Method and kit for detecting M protein in sample

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a method and a kit for detecting M protein in a sample. According to the kit for detecting or typing the M protein in the sample and the detection method provided by the invention, the problem of carrying out accurate qualitative and typing on the low-abundance M protein in the sample to be detected is solved; the detection method provided by the invention is higher in sensitivity, better in accuracy, simpler to operate and easier to realize automation, and does not have the subjectivity problem of manual interpretation; compared with other mass spectrometry methods, such as a liquid chromatography-tandem mass spectrometry method, the detection cost is lower, the operation is simpler, automation is easier to realize, the detection flux is higher, and popularization in clinical diagnosis and treatment is easier to realize.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection technology, and particularly relates to a method and a kit for detecting M protein in a sample. Background Art

[0002] Clinically, the detection of urine M protein is similar to the detection of serum M protein. Urine protein electrophoresis (uPE) and urine immunofixation electrophoresis (uIFE) are usually used to analyze whether M protein is present in the urine. The detection of urine FLC is also performed by uIFE, and the antibodies used are specific for FLC. Because uIFE first separates the protein in the urine through electrophoresis technology, and then immunoprecipitates, fixes and stains the electrophoretically separated protein with specific antibodies, manual analysis of the bands is used to determine whether M protein (monoclonal FLC or total M protein) is present in the urine. This method is based on the principle of electrophoresis and the protein content in urine is much lower than that in serum. Therefore, uIFE has poor detection sensitivity for monoclonal FLC or total M protein in urine. In addition, the manual interpretation of electrophoresis bands itself is highly subjective, and accurate test results are often not given for urine samples with low levels of intact immunoglobulins or FLC. In addition, in clinical practice, serum immunofixation electrophoresis usually only uses A, G, and M heavy chain antibodies to type serum M proteins, and lacks D and E antibodies. Although type D M proteins account for a small proportion in actual clinical samples, there are also medical records. Therefore, the sIFE method used in clinical practice may miss the detection of type D M proteins.

[0003] A method for detecting urine and serum M protein with higher sensitivity and accuracy is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, one purpose of the present invention is to provide a kit and a detection method for detecting or typing M protein in a sample. The present invention solves the problem of accurately characterizing and typing low-abundance M protein in the sample to be tested. Compared with the uIFE method, the detection method provided by the present invention has higher sensitivity, better accuracy, simpler operation, and is easier to automate, and there is no subjective problem of manual interpretation; compared with other mass spectrometry methods, such as liquid chromatography-tandem mass spectrometry, the detection cost is lower, the operation is simpler, it is easier to automate, the detection throughput is higher, and it is easier to promote in clinical diagnosis and treatment.

[0005] To this end, the first aspect of the present invention provides a kit for detecting or typing M protein in a sample. According to an embodiment of the present invention, the kit comprises:

[0006] Antibody-bound magnetic beads;

[0007] elution solution;

[0008] Reaction buffer;

[0009] Wherein, the antibody is the following antibody:

[0010] IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies;

[0011] Total kappa and total lambda light chain-specific antibodies;

[0012] and antibodies specific for kappa and lambda free light chains,

[0013] The elution solution contains an antigen-antibody complex dissociation reagent,

[0014] The mass ratio of the antibody to the magnetic beads is 1:(10-1000),

[0015] The particle size of the magnetic beads is 0.5-5 μm.

[0016] By optimizing the reagents contained in the kit, the inventors obtained a kit for detecting or typing M protein in samples. Using this kit, the problem of accurately detecting low-abundance M protein in the test sample was solved, including accurately identifying its form (free or intact) and simultaneously performing specific typing identification on each form of M protein. Compared with the uIFE method, the detection method provided by the present invention has higher sensitivity, better accuracy, simpler operation, and is easier to automate, and does not have the subjective problem of manual interpretation; compared with other mass spectrometry methods, such as liquid chromatography-tandem mass spectrometry, the detection cost is lower, the operation is simpler, it is easier to automate, the detection throughput is higher, and it is easier to promote in clinical diagnosis and treatment.

[0017] According to an embodiment of the present invention, the IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies are specific anti-human IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies or fragments thereof;

[0018] The total κ ​​type and total λ type light chain specific antibodies are specific anti-human total κ ​​type and total λ type light chain specific antibodies or fragments thereof;

[0019] The κ-type and λ-type free light chain specific antibodies are specific anti-human κ-type or λ-type free light chain antibodies or fragments thereof.

[0020] According to an embodiment of the present invention, the antibody is a monoclonal antibody or a polyclonal antibody or a fragment thereof.

[0021] According to an embodiment of the present invention, the antibody is a polyclonal antibody.

[0022] According to an embodiment of the present invention, the affinity constant KD value of the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies is 100-100,000 pM; the affinity constant KD value of the total κ ​​and total λ light chain-specific antibodies is 100-100,000 pM; and the affinity constant KD value of the κ and λ free light chain-specific antibodies is 100-100,000 pM.

[0023] According to an embodiment of the present invention, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:1:(1-10):1.

[0024] According to an embodiment of the present invention, the surface of the magnetic beads is modified with proteins or chemical groups, and the proteins or chemical groups are used to bind to the antibodies.

[0025] According to an embodiment of the present invention, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups.

[0026] According to an embodiment of the present invention, the protein is streptavidin.

[0027] According to an embodiment of the present invention, when the surface of the magnetic beads is modified with streptavidin, the antibody is linked to biotin.

[0028] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent includes at least one selected from an organic acid solution, an inorganic acid solution, and an alkaline solution.

[0029] The organic acid solution comprises at least one selected from the group consisting of formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid;

[0030] The inorganic acid solution comprises at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution;

[0031] The alkaline solution includes at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0032] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution.

[0033] According to an embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0034] According to a preferred embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.1-5%.

[0035] According to an embodiment of the present invention, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions.

[0036] According to an embodiment of the present invention, the reaction buffer is PBST solution.

[0037] According to an embodiment of the present invention, the kit further includes a cleaning solution.

[0038] According to an embodiment of the present invention, the cleaning solution includes at least one selected from PBS, PBST, and TBST solutions.

[0039] According to an embodiment of the present invention, the cleaning solution is PBST solution.

