Ionization control

Abstract

An elution buffer for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof or for eluting one or more predetermined antibodies or fragments from a target antigen, wherein the elution buffer has a pH of 1-5; and the elution buffer comprises a predetermined amount of an acid-stable mass spectrometry ionization control protein. The use of the elution buffer in the detection and quantification of analytes (e.g., by mass spectrometry) is also described.

Description

[0001] The present invention relates to elution buffers comprising a predetermined amount of an acid-stable ionization control for mass spectrometry, kits comprising such buffers and methods of preparing such buffers and kits.

[0002] BACKGROUND

[0003] Protein analysis by mass spectrometry has important clinical utility in in vitro diagnostics; however, analysis reproducibility remains a potential problem and peak intensity and m / z values can vary significantly between experiments.

[0004] Current methods for controlling analysis variability and reproducibility to enable routine use in in vitro diagnostics of human diseases include automated sample processing, extensive pre-separation strategies, immuno-capture, pre-structured target surfaces, standardized matrix (co-)crystallization, improved MALDI-TOF mass spectrometry (MS) instrument components, internal standard peptides, quality control samples, repeated measurements, and algorithms for normalization and peak detection (Albrethsen, J., 2007; Clin Chem, 53(5) 852-858).

[0005] However, in addition to the crystal formation between the matrix and the sample, these methods are influenced by other factors, thus not accurately reflecting spotting, crystallization, and ionization.

[0006] Previous attempts to control inter- and intra-experimental variability in MALDI-TOF MS generally utilized internal calibration peptides with physicochemical properties comparable to the protein of interest, incorporated into the sample at different concentrations, prior to comparing ion intensities of the calibration peptide and the analyte. However, problems with this approach include variability in peak intensity. It has been proposed to combine controls with iterative algorithms and / or repeated analysis as possible solutions to compensate for some analysis changes over time and improve reproducibility of protein analysis by MALDI-TOF MS (Albrethsen, J., Clin Chem 2007; 53(5): 852-858).

[0007] For example, one study utilized a method in which a synthetic peptide with the same primary sequence as a particular analyte was incorporated into a blood sample (Yi, J, et al., Methods Mol Biol 2011; 728: 161-75).

[0008] Matrix assisted laser desorption / ionization time of flight mass spectrometry analysis using internal controls also showed improved sensitivity in determining bacterial concentration in samples. Addition of cytochrome C as an internal control reduced signal intensity by 20-30% in samples with high concentration of bacteria, but improved signal intensity in some low concentration bacteria. In this case, the protein was incorporated into the matrix and then pre-mixed in a 2:1 matrix to analyte ratio (Gantt, SL, et al., J Am Soc Mass Spectrom 1999; 10(11): 1131-7).

[0009] In another example, ion suppression in atmospheric pressure matrix assisted laser desorption / ionization was investigated by incorporating an angiotensin II analogue as an internal standard into all fractions of 384 Prespotted AnchorChips. (Li, G, et al., Rapid Commun Mass Spectrom 2019; 33(4): 327-335). Signal intensity was then normalized to controls and peak clustering analysis was performed. Lower intensity peaks had reproducibility equivalent to higher intensity peaks.

[0010] Examples of other attempts to adjust variability between samples include post analysis adjustment by outlier removal and baseline removal by intensity scaling (Neubert et al., J Proteome Res 2008; 7(6) 2270-9).

[0011] An example of a significant utility of mass spectrometry for in vitro diagnostic use is in relation to a number of proliferative diseases associated with antibody producing cells.

[0012] Antibody molecules (also known as immunoglobulins) have a twofold symmetry and are generally composed of two identical heavy chains and two identical light chains, each chain containing a variable domain and a constant domain. The variable domains of the heavy and light chains combine to form an antigen binding site, such that both chains contribute to the antigen binding specificity of the antibody molecule. The basic tetrameric structure of an antibody comprises two heavy chains covalently linked by disulfide bonds. Each heavy chain in turn is linked to a light chain again via a disulfide bond. This results in a molecule that is essentially “Y” shaped.

[0013] In many such proliferative diseases, plasma cells proliferate to form a monoclonal tumor of identical plasma cells. This results in the production of large amounts of identical immunoglobulins and is known as a monoclonal gammopathy.

