A method for detecting endogenously synthesized immunoglobulins in the central nervous system based on MALDI-TOF MS

Abstract

The application belongs to the technical field of in-vitro detection, and particularly relates to a detection method of endogenous synthesis immunoglobulin in central nervous system based on MALDI-TOF MS. The application provides a kit for detecting and typing immunoglobulin in a sample and a method for detecting immunoglobulin in a sample, and solves the problems of low detection method flux, complicated experimental process, long time consumption, high experience requirement for detection personnel, and result interpretation being easily affected by electropherogram background of isoelectric focusing electrophoresis combined with immunofixed oligoclonal band electrophoresis (OCB) technology. The detection method provided by the application is more accurate, simpler to operate and easier to realize automation, has no subjective problems of manual interpretation, has lower detection cost, has higher detection flux, and is easier to popularize in clinical diagnosis and treatment.

Description

Technical Field

[0001] This invention relates to the field of in vitro detection technology, specifically to a method for detecting endogenous synthetic immunoglobulins in the central nervous system based on MALDI-TOF MS. Background Technology

[0002] Cerebrospinal fluid (CSF) is a transparent fluid found in the ventricular system and subarachnoid space. Secreted by the choroid plexus of the ventricles, it primarily functions as a buffer, nutrient transport, and waste removal agent. It surrounds and supports the brain and spinal cord, maintaining stable intracranial pressure and acting as a crucial "fluid barrier" for the central nervous system (CNS). The total protein content of CSF is approximately 0.15–0.45 g / L, with 80% of the protein originating from blood plasma. This protein primarily enters the CSF via ultrafiltration across the blood-brain barrier (BBB). The remaining proteins are synthesized and secreted by the choroid plexus epithelial cells, glial cells, and neurons within the CNS, and drain into the CSF through interstitial fluid drainage. Mass spectrometry-based proteomics analysis revealed that CSF contains thousands of proteins, mainly including high-abundance proteins such as albumin, immunoglobulins, α1-globulin, α2-globulin, and β-globulin, as well as low-abundance specific proteins such as prealbumin, β-amyloid, neurofilament protein, myelin basic protein, and S100 protein (Yang Linpeng, Fan Pengcheng, Jin Wanjun, et al. Research progress on proteomics technology and clinical application of cerebrospinal fluid [J]. Chinese Journal of Biotechnology, 2019, 35(09):1643-1649). Among them, immunoglobulins (Ig) are mainly derived from plasma ultrafiltration under normal physiological conditions, with a very small portion being synthesized endogenously within the sheath. However, under pathological conditions, the immune response of the CNS leads to abnormal expression of endogenous Ig, and the types and contents of Ig in CSF change. Previous studies have shown that abnormal expression of endogenous Ig is associated with various CNS diseases such as multiple sclerosis (MS), central nervous system infections, and autoimmune encephalitis (Niu Zhenyu, Jin Haiqiang, Hao Hongjun, et al. Analysis of oligoclonal band detection results in 3217 patients with neurological diseases [J]. Chinese Journal of Laboratory Medicine, 2024, 47(1):57-64). Furthermore, in clinical diagnosis, it has been found that changes in IgG in CSF of patients with CNS lesions generally precede abnormalities in imaging examinations. Therefore, the detection of endogenous synthetic Ig in CSF has important clinical value in the early diagnosis, treatment, and prognosis of CNS diseases.

[0003] Isoelectric focusing electrophoresis (IFE) combined with immunofixation oligoclonal band electrophoresis (OCB) is currently the gold standard for clinical detection of endogenous Ig synthesis in CSF. This method first separates proteins in the sample based on their isoelectric point differences. Then, labeled anti-human serum-IgG antibodies are added, and the specific binding of these antibodies identifies IgG proteins. Finally, the presence of endogenous IgG synthesis in CSF is determined by comparing the electrophoretic spectra of paired serum and CSF samples. In summary, this detection method has low throughput, is cumbersome and time-consuming, requires highly experienced personnel, and its results are easily affected by background interference from the electrophoretic spectra. Furthermore, it only reflects the synthesis of IgG immunoglobulins.