[0040] According to an embodiment of the present invention, the kit further comprises a matrix solution for MALDI-TOF MS detection.

[0041] According to an embodiment of the present invention, the matrix in the matrix liquid includes at least one selected from sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracene triol, and 3-indole acrylic acid.

[0042] According to an embodiment of the present invention, the matrix in the matrix liquid is sinapinic acid.

[0043] A second aspect of the present invention provides a method for detecting M protein in a sample. According to an embodiment of the present invention, the method comprises using the kit for detecting or typing M protein in a sample described in the first aspect to detect the sample to be tested, so as to determine whether the sample to be tested contains intact M protein and / or free light chains, or to perform typing on the intact M protein and / or free light chains.

[0044] A third aspect of the present invention provides a method for detecting M protein in a sample. According to an embodiment of the present invention, the method comprises:

[0045] S1: contacting the test sample and the antibody-bound magnetic beads in a reaction buffer, so that the intact M protein and / or free light chains contained in the test sample specifically bind to the antibodies bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with the intact M protein and / or free light chains;

[0046] S2: performing an elution treatment and a magnetic separation treatment on the antigen-antibody complex magnetic beads to elute and separate the enriched intact M protein and / or free light chains from the antibody-bound magnetic beads to obtain a test solution containing the intact M protein and / or free light chains;

[0047] S3: Spotting the test solution on a sample chip, performing crystallization treatment, and detecting using MALDI-TOF MS to determine whether the test sample contains intact M protein and / or free light chains, or typing the intact M protein and / or free light chains.

[0048] Wherein, the antibody is the following antibody:

[0049] IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies;

[0050] Total kappa and total lambda light chain-specific antibodies;

[0051] and antibodies specific for kappa and lambda free light chains.

[0052] The present invention provides a new method for detecting M protein, which comprehensively determines whether a sample to be tested (such as urine or serum) contains M protein by detecting FLC and intact immunoglobulin in the sample to be tested respectively. The detection of FLC in the sample to be tested is carried out by using magnetic beads bonded with κ-type and λ-type FLC-specific antibodies to enrich the two types of FLC in the sample, respectively, and then eluting and detecting them by MALDI-TOF MS. The results are automatically analyzed by software and the presence of monoclonal FLC and its specific type are determined. The detection of intact immunoglobulin in the sample to be tested is carried out by using magnetic beads bonded with five heavy chain-specific antibodies (IgA, G, D, E, M) and magnetic beads bonded with total κ-type and total λ-type light chain-specific antibodies to enrich the intact immunoglobulin in the sample to be tested, and then eluting and reducing and detecting them by MALDI-TOF MS. The results are automatically analyzed by software to determine whether the sample to be tested contains intact M protein or total M protein and its specific type.

[0053] According to an embodiment of the present invention, in the antibody-bound magnetic beads, the mass ratio of the antibody to the magnetic beads is 1:(10-1000).

[0054] According to an embodiment of the present invention, the particle size of the magnetic beads is 0.5-5 μm.

[0055] According to an embodiment of the present invention, the surface of the magnetic beads is modified with proteins or chemical groups, and the proteins or chemical groups are used to bind to the antibodies.

[0056] According to an embodiment of the present invention, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups.

[0057] According to an embodiment of the present invention, the protein is streptavidin.

[0058] According to an embodiment of the present invention, when the surface of the magnetic beads is modified with streptavidin, the antibody is linked to biotin.

[0059] According to an embodiment of the present invention, the IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies are specific anti-human IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies or fragments thereof;

[0060] The total κ ​​type and total λ type light chain specific antibodies are specific anti-human total κ ​​type and total λ type light chain specific antibodies or fragments thereof;

[0061] The κ-type and λ-type free light chain specific antibodies are specific anti-human κ-type or λ-type free light chain antibodies or fragments thereof.

[0062] According to an embodiment of the present invention, the antibody is a monoclonal antibody or a polyclonal antibody or a fragment thereof.

[0063] According to an embodiment of the present invention, the antibody is a polyclonal antibody.

[0064] According to an embodiment of the present invention, the affinity constant KD value of the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies is 100-100,000 pM; the affinity constant KD value of the total κ ​​and total λ light chain-specific antibodies is 100-100,000 pM; and the affinity constant KD value of the κ and λ free light chain-specific antibodies is 100-100,000 pM.

[0065] According to an embodiment of the present invention, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:1:(1-10):1.

[0066] According to an embodiment of the present invention, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions.

[0067] According to an embodiment of the present invention, the reaction buffer is PBST solution.

[0068] According to an embodiment of the present invention, in step S1 , when contacting, the volume ratio of the sample to be tested to the reaction buffer is 1:(1-200).

[0069] According to an embodiment of the present invention, in the reaction buffer, the mass concentration of the antibody-bound magnetic beads is 1-100 mg / mL.

[0070] According to an embodiment of the present invention, when the contact is performed, the reaction time is 10-40 minutes and the reaction temperature is 20-35°C.

[0071] According to an embodiment of the present invention, in step S2, the elution process is performed using an elution solution.

[0072] According to an embodiment of the present invention, the elution solution contains an antigen-antibody complex dissociation reagent.

[0073] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent includes at least one selected from an organic acid solution, an inorganic acid solution, and an alkaline solution.

[0074] The organic acid solution comprises at least one selected from the group consisting of formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid;

[0075] The inorganic acid solution includes at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution;

[0076] The alkaline solution includes at least one selected from sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0077] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution.

[0078] According to an embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0079] According to an embodiment of the present invention, the method further comprises, before step S2, washing the antigen-antibody complex magnetic beads enriched with intact M protein and / or free light chains;

[0080] According to an embodiment of the present invention, the cleaning process is performed using a cleaning liquid.

[0081] According to an embodiment of the present invention, the cleaning solution includes at least one selected from PBS, PBST, and TBST solutions.

[0082] According to an embodiment of the present invention, the cleaning solution is PBST solution.

[0083] According to an embodiment of the present invention, step S3 further includes mixing the solution to be tested with a matrix solution, spotting the mixed solution on a sample chip, and performing a crystallization process.

[0084] According to an embodiment of the present invention, the matrix in the matrix liquid includes at least one selected from sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracene triol, and 3-indole acrylic acid.