[0014] Diseases such as multiple myeloma and primary systemic amyloidosis (AL amyloidosis) account for approximately 1.5% and 0.3% of cancer deaths in the UK, respectively. Multiple myeloma is the second most common form of blood malignancy after non-Hodgkin's lymphoma. In the Caucasian population, the incidence is approximately 40 per million per year. Diagnosis of multiple myeloma is typically based on an excess of monoclonal plasma cells in the bone marrow, monoclonal immunoglobulins in the serum or urine, and the presence of associated organ or tissue damage such as hypercalcemia, renal insufficiency, anemia, or bone lesions. Normally, bone marrow contains about 1% plasma cells, while in multiple myeloma, this content is usually greater than 10%, frequently greater than 30%, but can exceed 90%.

[0015] Alzheimer's disease (AL) is a protein conformation disorder characterized by the accumulation of monoclonal free light chain fragments as amyloid deposits. These patients typically develop heart or kidney failure, but peripheral nerve and other organ involvement may also occur.

[0016] Many other diseases can be identified by the presence of monoclonal immunoglobulins in a patient's bloodstream or actually in their urine. These include plasmacytomas and extramedullary plasmacytomas, which are plasma cell tumors that occur outside the bone marrow and can develop in any organ. When present, the monoclonal protein is usually IgA. Multiple solitary plasmacytomas can occur with or without evidence of multiple myeloma. Waldenström macroglobulinemia (Waldenström macroglobulinemia) Macroglobulinaemia (MBI) is a low-grade lymphoproliferative disorder associated with the production of monoclonal IgM. Approximately 1,500 new cases are diagnosed annually in the United States and about 300 in the United Kingdom. Serum IgM quantification is important for both diagnosis and surveillance. In the UK, B-cell non-Hodgkin lymphoma accounts for approximately 2.6% of all cancer deaths, and monoclonal immunoglobulins have been identified in the serum of approximately 10–15% of patients using standard electrophoresis methods. In B cells, monoclonal proteins associated with chronic lymphocytic leukemia have been identified by free light chain immunoassay.

[0017] Additionally, there exists a condition known as MGUS. These are undetermined monoclonal gammopathy. The term refers to the unexpected presence of intact monoclonal immunoglobulins in individuals without evidence of multiple myeloma, AL amyloidosis, Waldenström macroglobulinemia, etc. MGUS can be found in 1% of the population over 50 years of age, 3% of the population over 70 years of age, and up to 10% of the population over 80 years of age. Most of these are IgG or IgM-related, although less commonly IgA-related or biclonal. While most people with MGUS die from unrelated diseases, MGUS can transform into malignant monoclonal gammopathy.

[0018] In at least some cases of the above highlighted diseases, the disease is present with an abnormal concentration of monoclonal immunoglobulins or free light chains. When the disease produces abnormal replication of plasma cells, this often results in the production of more immunoglobulins by that type of cell, as the "monoclonal" is reproduced and appears in the blood.

[0019] Sensitive assays have been developed that can detect free kappa light chains and separately detect free lambda light chains. The method uses polyclonal antibodies against free kappa or free lambda light chains. The possibility of producing such antibodies as one of many different possible specificities is also discussed in WO 97 / 17372. This document discloses a method of tolerizing an animal to allow it to produce the desired antibodies with more specificity than can be produced by the prior art. The free light chain assay uses antibodies that bind to free lambda or free kappa light chains. The concentration of free light chains is determined by nephelometry or turbidimetry.

[0020] The amount or type of characterization of free light chains (FLC), heavy chains or subclasses, or light chains of the type bound to heavy chains or subclasses is important in a wide range of diseases, including B-cell diseases such as multiple myeloma and other immune-mediated diseases, including B-cell diseases such as monoclonal gammopathies (of which multiple myeloma is an example) and other immune-mediated diseases, including hypergammaglobulinemia and hypogammaglobulinemia.

[0021] WO 2015 / 154052, incorporated herein in its entirety, discloses a method of detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof using MS. A sample comprising immunoglobulin light chains, heavy chains, or mixtures thereof is immuno-purified and subjected to mass spectrometry to obtain a mass spectrum of the sample. This can be used to detect monoclonal proteins in a sample from a patient. It can also be used for fingerprinting, isotyping, and identifying monoclonal antibodies.

