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 to 5; and the elution buffer comprises a predetermined amount of an acid-stable mass spectrum ionization control protein. Use of an elution buffer in the detection and quantification of an analyte (e.g., by mass spectrometry) is also described.

Description

[0001] The present invention relates to an elution buffer containing a predetermined amount of an acid-stable ionization control for mass spectrometry, a kit containing such a buffer, and a method for preparing such a buffer and kit.

[0002] background

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

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

[0005] However, in addition to crystal formation between the matrix and the sample, these methods are affected by other factors and therefore cannot accurately reflect spotting, crystallization, and ionization.

[0006] Previous attempts to control inter- and intra-experimental variability in MALDI-TOF MS have typically utilized internal calibration peptides with physicochemical properties comparable to the target protein, incorporated into the sample at varying concentrations before comparing the ionic strengths of the calibration peptides and the analyte. However, this approach suffers from issues including peak intensity variability. Combining controls with iterative algorithms and / or performing repetitive analyses has been proposed as a possible solution to compensate for some of the analytical variations over time and improve the reproducibility of protein analyses via 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 having the same primary sequence as a specific analyte was incorporated into a blood sample (Yi, J et al.). Methods Mol Biol 2011; 728: 161-75).

[0008] Using an internal control in matrix-assisted laser desorption / ionization time-of-flight mass spectrometry also showed improved sensitivity in determining bacterial concentrations in samples. The addition of cytochrome C as an internal control reduced signal intensity by 20-30% in samples with high bacterial concentrations, but improved signal intensity in some samples with low bacterial concentrations. In this case, proteins were incorporated into the matrix and then premixed at a matrix-analyte ratio of 2:1 (Gantt, SL et al.). , J Am Soc Mass Spectrom 1999; 10(11):1131-7).

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

[0010] Other examples of attempts to modulate quantitative variability between samples include post-analytical conditioning via outlier removal and baseline removal via intensity calibration (Neubert et al.). , J Proteome Res 2008; 7(6) 2270-9).

[0011] An example of the significant in vitro diagnostic applications of mass spectrometry is its association with many proliferative diseases involving antibody-producing cells.

[0012] Antibody molecules (also known as immunoglobulins) exhibit dual symmetry and are typically composed of two identical heavy chains and two identical light chains, each containing a variable domain and a constant domain. The variable domains of the heavy and light chains bind to form antigen-binding sites, ensuring that both chains contribute to the antigen-binding specificity of the antibody molecule. The basic tetrameric structure of an antibody consists of two heavy chains covalently linked by disulfide bonds. Each heavy chain is then again linked to a light chain via disulfide bonds. This produces a essentially "Y"-shaped molecule.

[0013] In many of these proliferative disorders, plasma cells proliferate to form monoclonal tumors of the same plasma cells. This results in the production of large amounts of the same immunoglobulins and is known as monoclonal gammaglobulinosis.

[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 people 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's macroglobulinaemia is a low-grade lymphoproliferative disorder associated with the production of monoclonal IgM. There are approximately 1,500 new cases annually in the United States and approximately 300 new cases annually in the United Kingdom. Serum IgM quantification is important for both diagnosis and surveillance. In the United Kingdom, 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, chronic lymphocytic leukemia monoclonal proteins 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 of the diseases highlighted above, there is an abnormal concentration of monoclonal immunoglobulins or free light chains. When a disease causes abnormal replication of plasma cells, this usually results in these cells producing more immunoglobulins as the “monoclonus” proliferates and appears in the blood.

[0019] Sensitive assays capable of detecting free κ light chains and free λ light chains alone have been developed. This method uses polyclonal antibodies against either free κ or free λ light chains. The possibility of generating such antibodies as one of many different possible specificities is also discussed in WO97 / 17372. This document discloses methods for producing desired antibodies with higher specificity than those achievable with existing technologies in tolerable animals. The free light chain assay uses antibodies to bind to either free λ or free κ light chains. The concentration of the free light chains is determined by nephelometry or turbidimetry.

[0020] Characterization of the quantity or type of free light chains (FLCs), heavy chains or subclasses, or light chains 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 gammaglobulinemia (of which multiple myeloma is an example) and other immune-mediated diseases, including hypergammaglobulinemia and hypogammaglobulinemia.

