fluids. The method comprises: 1) collecting a sample (e.g., biological fluid) from a subject; 2) proteolytically digesting FLC with an enzyme under non-reducing conditions to produce FLC dimer peptides; 3) performing mass spectrometry analysis to identify and quantify FLC dimer peptides.

[0073] The term "non-reducing conditions" as used herein means that the digestion is performed under conditions that maintain the disulfide bridges between the light chains, for example, in the absence of a reducing agent such as β-mercaptoethanol (β-ME), dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP).

[0074] The methods disclosed herein can be used to detect κLC dimers and / or λLC dimers. In some embodiments, FLC can be denatured before proteolytic cleavage. For example, FLC can be denatured by heating the sample or using a denaturant such as urea or guanidine hydrochloride. In an embodiment, the sample is treated with urea at a concentration of at least 4M, preferably at least 6M, such as 8M. The treatment can be performed for a time sufficient to denature the sample, such as at least 30 minutes or 1 hour.

[0075] In some embodiments, the method includes chemically modifying the free sulfhydryl groups of cysteine ​​prior to proteolytic cleavage. For example, the free sulfhydryl groups of cysteine ​​can be modified by treatment with agents such as N-ethylmaleimide (NEM) or iodoacetamide. In some embodiments, the chemical modification is accomplished by NEM to protect the free sulfhydryl groups from random oxidation processes (i.e., disulfide shuffling). In some embodiments, the method does not include any chemical modification.

[0076] In some embodiments, the method includes separation or enrichment of FLC before proteolytic cleavage. For example, separation of FLC can be achieved by any suitable protein purification technique, such as affinity chromatography, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography (HIC), Melon gel and / or gel electrophoresis. In another embodiment, FLC can be enriched using a matrix that specifically binds to FLC. A matrix that can specifically bind to κ and / or λ light chains is commercially available, for example, CaptureSelect from ThermoFisher. TM κXP Affinity Matrix and CaptureSelect TM Lambda XP affinity matrix.

[0077] FLC in the sample can be proteolytically cleaved with a suitable agent to generate digested peptides. Agents for cleaving proteins include chemical agents such as cyanogen bromide (CNBr) cleaved at methionine (Met) residues; BNPS-skatole cleaved at tryptophan (Trp) residues; formic acid cleaved at aspartic acid-proline (Asp-Pro) peptide bonds; hydroxylamine cleaved at asparagine-glycine (Asn-Gly) peptide bonds, and 2-nitro-5-thiocyanatobenzoic acid (NTCB) cleaved at cysteine ​​(Cys) residues, and enzymes (e.g., proteases). In an embodiment, FLC in the sample is proteolytically cleaved with any suitable enzyme (e.g., proteases) or a combination thereof. Enzymes that can be used for proteolytic cleavage include trypsin, pepsin, chymotrypsin, AspN, LysargiNase, LysC, LysN, GluC, ArgC, Pro / Ala proteases, Sap9, KEX2, or any combination thereof. Digestion can also be performed using a combination of one or more chemical agents and one or more proteases.

[0078] In some embodiments, FLC dimer peptides are quantified by spiking with known amounts of labeled synthetic peptides. The synthetic peptides may contain heavy isotope labeled amino acids, substituted amino acids, or chemically modified amino acids to establish a mass difference that can distinguish the labeled peptides from unlabeled endogenous peptides in a mass spectrum. For example, the arginine, lysine, or valine residues of the synthetic peptides may contain heavy isotopes, such as 13 C. 15 N and / or 2 H.

[0079] In some embodiments, the method involves determining the ratio of endogenous FLC dimer peptides to serum protein peptides in a sample. Examples of serum proteins are apolipoprotein B, transferrin and human serum albumin.

[0080] In an embodiment, FLC dimer peptide quantification is carried out by liquid chromatography-mass spectrometry (LC-MS) coupled with parallel reaction monitoring (PRM). Selected or multiple reaction monitoring (SRM or MRM) can also be used for quantitative specific fragment ions of FLC dimer peptide. Mass spectrometry can be carried out on a triple quadrupole mass spectrometer, an ion trap mass spectrometer, a time-of-flight mass spectrometer, an orbital trap mass spectrometer or a hybrid mass spectrometer.