[0022] MS is used to separate, for example, lambda and kappa chains in a sample by mass and charge. It can also be used to detect heavy and light chain components of immunoglobulins by, for example, reducing the disulfide bond between the heavy and light chains using a reducing agent. MS is also described in WO 2015 / 131169, incorporated herein in its entirety.

[0023] Purification of immunoglobulins in a sample in a diagnostic procedure typically uses antibodies against intact antibodies and / or free light chains, such as anti-IgG, anti-IgA, anti-IgM, anti-IgD, anti-IgE, anti-total kappa, anti-total lambda antibodies, or anti-free light chain antibodies, such as anti-free kappa or anti-free lambda light chain antibodies. It is important to have calibrators to ensure that the purification and detection processes are performed correctly.

[0024] WO2017 / 144900 describes the use of a number of controls with either a heavier form of the analyte to be detected or a monoclonal of the analyte to be detected. That is, for example, IgA can be quantified in comparison to a predetermined amount of heavier IgA kappa.

[0025] This is because different proteins are expected to crystallize on the mass spectrometry matrix at different rates. This means that when the matrix is sampled by mass spectrometry, different amounts of the control proteins and analyte will be detected. Furthermore, their ionization rates are expected to be different. This will lead to inconsistencies in the amount of immunoglobulin detected. In addition, the problem of analytical reproducibility of mass spectrometry-based methods means that peak intensities can vary significantly between experiments and mass drift can occur which can affect the recorded m / z values. For example, MALDI-TOF ionization depends on a point-to-point variable process of crystal formation between the matrix (e.g. HCCA) and the sample.

[0026] The authors have surprisingly found that by using an independent marker in an acidic elution buffer after immunoprecipitation and before spotting, it is possible to control the analytical variability in ionization for mass spectrometry.

[0027] SUMMARY

[0028] Provided herein is an elution buffer for eluting one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof or for eluting one or more predetermined antibodies or fragments from a target antigen, wherein:

[0029] The elution buffer has a pH of 1-5, more preferably a pH of 1-3, or even more preferably a pH of 1.5-3.0; the elution buffer comprises a predetermined amount of an acid-stable mass spectrometry ionization control protein.

[0030] The elution buffer can be used to elute, for example, analytes bound to antibodies attached to a substrate. Alternatively, the target antigen can be attached to a substrate and the antigen-specific antibodies or fragments eluted from the target antigen.

[0031] Such an elution buffer is used to release analytes bound to analyte-specific antibodies. The inclusion of an ionization control in the buffer allows it to be supplied by the vendor and reduces user error caused by the user having to measure or prepare the amount of ionization control material to be used separately.

[0032] The ionization control can also be used as a "lock mass calibrator" in mass spectrometry methods including, for example, MALDI and electrospray mass spectrometry. Such a lock mass calibrator is an ion with a known m / z value derived from the ionization control that allows real-time recalibration within the spectrum by correcting for m / z shifts caused by instrument and internal MALDI-target plate drift.

[0033] The sample containing the analyte to be analysed can be a biological sample such as blood, saliva, serum, plasma, cerebrospinal fluid or urine, more typically blood, serum or plasma.

[0034] The sample can be from a subject exhibiting hypogammaglobulinemia or hypergammaglobulinemia. The subject can have a proliferative disease associated with antibody producing cells such as a monoclonal gammopathy. These include myeloma and primary systemic amyloidosis, plasmacytoma, Waldenstrom's macroglobulinemia and MGUS.

[0035] An ionisation control protein compatible with the predetermined analyte is selected.

[0036] The ionisation control protein can be substantially stable in the elution buffer for at least 30 days, more preferably at least 60 days, typically at least 4 months or at least 6 months.

[0037] A control that is not suitable for long term storage can lead to physical stability problems which can result in the protein precipitating or otherwise being affected in the elution buffer, thus leading to poor crystallisation on the target or poor ionisation, both of which can affect the m / z peak height or area.

[0038] Damage to the control itself which results in a change in mass or ionisation state can also change the measured m / z.

[0039] The control protein can be stable at, for example, 22°C or lower, or 4°C. It can be pH stable, UV stable or light stable.

[0040] At least one mass spectrometric m / z peak value of the ionisation control protein can be substantially stable (as defined above).