[0085] According to an embodiment of the present invention, the matrix in the matrix liquid is sinapinic acid.

[0086] According to an embodiment of the present invention, the volume ratio of the test solution and the matrix solution when mixed is 1:(1-10).

[0087] According to an embodiment of the present invention, the method includes:

[0088] (1) when detecting whether the sample to be tested contains M protein, based on the MALDI-TOF MS detection result obtained in step S3, whether a peak signal exists within the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa, so as to determine whether the sample to be tested contains M protein; or

[0089] (2) When it is necessary to type the M protein contained in the sample to be tested, the type of the intact M protein and / or free light chain contained in the sample to be tested is determined based on the type of the antibody in the antibody-bound magnetic beads used in step S1 and the MALDI-TOF MS detection results obtained in step S3, and based on whether a peak signal exists within the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa.

[0090] Wherein, in case (1), in step S1, a dilution of the sample to be tested is prepared and divided into five portions, the first portion is contacted with magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies; the second and third portions are contacted with magnetic beads bonded to total κ ​​and total λ light chain specific antibodies, respectively; the fourth and fifth portions are contacted with magnetic beads bonded to κ and λ free light chain specific antibodies, respectively;

[0091] Alternatively, the test sample dilution solution is divided into three portions, the first portion is contacted with magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies; the second portion is contacted with magnetic beads bonded to total κ ​​and total λ light chain specific antibodies; and the third portion is contacted with magnetic beads bonded to κ and λ free light chain specific antibodies;

[0092] In case (2), in step S1, each type of antibody-bound magnetic beads is contacted with the sample to be tested respectively.

[0093] According to an embodiment of the present invention, the sample to be tested is a serum sample, a urine sample, a cerebrospinal fluid sample or a whole blood sample. The detection method of the present invention is particularly suitable for the detection and typing of M protein in urine and serum samples.

[0094] The present invention provides a method and kit for detecting or typing M protein in a sample, which solves the problem of accurately detecting low-abundance M protein in the sample to be tested, including accurately identifying its form (free or intact) and simultaneously identifying the specific typing of each form of M protein. Compared with the uIFE method, the detection method provided by the present invention has higher sensitivity, better accuracy, simpler operation, and is easier to automate, and does not have the subjective problem of manual interpretation; compared with other mass spectrometry methods, such as liquid chromatography-tandem mass spectrometry, the detection cost is lower, the operation is simpler, it is easier to automate, the detection throughput is higher, and it is easier to promote in clinical diagnosis and treatment.

[0095] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0097] Figures 1a-1c The difference in the enrichment ability of κ-FLC-specific antibody magnetic beads bound with 1 / 10, 1 / 100, and 1 / 1000 antibody-magnetic-bead mass ratios for FLC in negative urine samples is shown.

[0098] Figure 2 The detection limit of the present invention's detection method for monoclonal FLC in urine samples is shown;

[0099] Figures 3a-3d The results of the detection method of the present invention on urine samples that were negative for FLC and positive for intact M protein by clinical uIFE detection were shown, wherein: Figure 3a 3b and 3c are the results of the test of two FLC antibodies on this sample. Figure 3d This is the test result of the complete M protein typing of the sample in this protocol;

[0100] Figures 4a-4c The results of the detection method of the present invention on serum samples that were IgD positive and FLC monoclonal negative by clinical uIFE and immunoturbidimetric detection were shown, wherein, Figure 4a This is the test result of the complete M protein typing of this sample. Figure 4b This is an overlay of the results of the well detection of IgD heavy chain antibodies and two total light chain antibodies. Figure 4c This is an overlay of the results of two FLC antibody tests on this sample. DETAILED DESCRIPTION

[0101] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0102] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0103] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0104] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present invention belongs.

[0105] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0106] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0107] According to a specific embodiment of the present invention, the present invention provides a kit for detecting or typing M protein in a sample, comprising:

[0108] Antibody-bound magnetic beads;

[0109] elution solution;

[0110] Reaction buffer;

[0111] Wherein, the antibody is the following antibody:

[0112] IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies;

[0113] Total kappa and total lambda light chain-specific antibodies;

[0114] and antibodies specific for kappa and lambda free light chains,

[0115] The elution solution contains an antigen-antibody complex dissociation reagent,

[0116] The mass ratio of the antibody to the magnetic beads is 1:(10-1000),

[0117] The particle size of the magnetic beads is 0.5-5 μm.

[0118] It should be noted that the free light chains and FLC (free light chains) in the present invention have the same meaning and can be interchanged.

[0119] The "total κ ​​light chain-specific antibody" in the present invention refers to a specific antibody that can bind to both free and bound κ light chains; and the "total λ light chain-specific antibody" refers to a specific antibody that can bind to both free and bound λ light chains.

[0120] According to a specific embodiment of the present invention, the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies are specific anti-human IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies or fragments thereof; the total κ ​​and total λ light chain-specific antibodies are specific anti-human total κ ​​and total λ light chain-specific antibodies or fragments thereof; the κ and λ free light chain-specific antibodies are specific anti-human κ or λ free light chain antibodies or fragments thereof. According to a specific embodiment of the present invention, the antibodies are monoclonal antibodies or polyclonal antibodies or fragments thereof. According to a preferred embodiment of the present invention, the antibodies are polyclonal antibodies.

[0121] According to a specific embodiment of the present invention, the affinity constant KD value of the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies is 100-100,000 pM; the affinity constant KD value of the total κ ​​and total λ light chain-specific antibodies is 100-100,000 pM; and the affinity constant KD value of the κ and λ free light chain-specific antibodies is 100-100,000 pM. For example, in the kit, the affinity constant KD values ​​of the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, total κ ​​and total λ light chain-specific antibodies, and κ and λ free light chain-specific antibodies can be any value between 100 and 100,000 pM, including but not limited to 100 pM, 500 pM, 1000 pM, 5000 pM, 10,000 pM, 50,000 pM, and 100,000 pM, etc.

[0122] According to a specific embodiment of the present invention, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:1:(1-10):1. For example, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:1:(1-10):1, including but not limited to 1:1:1:1:1:1:1:1:1:1, 1:1:1:1:1:1:1:1:2:1, 1:1:1:1:1:1:1:1:1:5:1, 1:1:1:1:1:1:1:1:1:10:1, etc.