[0021] WO2015 / 154052 (incorporated herein in its entirety) discloses a method for detecting immunoglobulin light chains, immunoglobulin heavy chains, or mixtures thereof using MS. A sample containing immunoglobulin light chains, heavy chains, or mixtures thereof is immunopurified and then analyzed by mass spectrometry to obtain a mass spectrum of the sample. This can be used to detect monoclonal proteins in samples from patients. It can also be used for fingerprinting, isotyping, and identification of monoclonal antibodies.

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

[0023] In diagnostic procedures, the purification of immunoglobulins from samples 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 κ, anti-total λ antibodies, or anti-free light chain antibodies, such as anti-free κ or anti-free λ light chain antibodies. It is important to have a calibrator to ensure proper purification and detection procedures.

[0024] WO2017 / 144900 describes numerous controls using a heavier form of the analyte to be detected or a monoclonal form of the analyte to be detected. That is, for example, IgA can be quantified by comparison with a predetermined amount of a heavier form of IgA κ.

[0025] This is because different proteins are expected to crystallize at different rates on the mass spectrometry matrix. This means that when the matrix is ​​sampled by mass spectrometry, different amounts of control proteins and analytes will be detected. Furthermore, their ionization rates are expected to be different. This will lead to inconsistencies in the amount of immunoglobulins detected. Additionally, the reproducibility issues of mass spectrometry-based methods mean that peak intensities can vary significantly between experiments, and mass drift can occur, potentially affecting 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 unexpectedly discovered that analytical variability in ionization used in mass spectrometry could be controlled by using separate markers in the acidic elution buffer after immunoprecipitation and before spotting.

[0027] Overview

[0028] This article provides elution buffers 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 1-3, or even more preferably 1.5-3.0; the elution buffer contains a predetermined amount of acid-stable mass spectrometry ionization control protein.

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

[0031] These elution buffers are used to release analytes that have bound to analyte-specific antibodies. Including the 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 separately.

[0032] Ionization controls can also be used as “lock-in mass spectrometry calibrators” in mass spectrometry methods, including, for example, MALDI and electrospray mass spectrometry. Such lock-in mass spectrometry calibrators are ions with known m / z values ​​obtained from ionization controls, which allow for real-time recalibration within the spectrum by correcting for m / z shifts caused by instrument and internal MALDI target plate drift.

[0033] Samples containing the analyte to be analyzed can be biological samples, such as blood, saliva, serum, plasma, cerebrospinal fluid, or urine, with blood, serum, or plasma being more typical.

[0034] Samples can come from subjects exhibiting either hypogammaglobulinemia or hypergammaglobulinemia. Subjects may have proliferative disorders associated with antibody-producing cells, such as monoclonal gammaglobulinopathy. These include myeloma and primary systemic amyloidosis, plasmacytoma, Waldenström macroglobulinemia, and MGUS.

[0035] Select an ionization control protein that is compatible with the intended analyte.

[0036] The ionized 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] Prolonged storage of unsuitable controls may lead to physical stability issues, which could cause proteins to precipitate in the elution buffer or otherwise be affected, resulting in poor crystallization or poor ionization on the target, both of which can affect m / z peak height or area.

[0038] Damage to the control itself that causes changes in mass or ionization state can also alter 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 spectrum m / z peak of the ionized control protein can be substantially stable (as defined above).

[0041] An ionization control protein with at least one mass spectral peak containing an m / z value can be selected, said mass spectral peak not substantially overlapping with the mass spectral peak of said or each predetermined analyte. Uniform ionization is typically chosen, and generally does not substantially affect the intensity of the mass spectral signal.

[0042] An ionized control protein may be selected that has at least one mass spectrometric m / z peak within a predetermined mass spectrometry window for detecting or quantifying one or more peaks from the at least one predetermined analyte, or within a range of m / z observed by a mass spectrometer.

[0043] Samples can be treated with reducing agents, typically after elution but before mass spectrometry. This is particularly useful when immunoglobulin light chains in a sample are bound to heavy chains. The use of reducing agents decouples the light and heavy chains, allowing for the individual detection of the light chains by mass spectrometry. Reducing agents can also be used to separate other analyte proteins to isolate present subunits.