[0041] The ionisation control protein can be selected to have at least one mass spectrometric peak with an m / z value which does not substantially overlap with a mass spectrometric peak of the or each predetermined analyte. It is typically selected to ionise consistently and typically does not substantially affect the intensity of the mass spectrometric signal.

[0042] The ionisation control protein can be selected to have at least one mass spectrometric m / z peak value within a predetermined mass spectrometric window for detecting or quantifying one or more peaks from the at least one predetermined analyte or within an m / z range observed by the mass spectrometer.

[0043] The sample can be treated with a reducing agent, typically after elution but before mass spectrometry is performed. This is particularly useful when the immunoglobulin light chains in the sample are bound to the heavy chains. The use of a reducing agent decouples the light chains from the heavy chains and allows the light chains to be detected separately by mass spectrometry. A reducing agent can also be used to separate other analyte proteins to separate subunits present.

[0044] Decoupling can be achieved by treating the total immunoglobulin with a reducing agent such as DTT (2,3 dihydroxybutane-1,4-dithiol), DTE (2,3 dihydroxybutame-1,4-dithiol), mercaptoacetate, cysteine, sulfite, bisulfite, sulfide, disulfide, TCEP (tris(2-carboxyethyl)phosphine), 2-mercaptoethanol and salt forms thereof. In some embodiments, the reduction step is performed at an elevated temperature, for example at about 30°C to about 65°C, such as at about 55°C, to denature the proteins.

[0045] The decoupling step is typically performed after immunopurification or other enrichment of the immunoglobulin in the sample, or as part of the elution step after immunopurification of the sample.

[0046] The antibody used for immunopurification can be an intact antibody or a fragment thereof, such as Fab, F(ab) and F(ab') 2 fragment, or a single chain antibody. The antibody or fragment thereof can be cross-linked, for example as described in WO2017144903 (incorporated herein in its entirety).

[0047] Any acidic buffer (pH 1-5, more preferably pH 1-3 or pH 1.5-3) can be used as long as it does not interfere with the mass spectrometry, such as MALDI-TOF, ionization process.

[0048] The elution buffer can comprise an organic acid such as citric acid, acetic acid, formic acid, uric acid, propionic acid and inorganic acids such as hydrochloric acid. Acidic buffers or solutions containing salts can be avoided, especially at higher concentrations, as at high concentrations these can interfere with ionization or crystallization.

[0049] For example, the elution buffer of the application can comprise an elution buffer selected from:

[0050] (a) 5% v / v aqueous acetic acid;

[0051] (b) 0.1 M glycine at pH 2.0-3.0 or 0.2 M glycine at pH 2-6

[0052] The buffer comprising 5% acetic acid preferably has a pH of about 2.

[0053] The elution buffer can comprise 1-100 ng / ul of an ionization control protein, more preferably 1-10 ng / ul.

[0054] The reducing agent can be used in combination with the elution buffer and can also comprise tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol or cysteine. The reducing agent can be pre-weighed or provided to provide a final concentration in the range 10-100 mM, or more preferably about 20 mM.

[0055] The ionization control protein can comprise at least 30 amino acids or at least 50 amino acids and / or can have a mass of at least 3 kDa or be used to elute one or more predetermined antibodies or fragments from a target antigen kDa.

[0056] The ionization control protein advantageously has a different mass range or ion gate, or has multiple charge states, to enable use within the assay window of the analyte. The ionization control protein or peptide can be naturally occurring or synthetic.

[0057] Suitable proteins for use as ionization controls can include aprotinin, alpha 1 acid glycoprotein, beta 2 glycoprotein or prealbumin (also known as transthyretin). More preferably, the ionization control can comprise aprotinin or transthyretin. Aprotinin is a serine protease inhibitor derived from bovine pancreas. It is readily available as a pure protein and a drug from commercial sources; TRASYLOL. (CAS number: 9087-70-1, molar mass 6511.5 Da. UniProtKB Accession No. P00974. Isoelectric point pH 10.5). Stable at high temperatures in neutral or acidic media. Transthyretin (TTR, prealbumin or TBPA) is a transport protein found in serum and cerebrospinal fluid which carries the thyroid hormone thyroxine and retinol binding protein bound to retinol. It is a homotetramer or dimer of dimers of quaternary structure of 55 kDa. The human protein has UniProtKB Accession No. P02766.