[0081] In embodiments, the sample is a biological sample such as a biological fluid. In some embodiments, the biological sample is a blood-derived sample (e.g., blood, serum, plasma), urine, saliva, tears, urogenital secretions, nasal secretions, bronchoalveolar lavage fluid, synovial fluid or cerebrospinal fluid (CSF). The sample can be a biological sample obtained from any animal, including non-human primates or humans. In embodiments, the sample is a biological sample from people. In embodiments, the biological sample is serum. In another embodiment, the biological sample is CSF.

[0082] Method disclosed herein can be used for detecting and quantitatively FLC dimer in the subject sample of any disease characterized by abnormal generation or overexpression of FLC such as plasma cell dyscrasia, autoimmune disease, chronic kidney disease (CKD) and inflammatory disease. The example of plasma cell dyscrasia characterized by abnormal generation of FLC includes multiple myeloma, plasma cell leukemia (PCL), solitary plasmacytoma (SP), B-cell non-Hodgkin lymphoma (B-NHL), monoclonal gammopathy of unknown significance (MGUS), smoldering multiple myeloma (SMM), POEMS syndrome, amyloid light chain (AL) amyloidosis, non-amyloid light chain deposition disease (NALCDD) and Waldenstrom's macroglobulinemia. The example of autoimmune disease characterized by abnormal generation of FLC includes multiple sclerosis (MS) and systemic lupus erythematosus (SLE). The example of inflammatory disease characterized by abnormal generation of FLC includes asthma.

[0083] In some embodiments, the method monitors the patient's response to treatment by analyzing the patient's samples at different time points. The method may include collecting samples from the patient before treatment, collecting one or more samples after treatment begins, performing mass spectrometry analysis on the initial sample and one or more subsequent samples to quantify the amount of FLC dimer in the sample, and comparing the amount of FLC dimer in the sample before and after treatment begins. This method is particularly applicable to monitoring type-only light chain multiple myeloma, a disease in which malignant plasma cells only produce LC without heavy chains.

[0084] The method can also be used to predict relapse (e.g., multiple myeloma patients) or disease flare-ups (e.g., multiple sclerosis patients). By measuring the amount of FLC dimer in a sample from a patient over time, if the amount of FLC dimer in the patient sample begins to increase, relapse can be predicted. Early detection of relapse can help guide decisions to change treatment methods, thereby enabling patients to obtain better treatment outcomes.

[0085] This method can be used in conjunction with other techniques to diagnose conditions such as AL amyloidosis and multiple sclerosis, for example by comparing the amount of FLC dimer in a patient's biological fluid to the amount of FLC dimer in biological fluid taken from healthy control subjects.

[0086] Example

[0087] The present disclosure is illustrated in more detail by the following non-limiting examples.

[0088] Example 1: Enzymatic digestion of recombinant FLC to produce unique dimeric peptides

[0089] κ FLC forms dimers via a disulfide bond between constant region cysteine ​​107 of each monomer chain. λ FLC dimers are formed via a disulfide bond between constant region cysteine ​​105. Therefore, enzymatic digestion of dimerized FLC under non-reducing conditions can release peptides unique to κ or λ FLC dimers.

[0090] To discover and characterize FLC dimer peptides, human recombinant FLC (rFLC) κ (Pr00115) or rFLCλ (Pr00116) from Absolute Antibodies were used. PAGE under non-reducing conditions showed the presence of monomeric (25 kDa) and dimeric (50 kDa) species in both reagents. In addition, de novo protein sequencing confirmed the identity of the commercial protein and decoded its complete amino acid sequence.