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

[0045] The uncoupling step is usually performed after immunopurification or other enrichment of immunoglobulins in the sample, or as part of the elution step after immunopurification of the sample.

[0046] The antibodies used for immunopurification can be complete antibodies or fragments thereof, such as Fab, F(ab), and F(ab'). 2 Fragments, or single-chain antibodies. Antibodies or their fragments may be cross-linked, as described, for example, in WO2017144903 (which is incorporated herein by reference in its entirety).

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

[0048] Elution buffers can contain organic acids such as citric acid, acetic acid, formic acid, uric acid, and propionic acid, as well as inorganic acids such as hydrochloric acid. Acidic buffers or solutions containing salts should be avoided, especially at higher concentrations, as these may interfere with ionization or crystallization at high concentrations.

[0049] For example, the elution buffer of the present invention may contain elution buffers selected from the following:

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

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

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

[0053] The elution buffer may contain 1-100 ng / μl of ionized control protein, more preferably 1-10 ng / μl.

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

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

[0056] Ionized control proteins advantageously possess different mass ranges or ion gates, or have multiple charge states, to enable their use within the assay window of the analyte. Ionized control proteins or peptides can be naturally occurring or synthetic.

[0057] Suitable proteins for use as ionization controls may include aprotinin, α1-acid glycoprotein, β2-glycoprotein, or prealbumin (also known as transthyretin). More preferably, ionization controls may include aprotinin or transthyretin. Aprotinin is a serine protease inhibitor derived from bovine pancreas. It is readily available from commercial sources as a purified protein and a pharmaceutical; TRASYLOL. (CAS No.: 9087-70-1, molar mass 6511.5 Da. UniProtKB Registry No. P00974. Isoelectric point pH 10.5). It is stable in neutral or acidic media at high temperatures. Transthyretin (TTR, prealbumin, or TBPA) is a transporter protein found in serum and cerebrospinal fluid that carries the thyroid hormone thyroxine and a retinol-binding protein that binds to retinol. It is a 55 kDa homotetramer or dimer with a quaternary structure. The human protein has UniProtKB Registry No. P02766.

[0058] However, other essentially acid-stable proteins can be used in different mass ranges (ion gates), or one or more protein charge states may be suitable for a specific m / z measurement window. For example, in a measurement window of m / z 5–30 kDa, α1 acid glycoprotein (+1–21560), β2 glycoprotein I (+1–36255), or prealbumin monomer (+1–13760) would be appropriate.

[0059] By utilizing the additional plates and gate reversal in the instrument, larger mass windows, such as dual-ion gating methods or wide mass windows, can be targeted.

[0060] This document also provides a kit for analyzing one or more analytes by mass spectrometry, comprising an elution buffer as defined above and one or more analyte-specific antibodies or fragments thereof that are specific to the one or more predetermined analytes.

[0061] The analyte or antigen-specific antibody may be a protein or peptide, preferably a serum protein or peptide.

[0062] Antigen-specific antibodies include anti-streptolysin O, anti-tetanus toxoid immunoglobulin, and Haemophilus influenzae (Hib). Haemophilus influenzae ) specific immunoglobulins, diphtheria toxoid specific immunoglobulins, Streptococcus pneumoniae ( Streptococcus pneumoniae ) specific immunoglobulins, Salmonella typhi ( Salmonella typhi ) Specific immunoglobulin or varicella-zoster virus ( Varicella zoster Virus-specific immunoglobulins.

[0063] If the analyte is a serum protein, the serum protein may contain one or more complement proteins, such as C1, C2, C3, C4, or components thereof, such as components C3a, C3b, C3c.

[0064] Serum proteins may include immunoglobulins or fragments thereof, albumin, β2-microglobulin, α1-microglobulin, cysteine ​​protease inhibitor C, microalbumin, α1-acid glycoprotein, α1-antitrypsin, α2-macroglobulin, antistreptolysin O, antitetracycline toxoid immunoglobulin, apolipoprotein A, apolipoprotein B, ceruloplasmin, C-reactive protein, haptoglobulin, prealbumin, rheumatoid factor, or total serum protein transferrin.