[0058] However, other substantially acid stable proteins can be used in different mass ranges (ion gates) or where one or more protein charge states are suitable for a particular m / z assay window. For example, in an assay window of m / z 5-30 kDa, alpha 1 acid glycoprotein (+1 ~21560), beta 2 glycoprotein I (+1 36255) or prealbumin monomer (+1 ~13760) would be suitable.

[0059] By exploiting the use of additional plates and inversion of gates in the instrument, larger mass windows can be targeted, for example a dual ion gate method or a wide mass window.

[0060] Also provided herein is a kit for the analysis of one or more analytes by mass spectrometry comprising an elution buffer as defined above and one or more analyte specific antibodies or fragments thereof specific for the one or more predetermined analytes.

[0061] The analyte or antigen specific antibodies can be proteins or peptides, more preferably serum proteins or peptides.

[0062] Antigen-specific antibodies include anti-streptolysin O, anti-tetanus toxoid immunoglobulin, Haemophilus influenzae-specific immunoglobulin, diphtheria toxoid-specific immunoglobulin, Streptococcus pneumoniae-specific immunoglobulin, Salmonella typhi-specific immunoglobulin, or Varicella zoster virus-specific immunoglobulin.

[0063] If the analyte is a serum protein, the serum protein can comprise one or more complement proteins, for example the serum protein can comprise one or more complement protein components, such as CI, C2, C3, C4, or components thereof, for example components C3a, C3b, C3c.

[0064] The serum protein can comprise an immunoglobulin or fragment thereof, albumin, beta 2-microglobulin, alpha 1 -microglobulin, cystatin C, microalbumin, alpha 1 -acid glycoprotein, alpha 1 -antitrypsin, alpha 2-macroglobulin, anti-streptolysin O, anti-tetanus toxoid immunoglobulin, apolipoprotein A, apolipoprotein B, ceruloplasmin, C-reactive protein, haptoglobin, prealbumin, rheumatoid factor, or total serum protein transferrin.

[0065] The analyte can be a monoclonal antibody, such as a therapeutic monoclonal antibody. The analyte-specific antibodies that can be included in the kit can be one or more of the following: anti-IgA, anti-IgG, anti-IgM, anti-IgD, anti-IgE, anti-total light chain, anti-free light chain, anti-lambda light chain, anti-kappa light chain, anti-lambda free light chain, anti-kappa free light chain, anti-heavy chain subclass, anti-heavy chain class-light chain type, or anti-heavy chain subclass-light chain type specific antibodies; more preferably anti-IgG, anti-IgA, anti-IgM, anti-kappa, and / or anti-lambda specific antibodies.

[0066] The antibodies or fragments thereof specific for one or more predetermined analytes can also be bound to a substrate; for example, the antibodies or fragments thereof can be bound to latex beads. The target antigens can also be attached to a substrate such as latex beads.

[0067] The kit can also comprise a predetermined amount of a control analyte.

[0068] The kit can comprise one or more of the following: a sample dilution buffer, an immunocapture reagent or beads, a wash buffer, an elution buffer containing an optional reducing agent, a mass spectrometry matrix, a mass spectrometry matrix solvent, a MALDI target, and a mass spectrometer mass calibrator.

[0069] The reducing agent of the kit can comprise tris(2-carboxyethyl)phosphine, dithiothreitol, 2-mercaptoethanol, or cysteine, and can be as defined above.

[0070] The reducing agent is preferably pre-weighed or provided to provide a final concentration in the range of 10-100 mM, or more preferably about 20 mM.

[0071] The kit can additionally comprise a standard serum protein control. For example, the kit can comprise an antibody against the antibody specific for human.

[0072] Also provided herein is a method of detecting or quantifying an analyte comprising immunopurifying a predetermined analyte, eluting the analyte with an elution buffer according to the present application, and detecting the analyte and the ionization control protein by mass spectrometry.

[0073] The method is not limited to any particular mass spectrometry method; however, the mass spectrometry method can comprise liquid chromatography mass spectrometry (LC-MS) or MALDI-TOF mass spectrometry. More preferably, the mass spectrometry method can comprise MALDI-TOF mass spectrometry.

[0074] The immunoassay to which the present application applies has three main steps; 1) immunocapture of the analyte, 2) elution of the analyte, 3) optional reduction of the analyte, and 4) spotting of the analyte onto a MALDI-TOF target plate.