[0091] Recombinant FLCκ or rFLCλ was first reacted with N-ethylmaleimide (NEM) to block free thioether groups, thereby preventing random disulfide rearrangement. 100 μg of recombinant FLC was reacted with 100 μg of NEM in 0.1 M PBS (pH 7.2) in the presence of 4 M urea at room temperature for 2 hours. Then, the cells were lysed with Zeba according to the manufacturer's instructions. TMSpin Desalting Columns 7kDaMWCO removes excess NEM. Take 10μg of NEM-treated FLC and enzymatically digest it with LysC, LysargiNase, trypsin or chymotrypsin protease (MS grade). The enzyme to protein ratio of all proteases is 1:50. Digestion was carried out in 0.1M Tris buffer (pH 8.0) at 37°C for 18 hours. Then, according to the manufacturer's instructions, 0.5μg of digested protein was loaded onto EV-2001C18 Evotips (Evosep, Odense C, Denmark). The peptides were separated on a 15cm C18 column (PepSep, ReproSil 3μm C18 beads, 100μm ID) using a patented 0.1% FA / ACN Evosep gradient for 44min. The eluted peptides were injected online into Q Exactive TM Hybrid Quadrupole-Orbitrap TM Mass spectrometer (ThermoFisher Scientific). For ionization, the stainless steel emitter (ID 30 μm, OD 150 μm, PepSep, Marslev, Denmark) was maintained at 2 kV. Peptide selection used DDA mode with the following parameters: MS mass spectrum acquisition resolution of 70000, scan range of 400-2000 m / z, AGC target of 3e6, and maximum IT of 100 ms. MS / MS scans were performed in centroid mode with an orbital trap resolution of 17500, a quadrupole separation window of 2.4 m / z, an AGC target of 3e6, a maximum IT of 100 ms, and a collision energy of 27% for HCD fragmentation. The dynamic exclusion time was set to 7 s.

[0092] The DDA data were submitted to pLink software for identification of disulfide-linked peptides (32). The following parameters were used for pLink analysis: SS linker, 4 number of missed cleavages, peptide mass range 400-6000, peptide length 3-60, precursor and fragment tolerance of 20 ppm, and N-ethylmaleimide as a variable modification.

[0093] Each digestion protease produced unique κ and λ dimer peptides, see Table 1. Only symmetrical specific cleavage peptides are shown. The identity of each peptide was confirmed by MS / MS spectra. The dimer peptides SFNRGEC (SEQ ID NO: 1) and TVAPTECS (SEQ ID NO: 2) produced by LysC from κ and λ FLC, respectively, and the dimer peptide KTVAPTECS (SEQ ID NO: 4) produced by LysargiNase digestion of λ FLC were selected for further study.

[0094] Table 1 FLC dimer peptides produced by various digestive enzymes under non-reducing conditions

[0095]

[0096]

[0097] * indicates miscleaved peptide

[0098] The MS / MS spectrum of the precursor at m / z 541.22 confirmed the identity of the disulfide-linked peptide SFNRGEC generated by LysC protease cleavage at the C-terminus of κFLC ( Figure 1A The fragmentation of the precursor at m / z 806.35 confirmed the identity of the disulfide-linked peptide TVAPTECS (SEQ ID NO: 2) generated by LysC digestion of λFLC ( Figure 2A ). The spectra of cross-linked peptides were annotated using the pLabel tool of the pLink software. All major peaks were attributed to specific a-, b-, or y-fragment ions. Note that the y ion contains a disulfide bond. Other observed peaks represent internal fragment ions. When the pure synthetic disulfide-linked peptides SFNRGEC (SEQ ID NO: 1) or TVAPTECS (SEQ ID NO: 2) were analyzed by mass spectrometry, the same spectra were obtained (at Figure 1B and Figure 2B ).