[0123] According to a specific embodiment of the present invention, the surface of the magnetic beads is modified with a protein or chemical group, and the protein or chemical group is used to bind to the antibody. It should be noted that there are no special restrictions on the protein or chemical group. All proteins or groups that can be used for in vitro detection to connect proteins to magnetic beads are covered by the protection scope of the present invention. For example, the chemical group includes but is not limited to at least one of amino, carboxyl, epoxy, and hydroxyl groups; the protein includes but is not limited to streptavidin. According to a specific embodiment of the present invention, when the surface of the magnetic beads is modified with streptavidin, the antibody is linked to biotin.

[0124] According to a specific embodiment of the present invention, the particle size of the magnetic beads in the kit is 0.5-5 μm. For example, in the kit, the particle size of the magnetic beads can be any value within the range of 0.5-5 μm, including but not limited to 0.5 μm, 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, etc.

[0125] Regarding the particle size of the magnetic beads, the inventors have found that a particle size of 0.5-5 μm can further improve the sensitivity and accuracy of the detection results. Preferably, the particle size of the magnetic beads is 2.5 μm.

[0126] According to a specific embodiment of the present invention, when the mass ratio of the antibody to the magnetic beads is 1:(10-1000), the antibody-magnetic beads bonded at this ratio can further improve the detection sensitivity of the sample M protein. In the kit, the mass ratio of the antibody to the magnetic beads can be any value within the range of 1:(10-1000), for example, 1:10, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:1000, etc. Preferably, the mass ratio of the antibody to the magnetic beads is 1:100.

[0127] According to a specific embodiment of the present invention, the antigen-antibody complex dissociation reagent is not particularly limited, and all reagents that can be used to dissociate the antigen-antibody complex and have no effect on the antigen-antibody itself are covered within the scope of protection of the present invention. According to a specific embodiment of the present invention, the antigen-antibody complex dissociation reagent includes but is not limited to at least one of an organic acid solution, an inorganic acid solution, and an alkaline solution, and the organic acid solution includes at least one selected from formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, hydroxyacetic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid; the inorganic acid solution includes at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution; the alkaline solution includes at least one selected from sodium hydroxide solution, potassium hydroxide solution, trishydroxymethylaminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0128] According to a specific embodiment of the present invention, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution having a mass concentration of 0.05-5%.

[0129] According to a specific embodiment of the present invention, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions. Preferably, the reaction buffer is PBST solution.

[0130] According to a specific embodiment of the present invention, the kit further comprises a cleaning solution; the cleaning solution comprises at least one selected from PBS, PBST, and TBST solutions. Preferably, the cleaning solution is PBST solution.

[0131] It should be noted that when the reaction buffer and the cleaning solution in the kit are the same reagent, for example, both are PBST solutions, then the reagent in the kit can be used as both the reaction buffer and the cleaning solution.

[0132] According to a specific embodiment of the present invention, the kit further comprises a matrix solution for MALDI-TOF MS detection.

[0133] It should be noted that there are no particular limitations on the composition of the matrix liquid; all types of matrix liquids suitable for MALDI-TOF MS detection are encompassed within the scope of the present invention. According to a specific embodiment of the present invention, the matrix in the matrix liquid can be selected from at least one of sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracenetriol, and 3-indoleacrylic acid. According to a preferred embodiment of the present invention, the matrix in the matrix liquid is sinapinic acid.

[0134] According to a specific embodiment of the present invention, the present invention provides an M protein detection kit based on time-of-flight mass spectrometry technology, comprising:

[0135] Reagent components include magnetic beads bonded with IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, total κ ​​and total λ light chain-specific antibodies, as well as κ and λ free light chain-specific antibodies, sample diluent (reaction buffer), washing solution, eluent, matrix solution, and other reagent components.

[0136] According to a specific embodiment of the present invention, the present invention provides a method for detecting M protein in a sample, comprising using the aforementioned kit for detecting or typing M protein in a sample to detect the sample to be tested, so as to determine whether the sample to be tested contains complete M protein and / or free light chains, or to type the complete M protein and / or free light chains.

[0137] According to a specific embodiment of the present invention, the present invention provides a method for detecting M protein in a sample, the method comprising:

[0138] S1: contacting the test sample and the antibody-bound magnetic beads in a reaction buffer, so that the intact M protein and / or free light chains contained in the test sample specifically bind to the antibodies bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with the intact M protein and / or free light chains;

[0139] S2: performing an elution treatment and a magnetic separation treatment on the antigen-antibody complex magnetic beads to elute and separate the enriched intact M protein and / or free light chains from the antibody-bound magnetic beads to obtain a test solution containing the intact M protein and / or free light chains;

[0140] S3: Spotting the test solution on a sample chip, performing crystallization treatment, and detecting using MALDI-TOF MS to determine whether the test sample contains intact M protein and / or free light chains, or typing the intact M protein and / or free light chains.

[0141] Wherein, the antibody is the following antibody:

[0142] IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies;

[0143] Total kappa and total lambda light chain-specific antibodies;

[0144] and antibodies specific for kappa and lambda free light chains.

[0145] According to a specific embodiment of the present invention, in step S1, when contacting, the volume ratio of the sample to be tested to the reaction buffer is 1:(1-200).

[0146] According to a specific embodiment of the present invention, the mass concentration of the antibody-bound magnetic beads in the reaction buffer is 1-100 mg / mL. For example, the mass concentration of the antibody-bound magnetic beads in the reaction buffer can be 1 mg / mL, 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, etc.

[0147] According to a specific embodiment of the present invention, when contacting, the reaction time is 10-40 minutes and the reaction temperature is 20-35°C.

[0148] According to a specific embodiment of the present invention, in step S2, the elution treatment is performed using an elution solution. According to a specific embodiment of the present invention, the elution solution contains an antigen-antibody complex dissociation reagent, and the type of the antigen-antibody complex dissociation reagent is as described above.

[0149] According to a specific embodiment of the present invention, the method further comprises, before step S2, washing the antigen-antibody complex magnetic beads enriched with intact M protein and / or free light chains.

[0150] According to a specific embodiment of the present invention, the cleaning process is performed using a cleaning liquid, and the type of the cleaning liquid is as described above.