[0004] Therefore, there is an urgent need to develop a simple, time-saving method for detecting endogenous synthetic immunoglobulins in cerebrospinal fluid that reduces reliance on operators. Summary of the Invention

[0005] This invention aims to at least partially address one of the technical problems in related technologies. Therefore, one objective of this invention is to provide a kit and method for the detection and typing of endogenous immunoglobulins expressed in the central nervous system based on MALDI-TOF MS. This kit and method will solve the problems of long detection time, low throughput, and lack of typing in traditional detection methods, while providing simpler and more intuitive spectrum discrimination, reducing reliance on operators, and improving the accuracy of detection results. Addressing the limitation of clinical methods that only detect IgG, we have added IgA and IgM antibodies, enabling subtype discrimination while detecting endogenous Ig synthesis.

[0006] Therefore, in a first aspect, the present invention provides a kit for the detection and typing of endogenous immunoglobulins expressed in the central nervous system based on MALDI-TOF MS, comprising: Magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibodies; Reaction buffer solution; Elution solution; reducing agent; Matrix solution; The mass ratios of the anti-human immunoglobulin heavy chain IgG antibody, anti-human immunoglobulin heavy chain IgA antibody, and anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads are each independently 1:(20-200). The magnetic beads have a particle size of 0.2-5 μm.

[0007] Currently, the widely used clinical method of isoelectric focusing electrophoresis (IFE) combined with immunofixation has limitations such as low throughput, cumbersome and time-consuming experimental procedures, high requirements for the experience of the testing personnel, susceptibility of result interpretation to background interference from electrophoretic spectra, and the ability to only reflect the synthesis of IgG immunoglobulins. To address these issues, this invention provides a kit based on MALDI-TOF MS for the detection and typing of endogenous immunoglobulins expressed in the central nervous system. This kit leverages the high throughput and high sensitivity of MS to solve the problems of long processing times and low throughput associated with traditional methods. Furthermore, it offers simpler and more intuitive spectra interpretation, reduces reliance on operator skills, and improves the accuracy of the test results. Addressing the limitation of clinical methods that only detect IgG, the kit of this invention enables mass spectrometry detection of IgG, IgA, and IgM subtypes simultaneously. It can distinguish subtypes while detecting endogenous Ig synthesis, and the MALDI-TOF mass spectrum provides a more direct and richer reflection of the protein content in the sample, offering multiple data supports for the accurate classification of CNS-related diseases and the study of their pathogenesis.

[0008] According to an embodiment of the present invention, the affinity constants (KD) of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 9.9 × 10⁻⁶. -12 M-1×10 -9 M.

[0009] According to an embodiment of the present invention, the surface of the magnetic beads is modified with chemical groups, which are used to bind to the antibody.

[0010] According to embodiments of the present invention, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups.

[0011] According to an embodiment of the present invention, the elution solution comprises at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions; The organic acid solution includes at least one selected from 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 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, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0012] According to a preferred embodiment of the present invention, the elution solution is a trifluoroacetic acid solution.

[0013] According to an embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.05-5%.

[0014] According to an embodiment of the present invention, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions.

[0015] According to an embodiment of the present invention, the reaction buffer is a PBST solution.

[0016] According to an embodiment of the present invention, the kit further includes a cleaning solution.

[0017] According to an embodiment of the present invention, the cleaning solution includes at least one selected from PBS, PBST, and TBST solutions.

[0018] According to an embodiment of the present invention, the cleaning solution is a PBST solution.

[0019] According to an embodiment of the present invention, the reducing agent includes at least one selected from TCEP, DTT, β-mercaptoethanol, and glutathione.

[0020] According to an embodiment of the present invention, the reducing agent is TCEP.

[0021] According to an embodiment of the present invention, the matrix in the matrix solution includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid.

[0022] According to an embodiment of the present invention, the matrix solution is sinapic acid; according to a specific embodiment of the present invention, the matrix solution is a 20 mg / mL sinapic acid solution.

[0023] A second aspect of the present invention provides a method for detecting endogenous immunoglobulins expressed in the central nervous system. The method includes using the kit described in the first aspect to detect and classify a sample to be tested in order to determine whether the central nervous system abnormally expresses endogenous immunoglobulins and the subtypes of the expressed endogenous immunoglobulins, wherein the sample to be tested is a cerebrospinal fluid sample.