[0099] Example 2: Quantification of kappa and lambda LC dimers in spiked control serum

[0100] In order to quantify the dimeric FLC peptide in human serum, the recombinant FLC was dried in CentriVap and then reconstituted in control human serum (H4522, Sigma-Aldrich) at a final concentration of 2 g / L. The spiked serum was then serially diluted into the control serum in 2-fold increments until the final dilution was 64 times. The control serum without recombinant FLC was used as a negative control (0 g / L). The spiked serum was then treated with an equal amount of NEM and digested with LysC or LysargiNase as described in Example 1. The digest equivalent to 1 μg was loaded onto Evotips and separated on a 15 cm C18 column for 44 min as described above. For dimer peptide quantification, targeted MS / MS spectra (PRMs) were collected in centroid mode on a Q Exactive Orbitrap with the following parameters: orbitrap resolution 17500, quadrupole separation window 2 m / z, AGC target 3e6, maximum IT 100 ms, and collision energy 27% for HCD fragmentation. PRM data were analyzed using Skyline 20 software (33). After LysC digestion, the triply charged parent ion at m / z 541.22 was monitored for quantification of κFLC dimers, while the doubly charged parent ion at m / z 806.35 was monitored for quantification of λFLC. Figure 3 shows the pure synthetic dimer peptide SFNRGEC (approximated by SFNRGE in Skyline). C(Oxi) SFNRGEC)( Figure 3A ) and TVAPTECS (similar to TVAPTE in Skyline C(Oxi) STVAPTECS)( Figure 3C ) and by spiked with 125 mg / L rFLCκ ( Figure 3B ) and λ( Figure 3D ) serum. The ion distribution pattern and retention time of the pure dimeric peptide and the dimeric peptide generated by LysC digestion of rFLC are similar.

[0101] To determine the limit of detection (LOD) and lower limit of quantification (LLoQ), dilution curves of κ and λ dimer peptides were generated. It was observed that the dimer peptides SFNRGEC(κFLC) and TVAPTECS(λFLC) generated by LysC digestion (Figure 4) and the dimer peptide KTVAPTECS( Figure 5 ) and the linear relationship between the peak area and rFLC concentration.

[0102] LOD was defined as the lowest rFLC concentration for which the ion distribution pattern was similar to that of the synthetic peptide and the mass error of a single transition was less than 10 ppm. LLoQ was defined as the lowest rFLC concentration for which the calculated value was within 80-120% of the expected value and the coefficient of variation (CV) of replicate injections was less than 20%.

[0103] The peak areas of all peptides showed good linear relationships. 2 >0.95 (Figures 4 and 5). The LLoQ for κ dimer was as low as 31.25 mg / L. λ dimer could be accurately quantified by monitoring TVAPTECS-TVAPTECS peptides as low as 125 mg / L, or KTVAPTECS-KTVAPTECS peptides as low as 62.5 mg / L. Since they were not detected in normal serum without rFLC, the LODs for κ or λ dimer peptides could not be determined. To make these figures prospective, it must be considered that the reference ranges for κ FLC and λ FLC in healthy individuals are 3.3-19.4 mg / L and 5.7-26.3 mg / L, respectively, while the median difference between involved and uninvolved FLC concentrations (dFLC) in AL amyloidosis is 180 mg / L (2,34). Therefore, it can be concluded that FLC dimers can be quantified using mass spectrometry methods with sufficient sensitivity to detect differences between healthy and diseased subjects.

[0104] Example 3: FLC dimers can be measured in serum from control subjects and serum from patients with multiple myeloma