[0151] According to a specific embodiment of the present invention, step S3 further comprises mixing the test solution with a matrix solution, spotting the mixed solution on a sample chip, and performing a crystallization process. The type of matrix in the matrix solution is as described above.

[0152] According to a specific embodiment of the present invention, the volume ratio of the test solution and the matrix solution when mixed is 1:(1-10).

[0153] According to a specific embodiment of the present invention, the method comprises:

[0154] (1) when detecting whether the sample to be tested contains M protein, based on the MALDI-TOF MS detection result obtained in step S3, whether a peak signal exists within the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa, so as to determine whether the sample to be tested contains M protein; or

[0155] (2) When it is necessary to type the M protein contained in the sample to be tested, the type of the intact M protein and / or free light chain contained in the sample to be tested is determined based on the type of the antibody in the antibody-bound magnetic beads used in step S1 and the MALDI-TOF MS detection results obtained in step S3, and based on whether a peak signal exists within the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa.

[0156] Wherein, in case (1), in step S1, a dilution of the sample to be tested is prepared and divided into five portions, the first portion is contacted with magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies; the second and third portions are contacted with magnetic beads bonded to total κ ​​and total λ light chain specific antibodies, respectively; the fourth and fifth portions are contacted with magnetic beads bonded to κ and λ free light chain specific antibodies, respectively;

[0157] Alternatively, the test sample dilution solution is divided into three portions, the first portion is contacted with magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies; the second portion is contacted with magnetic beads bonded to total κ ​​and total λ light chain specific antibodies; and the third portion is contacted with magnetic beads bonded to κ and λ free light chain specific antibodies;

[0158] In case (2), in step S1, each type of antibody-bound magnetic beads is contacted with the sample to be tested respectively.

[0159] According to a specific embodiment of the present invention, the sample to be tested is a serum sample, a urine sample, a cerebrospinal fluid sample or a whole blood sample.

[0160] According to a specific embodiment of the present invention, the present invention provides a method for detecting M protein in a sample, comprising:

[0161] 1) Sample dilution: Take a certain amount of fresh urine or serum sample and dilute it with buffer solution by a certain multiple;

[0162] The urine sample volume can be selected from 5 μL to 100 μL, preferably 20 μL;

[0163] The serum sample volume can be selected from 5 μL to 100 μL, preferably 10 μL;

[0164] The buffer solution can be PBS, PBST, TBST solution, etc., preferably PBST;

[0165] The dilution multiple can be selected in the range of 1 to 200 times, preferably 20 times.

[0166] 2) Enrichment of intact immunoglobulins and FLC:

[0167] Prepare nine dilutions for the same sample. Add a specific amount of magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, magnetic beads bonded to total κ ​​and λ light chain-specific antibodies, and magnetic beads bonded to κ and λ FLC-specific antibodies to each dilution. Mix thoroughly and incubate for a period of time. Alternatively, prepare three dilutions for the same sample, one of which contains magnetic beads bonded to the five heavy chain-specific antibodies (IgA, G, D, E, and M), and the other two contain magnetic beads bonded to κ and λ FLC-specific antibodies. Alternatively, prepare five dilutions for the same sample, one of which contains magnetic beads bonded to the five heavy chain-specific antibodies (IgA, G, D, E, and M), two of which contain magnetic beads bonded to total κ ​​and total λ light chain-specific antibodies, and the other two contain magnetic beads bonded to κ and λ FLC-specific antibodies.

[0168] Each of the above antibodies can be in the form of a complete antibody or a fragment thereof, and can be a monoclonal or polyclonal complete antibody or a fragment thereof, preferably a polyclonal complete antibody;

[0169] The particle size of the antibody-bound magnetic beads can be in the range of 0.5 μm to 5 μm, preferably 2.5 μm; the surface modification groups of the magnetic beads can be selected from streptavidin, amino, carboxyl, epoxy, etc., preferably streptavidin magnetic beads;

[0170] The mass ratio of the antibody to the magnetic beads can be selected from 1 / 10 to 1 / 1000, preferably 1 / 100. At this ratio, the antibody magnetic beads have the best detection sensitivity for various types of M proteins in urine samples.

[0171] The mass range of the antibody-bound magnetic beads added to the sample diluent can be 0.01 mg to 1 mg, with the preferred addition amount being 0.1 mg;

[0172] The incubation time of the antibody-bound magnetic beads and the sample can be selected from 10 min to 40 min, preferably 20 min, and the incubation temperature can be selected from 20° C. to 35° C., preferably 25° C.

[0173] 3) Sample cleaning: The enriched magnetic beads and the sample diluent are magnetically separated, and the magnetic beads are washed multiple times. After the washing is completed, the magnetic beads and the washing solution are magnetically separated, and the washing solution is discarded.

[0174] The magnetic bead washing solution can be deionized water, PBS, PBST, TBST solution, etc. The washing times can be 3 to 5 times. The preferred washing steps are: washing twice with 600 μL PBST solution and washing once with 600 μL deionized water.

[0175] 4) Sample elution: Use a certain amount of eluent to elute the M protein enriched on the magnetic beads, perform magnetic separation, and remove the eluent.

[0176] The eluent may be trifluoroacetic acid aqueous solution, acetic acid aqueous solution, formic acid aqueous solution, etc., and the concentration range may be 0.1% to 5%, preferably 0.1% trifluoroacetic acid aqueous solution.

[0177] The amount of eluent added can be selected in the range of 5 μL to 50 μL, preferably 10 μL.

[0178] 5) Sample Reduction: Add a certain amount of reducing agent solution to the sample eluate enriched with magnetic beads bonded with IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, and magnetic beads bonded with total κ ​​and total λ light chain-specific antibodies. Incubate at room temperature for 10 minutes to reduce the disulfide bonds between the captured intact immunoglobulin heavy and light chains to obtain free immunoglobulin light chains.

[0179] The reducing agent solution may be selected from dithiothreitol, tris(2-carboxyethyl)phosphine, etc., preferably a tris(2-carboxyethyl)phosphine aqueous solution;

[0180] 6) Spotting: The above-mentioned reduced eluents, the final eluent, and the unreduced eluent are mixed with the matrix solution at a volume ratio of 1:1 and then spotted on the sample chip. After drying and crystallization, the sample is placed in the mass spectrometry system for collection.