[0065] The analyte can be a monoclonal antibody, such as a therapeutic monoclonal antibody. 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 chains, anti-free light chains, anti-λ light chains, anti-κ light chains, anti-λ free light chains, anti-κ free light chains, anti-heavy chain subclasses, 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-κ, and / or anti-λ specific antibodies.

[0066] Antibodies or fragments thereof that are specific to one or more predetermined analytes can also bind to a substrate; for example, antibodies or fragments thereof can bind to latex beads. Target antigens can also be attached to a substrate such as latex beads.

[0067] The kit may also contain a predetermined amount of control analyte.

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

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

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

[0071] The kit may additionally include a standard serum protein control. For example, the kit may contain an antibody against a human-specific antibody.

[0072] This article also provides a method for detecting or quantifying an analyte, comprising immunopurifying a predetermined analyte, eluting the analyte with an elution buffer according to the invention, and detecting the analyte and the ionized control protein by mass spectrometry.

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

[0074] The immunoassay used in this invention has three main steps: 1) immunocapture of the analyte, 2) elution of the analyte, 3) optional reduction of the analyte, and 4) spotting the analyte onto a MALDI-TOF target plate.

[0075] The present invention provides that the ionization control protein can be included in the reagent used in step 2) so that the control and analyte can be combined prior to step 3) and advantageously spotted together in step 4. This is important because the ionization control is used to control for variability in step 3 and subsequent ionization in the MALDI-TOF mass spectrometer.

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

[0077] This document also provides a method for preparing an elution buffer according to the invention, wherein the elution buffer is used to elute one or more predetermined analytes from one or more analyte-specific antibodies or fragments thereof, wherein:

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

[0079] The method for preparing the elution buffer according to the present invention includes:

[0080] (a) Identify the analyte;

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

[0082] (c) Identify ionization control proteins with 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 with m / z peaks of a plurality of potential ionization control proteins having acid stability, and outputting the identification of one or more ionization control proteins having m / z range and acid stability for the analyte. Attached Figure Description

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

[0085] Figure 1 This is an example of MALDI-TOF mass spectrometry, showing the mass distribution of the analyte (κ light chain (κ)) after elution with acetic acid containing aprotinin. A single aprotininase peak (+1 charge) is observed, which does not interfere with the analyte (κ light chain) peak. The ion charge states are given in parentheses.

[0086] Figure 2 The results show that the signal of the relative ionized control protein remains stable in both the presence and absence of the analyte. The MALDI-TOF mass spectrum of the aprotinin obtained in the absence of the analyte (black line) is not significantly different from the spectrum of the aprotinin containing the κ light chain from normal human serum (NHS) (gray line and inset).

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

[0088] Figure 4 The analyte signal relative to aprotinin, used as an ionization control, remained stable over time. 5% acetic acid containing aprotinin was stored at 22°C and used periodically to elute the κ light chain. MALDI-TOF mass spectra were obtained at each time point, and the peak area ratio (± standard deviation) of aprotinin and κ light chain (+2) was determined. No significant change in the peak area ratio was observed over 8 weeks.

[0089] Figure 5: Transthyretin (TTR) as a MALDI-TOF ionization control. MALDI-TOF mass spectrometry shows the mass distribution of TTR after elution with acetic acid in the cases of mixing with and without polyclonal IgG. Figure 5A (B) TTR peaks were observed at 13827 m / z and 6914 m / z, neither of which interfered with any λ or κ polyclonal light chain peaks. The signal intensity of the TTR ionization control peak remained unchanged in the presence or absence of the analyte (B). The TTR signal peaks did not overlap with either the aprotinin peak (C) or the glycosylated κ free light chain (larger mass) peak (D). Ionic charge states are given in parentheses.

[0090] Prepare a solution containing 2 ng / ml of acetic acid in 5% acetic acid containing 20 mM tris(2-carboxyethyl)phosphine (TCEP) reducing agent. -1 Aprotinin was used as an elution buffer for ionization control in mass spectrometry analysis. Analytes were eluted from the immunocapture beads using 5% acetic acid, which simultaneously promoted the separation of immunoglobulin heavy and light chains. 20 mM TCEP was used as an acid-stabilized reducing agent to break the disulfide bonds that hold intact immunoglobulins together.