[0105] As described in Example 2, free light chain dimers can be detected in control mixed serum (H4522, Sigma). In order to maximize the release of dimer peptides by LysC under non-reducing conditions, several digestion parameters were iterated. That is, digestion was performed for 1, 4 or 22 hours in 4M and 8M urea. N-ethylmaleimide (NEM) was not used because the possibility of forming FLC dimers due to random disulfide rearrangements in serum was not great. In brief, the control serum H4522 was diluted 12 times with water to a total protein concentration of 5 μg / μl. Then, 2 μl of diluted serum (10 μg) was mixed with 32 μl of 0.1M Tris buffer (pH 8.0) containing 4 or 8M urea and 0.2 μg LysC (the ratio of enzyme to protein was 1:50). The digestion reaction was incubated at 35°C for 1, 4 or 22 hours, and then stopped by acidification with TFA. The digested protein equivalent to 0.8 μg was then loaded onto an EV2011 Evotip Pure C18 desalting trap column (Evosep, Odense C, Denmark) according to the manufacturer's instructions and separated on a 15 cm C18 column using a patented Evosep gradient of 0.1% FA / ACN for 44 min. Target dimer peptide quantification (PRM) was performed on an Orbitrap Exploris 240 mass spectrometer in profile mode with the following parameters: orbitrap resolution of 30,000, quadrupole separation window of 0.6 m / z with an m / z offset of 0.25, standard AGC target, dynamic maximum IT mode, and collision energy of 27% for HCD fragmentation. Ions at m / z 541.22 and 806.35 were monitored to quantify κ and λ FLC dimers, respectively. Figure 6 The results shown in show that optimal release of both κ and λ dimer peptides was achieved with 1 or 4 hours in 8 M urea. To simplify the procedure, a shorter reaction time of 1 hour was chosen for all future experiments.

[0106] Then the optimized digestion conditions were applied to measure FLC dimers in serum samples from patients with multiple myeloma (MM), a disease often accompanied by monoclonal immunoglobulin (M protein) and FLC overproduction. Serum samples of MM patients with IgGκ (1916) and IgAλ (2666) were purchased from the Institute for Myeloma and Bone Cancer Research, California, USA. Sample collection was approved by the Western Institutional Review Board (WIRB) and consent was obtained from each patient individually. The M-proteins contained in the diagnostic samples were 11.7 g / L (1916) and 44.7 g / L (2666) respectively, as measured by serum protein electrophoresis. FLC measurement is not available. The diluted serum samples were digested with LysC at 35 ° C for 1 hour in the presence of 8 M urea, and the digests were then loaded onto Evotips and analyzed with Exploris-240 as described above. To reduce the variability between runs, the signals of κ and λ FLC dimers were normalized according to the signals of two LysC peptides from human serum albumin (HSA). The internal standard peptides SLHTLFGDK and TPVSDRVTK were selected to match the signal intensity and elution profile of the target peptides. Other serum proteins such as transferrin can also be used as endogenous internal standards for normalization of FLC dimer signals. The normalized peak areas of κ and λ FLC dimers in control and MM serum are shown in Figure 7 The amount of κLC dimer in the serum of patient 1916 with IgGκM-protein was 1.7 times higher than that measured in the control pooled serum (compare Figure 7 The amount of λLC dimers in the serum of patient 2666 with IgAλM-protein was 13.5 times higher than that measured in the control pooled serum (compare Figure 7 ). Interestingly, the amount of κFLC dimer in the serum of patient 2666 was 2-fold lower than that in the control serum. The phenomenon of inhibition by uninvolved LCs, termed immunoparesis, has been described in detail in the literature (35).

[0107] The data presented in this example clearly demonstrate that the levels of kappa and lambda FLC dimers in MM patient sera are different from those in control sera, thus demonstrating that the detection methods described herein can be used for the diagnosis and monitoring of MM.

[0108] Example 4: Enrichment of κ and λ LC improves detection of κ and λ FLC dimers in control serum

[0109] To improve the signal from FLC dimers and thus increase the sensitivity of the assay, κ and λ FLC were enriched separately from control serum. Briefly, the FLC dimers were purified according to the manufacturer’s instructions (Magne TM Protein G Beads, Promega), first using G-beads to deplete the IgG portion of 50 μl to 100 μl of control mixed serum. The IgG-depleted serum was then incubated with Capture Select affinity beads specific for κ (κXP) or λ (λXP) LC. Equal amounts of IgG-depleted serum were incubated with protein L agarose, which specifically binds to human κLC. The bound κ or λ LC was eluted with 0.1 M glycine buffer (pH 2.5). The eluate was neutralized by adding 1 M Tris buffer (pH 8.0), dried in a CentriVap, reconstituted in 0.1 M Tris buffer (pH 8.0) containing 8 M urea, and digested with LysC (0.2 μg / reaction). For comparison, the diluted serum that was not enriched was also digested at the same time. The digest was loaded onto Evotips and then analyzed with Exploris-240 as described in Example 3.