[0181] The matrix liquid can be selected from sinapinic acid, α-cyano-4-hydroxycinnamic acid, dihydroxybenzoic acid, etc., preferably sinapinic acid.

[0182] Sample collection: Load the dried and crystallized sample chip into the mass spectrometer and set the mass spectrometer acquisition conditions, including: laser energy 5-25 μJ, detector voltage 0.4-0.7 kV, focus mass 20,000-25,000 Da, scan speed 0.5-1.5 mm / s, scan range 2,000-200,000 Da; linear positive ion mode.

[0183] Result interpretation: The automatic sample interpretation software determines whether the sample contains M protein based on the presence of a clear peak in the mass-to-charge ratio (m / z) range of 22kDa to 24.5kDa. The specific form and typing of the M protein are determined based on the type of antibody used for sample enrichment.

[0184] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0185] Example 1 Detection of M protein

[0186] 1. Sample Pretreatment

[0187] (1) Sample dilution: Take 20 μL of fresh urine sample and dilute it 20 times with PBST buffer;

[0188] Take 10 μL of fresh serum sample and dilute it 20 times with PBST buffer.

[0189] (2) Enrichment of intact immunoglobulins and FLC:

[0190] Prepare 9 dilutions for the same sample. Add a certain amount of magnetic beads bonded with IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, magnetic beads bonded with total κ ​​and total λ light chain-specific antibodies, and magnetic beads bonded with κ and λ FLC-specific antibodies to each dilution. Mix thoroughly and incubate for a period of time.

[0191] The aforementioned IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, total κ ​​and total λ light chain-specific antibodies, and κ and λ FLC-specific antibodies are polyclonal complete antibodies. The particle size of the magnetic beads bound to the antibodies is 2.5 μm; the surface modification group of the magnetic beads is streptavidin.

[0192] The mass ratio of antibody to magnetic beads is 1 / 100. At this ratio, the antibody magnetic beads have the best detection sensitivity for various types of M proteins in urine samples.

[0193] The amount of antibody-bound magnetic beads added to the sample diluent was 0.1 mg;

[0194] The antibody-bound magnetic beads were incubated with the sample for 20 min at a temperature of 25°C.

[0195] (3) Sample cleaning: The enriched magnetic beads and the sample diluent are magnetically separated, and the magnetic beads are washed multiple times. After the washing is completed, the magnetic beads and the washing solution are magnetically separated, and the washing solution is discarded.

[0196] The cells were washed twice with 600 μL of PBST solution and once with 600 μL of deionized water.

[0197] (4) Sample elution: Use 10 μL of elution solution 0.1% trifluoroacetic acid aqueous solution to elute the M protein enriched on the magnetic beads, perform magnetic separation, and remove the eluate.

[0198] (5) Sample reduction: Add a certain amount of reducing agent solution, i.e., tris(2-carboxyethyl)phosphine aqueous solution, to the sample eluate enriched with magnetic beads bonded with IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, and magnetic beads bonded with total κ ​​and total λ light chain-specific antibodies, and incubate at room temperature for 10 minutes to reduce the disulfide bonds between the captured intact immunoglobulin heavy and light chains to obtain free immunoglobulin light chains.

[0199] (6) Spotting: The reduced eluents and the unreduced eluents were mixed with the matrix solution at a volume ratio of 1:1 and then spotted on the sample chip. After drying and crystallization, the sample was placed in the mass spectrometry system for collection. The matrix solution was sinapinic acid.

[0200] 2. Sample collection:

[0201] The dried and crystallized sample chip was loaded into the mass spectrometer, and the mass spectrometer acquisition conditions were set, including: laser energy 10 μJ, detector voltage 0.5 kV, focus mass 22000 Da, scanning speed 1.0 mm / s, scanning range 2000-200000 Da; linear positive ion mode.

[0202] 3. Interpretation of results:

[0203] The automatic sample interpretation software determines whether the sample contains M protein based on the presence of a clear peak in the mass-to-charge ratio (m / z) range of 22kDa to 24.5kDa, and interprets the specific form and typing of the M protein based on the antibody type information used for sample enrichment.

[0204] Example 2

[0205] Ten monoclonal FLC-negative urine samples were mixed and serially diluted with 1xPBS at dilution ratios of 2, 10, and 100, respectively. 20 μL of the original mixture and the three dilutions were added to 380 μL of PBST. Each dilution was enriched with 0.1 mg of κ-FLC-specific antibody magnetic beads bound to 1 / 10, 1 / 100, and 1 / 1000 antibody magnetic beads (the affinity constant KD value of the κ-FLC-specific antibody was 10,000 pM). Subsequent sample washing, sample elution, and spot testing were performed (for the detection steps, refer to Example 1). Figure 1a 、 1b 1c and 1d respectively show the enrichment ability of κ-type FLC-specific antibody magnetic beads bonded with antibody magnetic beads at a mass ratio of 1 / 10, 1 / 100, and 1 / 1000 for FLC in negative urine samples. Among them, sample enrichment using antibody magnetic beads at a mass ratio of 1 / 100 can detect FLC in urine diluted at least 10 times.

[0206] Ten monoclonal FLC-negative urine samples were selected and mixed and packaged. Different concentrations of monoclonal κ-FLC were added to prepare urine samples containing 0.01 mg / mL, 0.05 mg / mL, and 0.1 mg / mL monoclonal κ-FLC. 20 μL of each dilution was added to 380 μL PBST. Each dilution was enriched with 0.1 mg of κ-FLC-specific antibody magnetic beads bonded at a 1 / 100 antibody magnetic bead mass ratio. Subsequent sample washing, sample elution, and spot testing were performed. Figure 2 The results showed that the present method has the ability to detect monoclonal FLC in urine samples as low as 0.01 mg / mL.

[0207] Example 3 Detection of Urine M Protein in Patients with Monoclonal Gammopathy

[0208] A urine sample with a clinical uIFE test result of FLC negative and intact M protein positive was tested. After the sample was diluted in separate tubes, it was enriched using five heavy chain antibodies in separate wells or separate wells, two total light chain antibodies, and two FLC antibodies in separate wells, and subsequent sample cleaning, sample elution and spot testing were performed (refer to Example 1). Among them, the ratio of magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, magnetic beads bonded to total κ ​​and total λ light chain-specific antibodies, and magnetic beads bonded to κ and λ free light chain-specific antibodies was 1:1:1:1:1:1:1:1:1:1.