[0075] The present application provides that the ionization control protein can be comprised in a reagent for step 2) in order to combine the control and the analyte prior to step 3) and advantageously to be spotted together in step 4. This is important since the ionization control is used to control variability in step 3) as well as subsequent ionization in the MALDI-TOF mass spectrometer.

[0076] The method can also provide the use of a kit according to the present application for analyzing one or more analytes by mass spectrometry, the kit comprising an elution buffer according to any of the preceding claims and one or more analyte specific antibodies or fragments thereof specific for one or more predetermined analytes.

[0077] Also provided herein is a method of preparing an elution buffer according to the present application, wherein the elution buffer is used for eluting one or more predetermined analytes from one or more analyte specific antibodies or fragments thereof, wherein:

[0078] the elution buffer has a pH in the range of 1-6, more preferably a pH in the range of 2-6, more preferably a pH in the range of 1-4, or even more preferably a pH in the range of 1.5-3.0; and the elution buffer comprises a predetermined amount of an acid stable mass spectrometry ionization control protein.

[0079] The method of preparing an elution buffer according to the present application comprises:

[0080] (a) identifying the analyte;

[0081] (b) identifying the m / z of at least one peak of the ionization control as compared to the m / z of one or more expected peaks of the analyte;

[0082] (c) identifying an ionization control protein having an m / z range and acid stability.

[0083] A computer-implemented method comprising: inputting an analyte, comparing one or more m / z peaks of the analyte to m / z peaks of a plurality of potential ionization control proteins having acid stability, and outputting for the analyte an identification of one or more ionization control proteins having an m / z range and acid stability. BRIEF DESCRIPTION OF DRAWINGS

[0084] The present application will now be described by way of example only and with reference to the following drawings.

[0085] Figure 1 is an example of a MALDI-TOF mass spectrum showing the mass distribution of the analyte (Kappa light chain (K)) after elution with aproticin-containing acetic acid. A single aproticin peak (+1 charge) is observed which does not interfere with the analyte (Kappa light chain) peaks. Ion charge states are given in brackets.

[0086] Figure 2 shows that the relative ionization control protein signal remains stable in the presence and absence of analyte. The MALDI-TOF mass spectrum of aproticin obtained in the absence of analyte (black line) shows no significant difference from the aproticin spectrum containing Kappa light chain from normal human serum (NHS) (grey line and inset).

[0087] Figure 3 shows that aproticin remains stable in 5% acetic acid. Kappa light chain was periodically eluted with aproticin-containing 5% acetic acid that had been stored at 22°C. MALDI-TOF mass spectra were obtained at each time point and the aproticin and Kappa light chain (+2) peak areas (± standard deviation) were determined. No depreciation of signal was observed for either protein over an 8 week period.

[0088] Figure 4 shows that the analyte signal relative to aproticin as ionization control remains stable over time. Aproticin-containing 5% acetic acid was stored at 22°C and used periodically to elute Kappa light chain. MALDI-TOF mass spectra were obtained at each time point and the aproticin and Kappa light chain (+2) peak area ratios (± standard deviation) were determined. No significant change in peak area ratio was observed over an 8 week period.

[0089] Figure 5: Trans-thyretin protein (TTR) as MALDI-TOF ionization control. MALDI-TOF mass spectra showing the mass distribution of TTR after elution with acetic acid with (Figure 5A) and without (Figure 5B) mixing with polyclonal IgG. The TTR peaks are observed at 13827 m / z and 6914 m / z, neither of which interfere with any of the lambda or kappa polyclonal light chain peaks. The signal intensity of the TTR ionization control peak is unchanged in the presence or absence of analyte (B). The TTR signal peak does not overlap with either the peak for aprotinin (C) or the peak for the glycosylated kappa free light chain (D), which is of greater mass. Ion charge states are given in parentheses.

[0090] A normal human serum sample (NHS) was diluted 1 : 10 and captured using paramagnetic microparticles containing antibodies specific for human kappa immunoglobulin light chain (per Step 1 above). Elution was performed with an acidic buffer solution containing both a reducing agent and aprotinin as an ionization control. The eluate was subsequently spotted in a sandwich with MALDI matrix (HCCA) onto a MALDI-TOF target plate and dried. Mass spectra were acquired in positive ion mode covering the m / z range of 5000 to 30,000, which included the singly charged (+1, m / z 22705), doubly charged (+2, m / z 11353), and triply charged (+3, m / z 7569) ions of the analyte (human kappa light chain; Table 1). -1 Aprotinin elution buffer for ionization control for mass spectrometry analysis. 5% acetic acid was used to elute the analyte from the immunocapture beads and simultaneously facilitate the separation of the immunoglobulin heavy chain from the light chain. 20 mM TCEP was used as an acid-stable reducing agent to break the disulfide bonds that hold the intact immunoglobulin together.