[0110] When κLC was enriched from 50 μl of serum using κ affinity resin, the κFLC dimer signal increased 186-fold compared to unenriched serum ( Figure 8 ). κLC was similarly enriched using protein L resin, and the κFLC dimer signal increased 155-fold. λLC was enriched from 100 μl of serum using λ affinity resin, and the λFLC dimer signal increased 113-fold compared to unenriched serum. Surprisingly, the λ affinity resin also binds κLC nonspecifically and vice versa. Binding, washing, and elution conditions can be optimized to minimize nonspecific binding and improve target enrichment.

[0111] The enrichment strategy described in this example can be used to improve the sensitivity of detecting FLC dimers in serum. In addition, enrichment can also be performed from other biological fluids such as saliva, tears, CSF and urine.

[0112] Example 5: Absolute quantification of λ LC dimers in serial samples from MM patients

[0113] Absolute quantification of FLC dimers can be achieved by spiking serum digests with known amounts of stable isotope labeled (SIL) peptides as single-point internal calibrators. The SIL dimer peptide SNFRGEC was synthesized with heavy isotope labeled arginine (13C6, 15N4), while the SIL dimer peptide TVAPTESC was synthesized with heavy valine (13C5, 15N). Synthesis was performed by GenScript, Piscataway, NJ, USA. Mass spectrometry confirmed the identity of the peptides. Net peptide amounts were derived from amino acid (AA) analysis.

[0114] SIL peptides were spiked into control serum digests in increasing amounts. Ions at m / z 547.89 and 812.36 were monitored for the κ and λ SIL dimer peptides, respectively. The RT and ion profiles for the unlabeled and SIL peptides were identical. Fig. 9 The linear relationship between SIL peptide concentration and MS peak area (R 2 =0.99). Importantly, the amount of unlabeled FLC dimer peptide in serum was not affected by the addition of SIL peptide. Based on the expected peak area of ​​FLC dimer peptide in control serum samples, the optimal target concentration of SIL peptide was 0.16-0.32 nM.

[0115] In order to show that SIL peptides can be used for absolute quantification of LC dimers in serum, MM patient 2666 in Example 3 was selected. The time span of the serial serum samples obtained from this patient was 2 years, during which the patient received treatment and achieved complete response (CR) and relapse (R). As described in Example 3, the serum samples were digested with LysC in the presence of 8M urea. The digest was then spiked with λSIL (final concentration of 0.286nM), loaded into Evotips, and analyzed with Exloris-240. The acquisition method was modified to include the SIL peptide mass. The peak area of ​​the unlabeled dimer peptide TVAPTESC was normalized according to the peak area of ​​the spiked SIL peptide. The λ dimer concentration at each time point was calculated based on the amount of the SIL peptide spiked, and a graph of vs. the time since the diagnosis of MM was drawn. Fig.10 The lambda FLC dimer monitoring curve depicted has a good correlation with the M-protein amount and overall disease state at each time point. The initial reduction of the lambda dimer amount reflects the response to treatment, and the subsequent increase is consistent with relapse. In addition, the increase in the amount of FLC dimer was observed from M16 to M20, which was 3 months earlier than the relapse time confirmed by traditional detection methods such as SPEP and IFE. Therefore, this embodiment shows the practicality of utilizing mass spectrometry to measure FLC dimer in monitoring MM disease state and detecting relapse. This detection method is more important for monitoring MM with only LC, because its selection is very limited clinically.