[0209] Figure 3aFigure 3b and 3c are the results of the combined test of five heavy chain antibodies on this sample. The results show that this sample is positive for the complete M protein and weakly positive for the λ-type FLC monoclonal. Figure 3d This is the specific typing result of the intact M protein in this sample, which was positive for IgG-λ M protein and weakly positive for IgA-λ M protein. Compared with the results given by clinical uIFE, this protocol can provide specific detection (screening) and typing results for both free and intact M protein in this urine sample.

[0210] Example 4 Comparative test of 10 clinical urine samples tested by uIFE

[0211] Ten urine samples collected after clinical uIFE testing were tested for M protein using this protocol, and the comparison results of the two methods are shown in Table 1. Among the 10 samples, there were 7 samples with completely consistent results between the detection method of the present invention and the uIFE test results, and the remaining 3 samples had a higher probability of detecting M protein using the protocol of the present invention (referred to as "this protocol" in Table 1), and this protocol can give specific typing results for the intact M protein.

[0212] Table 1

[0213]

[0214] Example 5 Detection of serum M protein in patients with monoclonal immunoglobulin

[0215] A serum sample that was IgD positive and FLC monoclonal negative by clinical uIFE and immunoturbidimetric testing was tested. After the sample was diluted in different tubes, it was enriched in different wells using five heavy chain antibodies, two total light chain antibodies, and two FLC antibodies, and subsequent sample washing, sample elution and spot testing were performed. Figure 4a This is the specific typing result of the complete M protein of the sample in this protocol. The test result is IgD-λ type M protein positive. Figure 4b This is an overlay of the results of the well detection of IgD heavy chain antibodies and two total light chain antibodies. The typing of the complete M protein of the serum can be clearly confirmed from the figure. Figure 4c This is an overlay of the results of two FLC antibody tests for this sample, which showed positive results for the monoclonal λ-type FLC. Compared to the results obtained by clinical uIFE, this protocol can provide specific detection (screening) and typing results for both free and intact M proteins in this serum sample.

[0216] Example 6 Comparative testing of 8 clinical serum samples tested by uIFE and immunoturbidimetry

[0217] Eight serum samples collected after clinical uIFE and immunoturbidimetric testing were tested for M protein using this protocol. The comparison results of the two methods are shown in Table 2. Among the eight samples, the results of two samples were completely consistent with those of the uIFE test. For the remaining six samples, the probability of detecting FLC-type M protein was higher with this protocol. In addition, this protocol can provide specific typing results including light chains for IgD-type M protein.

[0218] Table 2

[0219]

[0220] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0221] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting or typing M protein in a sample, characterized in that: include: Antibody-bound magnetic beads; elution solution; Reaction buffer; Wherein, the antibody is the following antibody: IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies; Total kappa and total lambda light chain-specific antibodies; and antibodies specific for kappa and lambda free light chains, The elution solution contains an antigen-antibody complex dissociation reagent, The mass ratio of the antibody to the magnetic beads is 1:(10-1000), The particle size of the magnetic beads is 0.5-5 μm.

2. The kit according to claim 1, wherein The IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies are specific anti-human IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies or fragments thereof; The total κ ​​type and total λ type light chain specific antibodies are specific anti-human total κ ​​type and total λ type light chain specific antibodies or fragments thereof; The κ-type and λ-type free light chain specific antibodies are specific anti-human κ-type or λ-type free light chain antibodies or fragments thereof; Optionally, the antibody is a monoclonal antibody or a polyclonal antibody or a fragment thereof; Optionally, the antibody is a polyclonal antibody; Optionally, the affinity constant KD value of the IgA, IgG, IgD, IgE, or IgM heavy chain-specific antibody is 100-100,000 pM; The affinity constant KD value of the total κ ​​type and total λ type light chain specific antibody is 100-100000 pM; The affinity constant KD value of the κ-type and λ-type free light chain-specific antibodies is 100-100000 pM; Optionally, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:1:(1-10):

1.

3. The kit according to claim 1, wherein The surface of the magnetic beads is modified with proteins or chemical groups, and the proteins or chemical groups are used to bind to the antibodies; Optionally, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups; Optionally, the protein is streptavidin; Optionally, when the surface of the magnetic beads is modified with streptavidin, the antibody is linked to biotin.

4. The kit according to claim 1, wherein The antigen-antibody complex dissociation reagent includes at least one selected from an organic acid solution, an inorganic acid solution, and an alkaline solution; The organic acid solution comprises at least one selected from the group consisting of formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid; The inorganic acid solution comprises at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution; The alkaline solution includes at least one selected from the group consisting of sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution; Optionally, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution; Optionally, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

5. The kit according to claim 1, wherein The reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions; Optionally, the reaction buffer is PBST solution.

6. The kit according to claim 1, wherein The kit further comprises a cleaning solution; Optionally, the cleaning solution comprises at least one selected from PBS, PBST, and TBST solutions; Optionally, the cleaning solution is PBST solution.

7. The kit according to claim 1, wherein The kit further comprises a matrix solution for MALDI-TOFMS detection; Optionally, the matrix in the matrix solution includes at least one selected from the group consisting of sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracenetriol, and 3-indoleacrylic acid; Optionally, the matrix in the matrix solution is sinapinic acid.

8. A method for detecting M protein in a sample, characterized in that: The method comprises using the kit for detecting or typing M protein in a sample according to any one of claims 1 to 7 to detect the sample to be tested, so as to determine whether the sample to be tested contains complete M protein and / or free light chains, or to type the complete M protein and / or free light chains.