[0024] A third aspect of the present invention provides a method for detecting endogenous immunoglobulins expressed in the central nervous system. According to embodiments of the present invention, the method includes: S1: The sample to be tested is divided into three parts, and each part is contacted with magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibody, magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibody, and magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibody in a reaction buffer, so that the IgG, IgA and IgM contained in the sample to be tested can specifically bind to the antibodies bonded to the magnetic beads, and three antigen-antibody complex magnetic beads are obtained. S2: The three portions of antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched IgG, IgA and IgM from the antibodies, and obtain a test solution containing IgG, IgA and IgM. S3: The test solution is reduced using a reducing agent in order to break down the immunoglobulins; S4: The test solution after reduction treatment is mixed with the matrix solution and spotted onto the sample chip for crystallization. MALDI-TOF MS is then used for detection to determine whether the central nervous system abnormally expresses the endogenous immunoglobulin and the subtype of the expressed endogenous immunoglobulin. In the antibody-bonded magnetic beads, the mass ratio of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads is independently 1:(20-200); The magnetic beads have a particle size of 0.2-5 μm; The sample to be tested is a cerebrospinal fluid sample.

[0025] The detection method provided by this invention can simultaneously detect IgG, IgA, and IgM subtypes, enabling high-throughput and rapid detection of the presence of immunoglobulin synthesis and its subtypes in cerebrospinal fluid. This allows for the determination of whether the central nervous system is abnormally expressing endogenous immunoglobulins. MALDI-TOF mass spectrometry provides a more direct and richer picture of the proteins in the sample, offering various data supports for the precise classification of central nervous system-related diseases and the study of their pathogenesis.

[0026] According to an embodiment of the present invention, the surface of the magnetic beads is modified with chemical groups, which are used to bind to the antibody.

[0027] According to embodiments of the present invention, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups.

[0028] According to an embodiment of the present invention, the affinity constants (KD) of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 9.9 × 10⁻⁶. -12M-1×10 -9 M.

[0029] According to an embodiment of the present invention, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions.

[0030] According to an embodiment of the present invention, the reaction buffer is a PBST solution.

[0031] According to an embodiment of the present invention, in step S1, when contact is performed, the volume ratio of the sample to be tested to the reaction buffer is 1:(20-100).

[0032] According to an embodiment of the present invention, in the reaction buffer, the mass concentrations of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 0.003-0.01 mg / mL.

[0033] According to an embodiment of the present invention, the reaction time during contact is 10-40 min and the reaction temperature is 20-30°C.

[0034] According to an embodiment of the present invention, in step S2, the elution process is performed using an elution solution.

[0035] According to an embodiment of the present invention, the elution solution contains an antigen-antibody complex dissociation reagent.

[0036] According to embodiments of the present invention, the antigen-antibody complex dissociation reagent includes at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions; The organic acid solution includes at least one selected from 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 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, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution.

[0037] According to an embodiment of the present invention, the antigen-antibody complex dissociation reagent is preferably a trifluoroacetic acid solution.

[0038] According to an embodiment of the present invention, the mass concentration of the trifluoroacetic acid solution is 0.05-5%, preferably 0.1%.

[0039] According to an embodiment of the present invention, the method further includes cleaning the antigen-antibody complex magnetic beads before step S2.

[0040] According to an embodiment of the present invention, the cleaning process is performed using a cleaning solution.

[0041] According to an embodiment of the present invention, the cleaning solution includes at least one selected from PBS, PBST, and TBST solutions.

[0042] According to an embodiment of the present invention, the cleaning solution is preferably a PBST solution.

[0043] According to an embodiment of the present invention, the reducing agent includes at least one selected from TCEP, DTT, β-mercaptoethanol, and glutathione.

[0044] According to a preferred embodiment of the present invention, the reducing agent is preferably TCEP.

[0045] According to an embodiment of the present invention, the matrix in the matrix solution includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid.

[0046] According to an embodiment of the present invention, the matrix solution is preferably sinapic acid, and the matrix solution is more preferably a 20 mg / mL sinapic acid solution.

[0047] According to an embodiment of the present invention, the volume ratio of the test solution after the reduction treatment to the matrix solution is 1:(1-10).

[0048] According to an embodiment of the present invention, the method further includes: (1) Using the method described above for detecting immunoglobulins in a sample, immunoglobulins from serum samples and cerebrospinal fluid samples from the same individual are detected simultaneously to obtain mass spectra of serum samples and cerebrospinal fluid samples. (2) Compare the mass spectra of the serum sample and the cerebrospinal fluid sample. If the mass spectra peaks of the two samples in the range of 20,000-30,000 m / z are inconsistent and there is a "sharp peak" in the cerebrospinal fluid sample, it is determined that the cerebrospinal fluid sample has endogenous immunoglobulin synthesis. Based on the different detection antibodies, the subtype of the synthesized endogenous immunoglobulin can be determined.