[0116] Example 6: Absolute quantification of FLC dimers in serum, saliva and CSF

[0117] To demonstrate the broad applicability of FLC dimer quantification using MS, we quantified and compared the amount of FLC dimers in various biological fluids such as serum, saliva, and CSF. As described in Example 3, pooled serum (H4522, Sigma) was diluted and digested with LysC. Saliva from healthy individuals was collected a few hours before eating in the morning using the passive salivation method. Freshly collected saliva was centrifuged at 16,000g before analysis. CSF from 2 healthy individuals was purchased from BioIVT, New York, NY, USA. 10 μl of saliva or 20 μl of CSF was dried in a CentriVap, reconstituted in 0.1 M Tris buffer (pH 8.0) containing 8 M urea, and then digested with LysC (0.2 μg / reaction) for 1H at 35°C. The digest was diluted and spiked with the κ dimer SIL peptide SFN at a concentration of 0.286 nM R GEC and λ dimer SIL peptide T at a concentration of 0.107 nM V APTECS. The SIL-spiked digests were loaded into Evotips and analyzed with the Exploris-240 as described in Example 3. The concentrations of κ and λ FLC dimers in the three fluids were compared ( Fig.11 ). FLC dimer levels were comparable in serum and saliva, whereas significantly lower levels of FLC dimer were detected in CSF. The relative abundance of kappa dimer to lambda dimer remained consistent across the fluids analyzed, ranging from 5:1 to 13:1.

[0118] Example 7: Absolute quantification of FLC dimers in control CSF and CSF from multiple sclerosis patients

[0119] Multiple sclerosis (MS) is often associated with an increase in the amount of oligoclonal Ig and FLC in CSF (17). Therefore, we quantified and compared the amount of FLC dimers in the CSF of control subjects and the CSF of patients with MS. CSF from healthy individuals and from patients with MS and other non-MS neurological symptoms (stroke, migraine, clinically isolated syndrome, and demyelinating disease) were purchased from BioIVT. Table 2 shows the demographic and clinical data of the patients.

[0120] Table 2: Patient demographics and clinical data

[0121]

[0122]

[0123] *, newly diagnosed; N / A, not applicable; NR, not reported; RRMS, relapsing-remitting multiple sclerosis

[0124] CSF was digested with LysC and analyzed with Exploris-240 as described in Example 6. By spiking known amounts of kappa and lambda SIL dimer peptides, SFN R GEC and T V APTECS was used to absolutely quantify κ and λ FLC dimers. Fig.12 The concentration of κ and λ dimers in CSF from healthy individuals, patients with multiple sclerosis, and patients with non-MS neurological disorders is shown. The concentration of κ FLC dimers in control subjects ranged from 0.057 to 0.081 mg / L. Five (70%) of the 7 analyzed CSF samples from patients with multiple sclerosis had κ FLC dimer concentrations 1.85 to 17.3 times higher than those in control CSF. The amount of κ dimers in subjects with non-MS neurological diseases is slightly lower. One of the two MS patients with low κ FLC dimer concentrations (patient #3) has had the disease for a long time (more than 25 years) and has been treated with disease-modifying drugs (Ogilvy), which may have inhibited the production of κ and λ FLC. The highest amount of κ FLC dimers (1.4 mg / L) was detected in newly diagnosed patient #8. Interestingly, the λ FLC dimer concentration of patient #8 was also 0.053 mg / L, the highest among all analyzed subjects. Overall, the concentration of λFLC dimers in the CSF of MS patients was comparable to that in control CSF ( Fig.12 ).

[0125] In conclusion, the FLC dimer detection method is suitable for measuring the amount of kappa and lambda FLC dimers in the CSF of control and diseased subjects. The data in this example indicate that MS patients can be distinguished from healthy controls and patients with other neurological diseases based on the amount of kappa FLC dimers in CSF.

[0126] Although the present invention has been described above by way of specific embodiments thereof, modifications may be made to the present invention without departing from the spirit and nature of the inventive subject matter defined in the appended claims. In the claims, the word "including" is used as an open-ended term, substantially equivalent to the phrase "including but not limited to". The singular forms "a", "an", and "the" include corresponding plural references unless the context clearly dictates otherwise.