[0091] A normal human serum sample (NHS) was diluted 1 : 10 and captured using paramagnetic microparticles containing antibodies specific for human kappa immunoglobulin light chain (per Step 1 above). Elution was performed with an acidic buffer solution containing both a reducing agent and aprotinin as an ionization control. The eluate was subsequently spotted in a sandwich with MALDI matrix (HCCA) onto a MALDI-TOF target plate and dried. Mass spectra were acquired in positive ion mode covering the m / z range of 5000 to 30,000, which included the singly charged (+1, m / z 22705), doubly charged (+2, m / z 11353), and triply charged (+3, m / z 7569) ions of the analyte (human kappa light chain; Table 1).

[0092] Table 1: Mass spectra acquired in positive ion mode

[0093]

[0094] The aprotinin intensity signal is clearly shown in Figure 1 Figure 5B as a distinct peak at m / z 6512 that does not interfere with or overlap any of the three peaks of the analyte. To show that the aprotinin signal is independent of the presence of the analyte, it was analyzed in the presence (+NHS) and absence (-NHS) of the latter. Figure 2 It is demonstrated that the aprotinin ionization control signal intensity is the same in either case.

[0095] To investigate the stability of the ionization control under acidic conditions, 50 ml aliquots of the preparation (supplemented with 2 ng ml -1 aprotinin 5% acetic acid) were stored at 22°C. Periodically, each aliquot was removed, supplemented with reducing agent (TCEP) and then used to elute analyte from anti-kappa microparticles for MALDI-TOF analysis. Figure 3 The peak areas for the kappa analyte and aprotinin are shown in Figure 2. The peak areas for both varied over the course of the experiment. This variation is due to known MALDI spot-to-spot sample inconsistencies, but there was no decay in the analyte or aprotinin signal over the 60 day period at 22°C. By extrapolation, this indicates that aprotinin is stable under acidic conditions for at least 6 months when stored at 4°C (using the Arrhenius equation). When the stability data is expressed as the ratio of the analyte signal peak to the ionization control signal peak, the variability is significantly minimized Figure 4 ). This illustrates the use of aprotinin (as an ionization control) to overcome ionization differences between different MALDI-TOF acquisitions.

[0096] Figure 5 shows another example of an ionization control, transthyretin. MALDI-TOF mass spectra were generated showing the mass (m / z) distribution of TTR (0.01 mg / ml) in elution buffer in the presence or absence of 0.1 mg / ml polyclonal IgG (Figure 5A and B). The TTR monomer peak was observed at 13827 m / z (+1 charge state) and 6914 m / z (+2 charge state), neither of which interfered with any of the lambda or kappa polyclonal light chain peaks from IgG. The signal intensity of the TTR ionization control peak was unchanged in the presence or absence of analyte (Figure 5B). The TTR signal peak did not overlap either the signal peak for aprotinin (Figure 5C) or the signal peak for glycosylated kappa free light chain (larger m / z) (Figure 5D).

Claims

1. An elution buffer for eluting one or more predetermined analytes from one or more immunoglobulin-specific antibodies or fragments thereof, said elution buffer comprising a predetermined amount of an acid-stable ionized control protein or peptide for mass spectrometry, wherein: The pH of the elution buffer is 1-5; and the ionized control protein or peptide is stable in the elution buffer for at least 30 days. The one or more predetermined analytes are κ light chains, λ light chains, κ free light chains, or λ free light chains, and The ionized control protein or peptide is selected having at least one mass spectrometric peak containing an m / z value, wherein the mass spectrometric peak does not overlap with the mass spectrometric peak of the analyte or each predetermined analyte. The ionized control protein or peptide mentioned therein is selected from aprotinin and transthyretin.

2. The elution buffer according to claim 1, wherein at least one mass spectrometry m / z peak of the ionized control protein or peptide is stable for at least 30 days.