[0091] Normal human serum samples (NHS) were diluted 1:10 and captured using paramagnetic microparticles containing antibodies specific to the human κ immunoglobulin light chain (following step 1 above). Elution was performed with an acidic buffer containing both a reducing agent and aprotinin (as an ionization control). The eluent was then spotted onto a MALDI-TOF target plate in a sandwich structure with a MALDI matrix (HCCA) and dried. Mass spectrometry was obtained in positive ion mode covering the range of 5000 to 30000 m / z, including single-charged (+1, m / z 22705), double-charged (+2, m / z 11353), and triple-charged (+3, m / z 7569) ions of the analyte (human κ light chain; Table 1).

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

[0093] aprotinin intensity signal in Figure 1 The distinct peaks at m / z 6512 are clearly visible and do not interfere with or overlap with any of the three peaks of the analyte. To demonstrate that the aprotinin is independent of the presence of the analyte, it was analyzed in the presence (+NHS) and absence (-NHS) of the analyte. Figure 2 This demonstrates that the signal intensity of the aprotinin ionization control is the same under any circumstances.

[0094] To investigate the stability of the ionization control under acidic conditions, 50 ml aliquots of the preparation were prepared (with 2 ng / ml supplement). -1 The aprotinin (5% acetic acid) was stored at 22°C. Periodically, each aliquot was removed, replenished with reducing agent (TCEP), and then used to elute the analyte from the aprotinin microparticles for MALDI-TOF analysis. Figure 3 The peak areas of the κ analyte and aprotinin are shown in the figure. The peak areas differed between the two during the experiment. This variation is due to known MALDI point-to-point sample inconsistencies, but the signals of either the analyte or aprotinin did not decay over 60 days at 22°C. Extrapolation indicates that aprotinin is stable for at least 6 months under acidic conditions when stored at 4°C (using the Arrheniuse equation). The variability was significantly minimized when stability data were expressed as the ratio of the analyte signal peak to the ionized control signal peak. Figure 4 This demonstrates the use of aprotinin (as an ionization control) in overcoming ionization differences between different MALDI-TOF acquisitions.

[0095] Figure 5 shows another example of an ionization control, namely transthyretin. MALDI-TOF mass spectra were generated, showing the mass (m / z) distribution of TTR (0.01 mg / ml) in the elution buffer with or without 0.1 mg / ml polyclonal IgG. Figure 5A And B). TTR monomer peaks were observed at 13827 m / z (+1 charge state) and 6914 m / z (+2 charge state), neither of which interfered with either of the λ or κ polyclonal light chain peaks from IgG. The signal intensity of the TTR ionization control peak remained unchanged in the presence or absence of the analyte. Figure 5B The TTR signal peak does not overlap with the aprotinin signal peak. Figure 5C It also does not overlap with the signal peak of the glycosylated κ free light chain (larger m / z). Figure 5D ).

Claims

1. 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 ionisation control protein.

2. The elution buffer according to claim 1, wherein the ionisation control protein is substantially stable in the elution buffer for at least 30 days.

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

4. The elution buffer according to claims 1 to 3, wherein the ionisation control protein is selected to have at least one mass spectrometry peak comprising an m / z value that is substantially non-overlapping with a mass spectrometry peak of the or each predetermined analyte.

5. The elution buffer according to claim 4, wherein the ionisation control protein is selected to have at least one mass spectrometry m / z peak within a predetermined mass spectrometry window used to detect or quantify one or more peaks from the at least one predetermined analyte.

6. The elution buffer according to any one of claims 1 to 5, comprising an elution buffer selected from: (a) 5% v / v aqueous acetic acid; (b) 0.1 M glycine at pH 2.0-3.0 or 0.2 M glycine at pH 2-6.

7. The elution buffer according to any one of the preceding claims, comprising 0.5 to 100 ng of ionisation control protein.

8. The elution buffer according to any one of the preceding claims, wherein the ionisation control protein comprises at least 30 amino acids.

9. The elution buffer according to any one of the preceding claims, wherein the ionisation control protein has a mass of at least 3 kDa.

10. The elution buffer according to any one of the preceding claims, wherein the ionisation control protein is selected from aprotinin, beta 2 glycoprotein, transthyretin and alpha 1 acid glycoprotein.

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