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Claims

1. A method for detecting immunoglobulin free light chain (FLC) dimers in a sample, comprising: (a) subjecting the sample to proteolytic digestion under non-reducing conditions to obtain a digested sample; and (b) performing mass spectrometry analysis on the digested sample to detect immunoglobulin free light chain dimer peptides, wherein the detection of immunoglobulin free light chain dimer peptides indicates the presence of immunoglobulin free light chain dimers in the sample.

2. The method according to claim 1, wherein: The sample is a serum sample, a cerebrospinal fluid sample or a saliva sample.

3. The method according to claim 1 or 2, wherein: The sample is suspected of containing free light chain dimers of the immunoglobulin.

4. The method according to any one of claims 1 to 3, wherein: The immunoglobulin free light chain dimers include dimers of kappa light chains.

5. The method according to any one of claims 1 to 4, wherein: The immunoglobulin free light chain dimers include dimers of lambda light chains.

6. The method according to any one of claims 1 to 5, wherein: Subjecting the sample to proteolytic digestion comprises contacting the sample with at least one endoprotease.

7. The method according to claim 6, wherein: The at least one endoprotease comprises trypsin, chymotrypsin, LysC, LysargiNase or any combination thereof.

8. The method according to any one of claims 1 to 7, wherein: The method further comprises subjecting the sample to a denaturing step prior to the proteolytic digestion.

9. The method according to claim 8, wherein: The denaturation step comprises contacting the sample with urea.

10. The method according to claim 9, wherein: The denaturation step comprises contacting the sample with urea at a concentration of at least 4 M for at least 30 minutes.

11. The method according to any one of claims 1 to 10, wherein: The method further comprises contacting the sample with a cysteine ​​modifying agent prior to the proteolytic digestion.

12. The method according to claim 11, wherein: The cysteine ​​modifying agents include N-ethylmaleimide (NEM).

13. The method according to any one of claims 1 to 12, wherein: The mass spectrometry was liquid chromatography coupled with parallel reaction monitoring.

14. The method according to claim 13, wherein: The mass spectrometry analysis was performed on a quadrupole orbital trap mass spectrometer.

15. The method according to any one of claims 1 to 14, wherein: The immunoglobulin FLC dimer is measured by spiking a known amount of a synthetic peptide containing a specific marker.

16. The method according to claim 15, wherein: The specific labels include heavy isotope labeled amino acids.

17. The method of any one of claims 1 to 16, further comprising enriching the sample for kappa and / or lambda light chains prior to subjecting the sample to proteolytic digestion.

18. The method according to claim 17, wherein: The enrichment comprises contacting the sample with one or more matrices that bind kappa and / or lambda light chains; and eluting the kappa and / or lambda light chains bound to the one or more matrices.

19. The method according to any one of claims 1 to 18, wherein: The immunoglobulin free light chain dimer peptide is and / or 20. The method according to any one of claims 1 to 19, wherein: The sample is a biological sample from a subject suffering from a disease or disorder associated with FLC production.

21. The method according to claim 20, wherein: The disease or disorder is an autoimmune disease, an inflammatory disorder, a viral infection or plasma cell dyscrasia (PCD).

22. The method of claim 21, wherein the PCD is monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), multiple myeloma (MM), amyloid light chain (AL) amyloidosis, or non-amyloid light chain deposition disease (NALCDD).

23. The method according to claim 20, wherein: The sample is a serum sample from a human subject suffering from multiple myeloma.

24. The method according to claim 23, wherein: The method further comprises quantifying the amount of immunoglobulin FLC dimer in the sample, and wherein the amount of immunoglobulin FLC dimer is correlated with tumor burden.

25. The method according to claim 20, wherein: The sample is from a human subject having or suspected of having multiple sclerosis.

26. The method according to claim 25, wherein: The sample is a cerebrospinal fluid sample.

27. The method according to claim 25, wherein: The method comprises comparing the amount of immunoglobulin FLC dimer in the sample with the amount of immunoglobulin FLC dimer in a control sample from a healthy subject.

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