3. The elution buffer according to claim 1, wherein the ionized control protein is selected having at least one mass spectrometric m / z peak within a predetermined mass spectrometry window for detecting or quantifying one or more peaks from the one or more predetermined analytes.

4. The elution buffer according to claim 1, comprising an elution buffer selected from: (a) 5% v / v aqueous acetic acid solution; (b) 0.1M glycine at pH 2.0-3.0 or 0.2M glycine at pH 2-5.

5. The elution buffer according to claim 1, comprising 0.5 to 100 ng / μL of an ionized control protein or peptide.

6. The elution buffer according to claim 1, wherein the ionized control protein or peptide comprises at least 30 amino acids.

7. The elution buffer according to claim 1, wherein the mass of the ionized control protein or peptide is at least 3 kDa.

8. The elution buffer according to claim 1, wherein the elution buffer further comprises a reducing agent.

9. The elution buffer according to claim 8, wherein the reducing agent is selected from tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), 2-mercaptoethanol, or cysteine.

10. A kit for analyzing one or more immunoglobulins or fragments thereof by mass spectrometry, comprising an elution buffer according to any one of claims 1 to 9 and one or more immunoglobulin-specific antibodies or fragments thereof.

11. The kit according to claim 10, wherein the immunoglobulin-specific antibody is anti-IgA, anti-IgG, anti-IgM, anti-IgD, or anti-IgE.

12. The kit according to claim 10, wherein the immunoglobulin-specific antibody is an anti-total light chain or an anti-heavy chain subclass.

13. The kit according to claim 12, wherein the immunoglobulin-specific antibody is an anti-λ light chain or an anti-κ light chain.

14. The kit according to claim 12, wherein the immunoglobulin-specific antibody is an anti-free light chain antibody.

15. The kit according to claim 14, wherein the immunoglobulin-specific antibody is an anti-λ free light chain or an anti-κ free light chain.

16. The kit according to claim 10, wherein the immunoglobulin-specific antibody is an anti-heavy chain / light chain type.

17. The kit according to claim 16, wherein the immunoglobulin-specific antibody is an anti-heavy chain subclass - light chain type.

18. The kit of claim 10, wherein the antibody or a fragment thereof binds to the substrate.

19. The kit according to claim 10, further comprising a predetermined amount of a control analyte.

20. The kit of claim 10, further comprising one or more of the following: sample dilution buffer, reducing agent, mass spectrometry matrix, mass spectrometry matrix solvent, MALDI target, and mass spectrometer mass calibrator.

21. The kit according to claim 10, further comprising a standard serum protein control.

22. A method for detecting or quantifying an immunoglobulin or a fragment thereof, comprising immunopurifying a predetermined immunoglobulin or a fragment thereof, eluting the immunoglobulin or a fragment thereof with an elution buffer according to any one of claims 1 to 9, and detecting the immunoglobulin or a fragment thereof and the ionized control protein or peptide by mass spectrometry.

23. The method of claim 22, wherein the mass spectrometry method is MALDI-TOF.

24. The method of claim 22, comprising using the kit of any one of claims 10 to 21.

25. A method for detecting or quantifying immunoglobulins or fragments thereof using the elution buffer of any one of claims 1 to 9, comprising: (a) Identify the immunoglobulin or a fragment thereof; (b) Identify the m / z of at least one peak of the ionized control protein or peptide compared with the m / z of one or more expected peaks of the immunoglobulin or its fragment; (c) Identify ionized control proteins or peptides having the stated m / z range and acid stability.

26. The method according to any one of claims 22 to 25 is used for analyzing one or more immunoglobulins or fragments thereof by mass spectrometry.

27. An elution buffer for eluting one or more predetermined immunoglobulins or fragments thereof from one or more immunoglobulin-specific antibodies or fragments thereof, or for eluting one or more predetermined antibodies or fragments thereof from a target immunoglobulin or fragment thereof, wherein the elution buffer contains an acid-stable ionization control for mass spectrometry, wherein: The pH of the elution buffer is 1-5; and The elution buffer contains a predetermined amount of acid-stable mass spectrometry ionization control protein or peptide. The ionized control protein or peptide is stable in the elution buffer for at least 30 days, and The ionized control protein or peptide mentioned therein is selected from aprotinin and transthyretin.

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