9. A method for detecting M protein in a sample, characterized in that: include: S1: contacting the test sample and the antibody-bound magnetic beads in a reaction buffer, so that the intact M protein and / or free light chains contained in the test sample specifically bind to the antibodies bound to the magnetic beads, thereby obtaining antigen-antibody complex magnetic beads enriched with the intact M protein and / or free light chains; S2: performing an elution treatment and a magnetic separation treatment on the antigen-antibody complex magnetic beads to elute and separate the enriched intact M protein and / or free light chains from the antibody-bound magnetic beads to obtain a test solution containing the intact M protein and / or free light chains; S3: Spotting the test solution on a sample chip, performing crystallization treatment, and detecting using MALDI-TOF MS to determine whether the test sample contains intact M protein and / or free light chains, or typing the intact M protein and / or free light chains. Wherein, the antibody is the following antibody: IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies; Total kappa and total lambda light chain-specific antibodies; and antibodies specific for kappa and lambda free light chains.

10. The method according to claim 9, characterized in that In the antibody-bound magnetic beads, the mass ratio of the antibody to the magnetic beads is 1:(10-1000); Optionally, the particle size of the magnetic beads is 0.5-5 μm; Optionally, the surface of the magnetic beads is modified with proteins or chemical groups, and the proteins or chemical groups are used to bind to the antibodies; Optionally, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups; Optionally, the protein is streptavidin; Optionally, when the surface of the magnetic beads is modified with streptavidin, the antibody is linked to biotin; Optionally, the IgA, IgG, IgD, IgE, or IgM heavy chain specific antibody is a specific anti-human IgA, IgG, IgD, IgE, or IgM heavy chain specific antibody or a fragment thereof; The total κ ​​type and total λ type light chain specific antibodies are specific anti-human total κ ​​type and total λ type light chain specific antibodies or fragments thereof; The κ-type and λ-type free light chain specific antibodies are specific anti-human κ-type or λ-type free light chain antibodies or fragments thereof; Optionally, the antibody is a monoclonal antibody or a polyclonal antibody or a fragment thereof; Optionally, the antibody is a polyclonal antibody; Optionally, the affinity constant KD value of the IgA, IgG, IgD, IgE, or IgM heavy chain-specific antibody is 100-100,000 pM; The affinity constant KD value of the total κ ​​type and total λ type light chain specific antibody is 100-100000 pM; The affinity constant KD value of the κ-type and λ-type free light chain-specific antibodies is 100-100000 pM; Optionally, in the kit, the ratio of the magnetic beads bonded to the IgA, IgG, IgD, IgE, and IgM heavy chain-specific antibodies, the magnetic beads bonded to the total κ ​​and total λ light chain-specific antibodies, and the magnetic beads bonded to the κ and λ free light chain-specific antibodies is 1:1:1:1:1:1:1:(1-10):1; Optionally, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions; Optionally, the reaction buffer is PBST solution.

11. The method according to claim 9, characterized in that In step S1, when contacting, the volume ratio of the sample to be tested to the reaction buffer is 1:(1-200); Optionally, in the reaction buffer, the mass concentration of the antibody-bound magnetic beads is 1-100 mg / mL; Optionally, when contacting, the reaction time is 10-40 minutes and the reaction temperature is 20-35°C.

12. The method according to claim 9, characterized in that In step S2, the elution treatment is performed using an elution solution; Optionally, the elution solution contains an antigen-antibody complex dissociation reagent, Optionally, the antigen-antibody complex dissociation reagent comprises at least one selected from an organic acid solution, an inorganic acid solution, and an alkaline solution; The organic acid solution comprises at least one selected from the group consisting of formic acid solution, acetic acid solution, trifluoroacetic acid solution, propionic acid solution, glycolic acid solution, amino acid solution, butyric acid solution, citric acid, succinic acid, ascorbic acid, lactic acid, malic acid, and tartaric acid; The inorganic acid solution comprises at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution; The alkaline solution includes at least one selected from the group consisting of sodium hydroxide solution, potassium hydroxide solution, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution; Optionally, the antigen-antibody complex dissociation reagent is a trifluoroacetic acid solution; Optionally, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

13. The method according to claim 11 or 12, characterized in that The method further comprises, before step S2, washing the antigen-antibody complex magnetic beads enriched with intact M protein and / or free light chains; Optionally, the cleaning process is performed using a cleaning liquid; Optionally, the cleaning solution comprises at least one selected from PBS, PBST, and TBST solutions; Optionally, the cleaning solution is PBST solution.

14. The method according to claim 9, characterized in that Step S3 further includes mixing the test solution with a matrix solution, spotting the mixed solution on a sample chip, and performing a crystallization process; Optionally, the matrix in the matrix solution includes at least one selected from the group consisting of sinapinic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthracenetriol, and 3-indoleacrylic acid; Optionally, the matrix in the matrix solution is sinapinic acid; Optionally, the volume ratio of the test solution and the matrix solution when mixed is 1:(1-10).

15. The method according to claim 9, characterized in that The method comprises: (1) when detecting whether the sample to be tested contains M protein, based on the MALDI-TOF MS detection result obtained in step S3, whether a peak signal exists within the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa, so as to determine whether the sample to be tested contains M protein; or (2) When it is necessary to type the M protein contained in the sample to be tested, according to the type of the antibody in the magnetic beads bonded to the antibody used in step S1, based on the MALDI-TOF MS detection result obtained in step S3, according to whether there is a peak signal in the mass-to-charge ratio m / z range of 22 kDa to 24.5 kDa, so as to determine the type of the intact M protein and / or free light chain contained in the sample to be tested, wherein, in case (1), in step S1, a dilution of the sample to be tested is prepared and divided into five parts, the first part is contacted with magnetic beads bonded with IgA type, IgG type, IgD type, IgE type, and IgM type heavy chain specific antibodies; the second and third parts are contacted with magnetic beads bonded with total κ ​​type and total λ type light chain specific antibodies, respectively; the fourth and fifth parts are contacted with magnetic beads bonded with κ type and λ type free light chain specific antibodies, respectively; Alternatively, the dilution of the test sample is divided into three portions, the first portion is contacted with magnetic beads bonded to IgA, IgG, IgD, IgE, and IgM heavy chain specific antibodies; the second portion is contacted with magnetic beads bonded to total κ ​​and total λ light chain specific antibodies; and the third portion is contacted with magnetic beads bonded to κ and λ free light chain specific antibodies; In case (2), in step S1, each type of antibody-bound magnetic beads is contacted with the sample to be tested respectively.

16. The method according to claim 9, characterized in that The sample to be tested is a serum sample, a urine sample, a cerebrospinal fluid sample or a whole blood sample.

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