[0049] According to an embodiment of the present invention, immunoglobulins in the sample to be tested are detected using a QuanTOF mass spectrometer.

[0050] According to an embodiment of the present invention, the parameters for detection using a QuanTOF mass spectrometer are as follows: spectrum storage range of m / z 5000~100000, focusing mass of 23000 Da, detection voltage of -0.59 kV, and laser energy of 9.4 μJ.

[0051] Beneficial effects: Currently, the widely used clinical method of isoelectric focusing electrophoresis (IFE) combined with immunofixation has limitations such as low throughput, cumbersome and time-consuming experimental procedures, high requirements for the experience of the testing personnel, susceptibility of result interpretation to background interference from electrophoretic spectra, and the ability to only reflect IgG immunoglobulin synthesis. To address these issues, this invention provides a method for detecting endogenous synthetic immunoglobulins in cerebrospinal fluid based on MALDI-TOF MS. This method utilizes the high throughput and high sensitivity of MS to solve the problems of long processing times and low throughput in traditional detection methods. Furthermore, the spectra interpretation is simpler and more intuitive, reducing reliance on operator skills and improving the accuracy of the results. Addressing the limitation of clinical methods that only detect IgG, mass spectrometry can simultaneously detect IgG, IgA, and IgM subtypes. It can distinguish subtypes while detecting endogenous Ig synthesis, and MALDI-TOF mass spectra provide a more direct and richer reflection of the protein composition in the sample, offering multiple data supports for precise subtyping and pathogenesis research of CNS-related diseases.

[0052] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figures 1-3 These are MALDI-TOF mass spectra of serum and cerebrospinal fluid IgG antibody detection in samples S / C-1~3; Figures 4-7 These are MALDI-TOF mass spectra of serum and cerebrospinal fluid samples for IgG antibody detection. Figures 8-9 This is a MALDI-TOF mass spectrum of abnormally synthesized endogenously IgG; Figures 10-12 These are MALDI-TOF mass spectra of serum and cerebrospinal fluid samples for IgA and IgM antibody detection. Detailed Implementation

[0054] 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 construed as limiting the present invention.

[0055] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0056] The endpoints and any values ​​of the ranges 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 endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0057] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0058] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0059] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0060] Reagent test kit According to a specific embodiment of the present invention, the present invention provides a kit for detecting and typing endogenous immunoglobulins expressed in the central nervous system based on MALDI-TOF MS, comprising: Magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibodies; Reaction buffer solution; Elution solution; reducing agent; Matrix solution; The mass ratios of the anti-human immunoglobulin heavy chain IgG antibody, anti-human immunoglobulin heavy chain IgA antibody, and anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads are each independently 1:(20-200). The magnetic beads have a particle size of 0.2-5 μm.

[0061] According to a specific embodiment of the present invention, the affinity constants (KD values) of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 9.9 × 10⁻⁶. -12 M-1×10 -9 M. Normally, if abnormally expressed immunoglobulins are present in the central nervous system, their content is low. This invention uses antibodies with strong affinity for enrichment, which can significantly improve the enrichment rate, thereby improving detection sensitivity and the accuracy of detection results.

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

[0063] According to a specific embodiment of the present invention, the surface of the magnetic beads is modified with chemical groups, which are used to bind to the antibody. It should be noted that there are no particular limitations on the chemical groups; all groups suitable for in vitro detection and used for magnetic bead linkage are covered within the scope of this invention. For example, the chemical groups include, but are not limited to, at least one of amino, carboxyl, epoxy, and hydroxyl groups. The present invention preferably uses carboxyl magnetic beads, thereby improving the antibody binding rate.

[0064] According to a specific embodiment of the present invention, the particle size of the magnetic beads in the kit is 0.2-5 μm. For example, in the kit, the particle size of the magnetic beads can be any value within the range of 0.2-5 μm, including but not limited to 0.2 μm, 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.

[0065] This invention preferably uses carboxyl magnetic beads with a particle size of 3.0 μm. The relatively large surface curvature of these beads allows for more upright antibody binding, resulting in less steric hindrance and better utilization of active sites, thus improving the antigen-antibody binding law. Furthermore, the moderate dispersion of these beads reduces sedimentation, leading to a more uniform binding system and a higher antibody binding rate. This enhances the efficiency and ability of antibody enrichment for immunoglobulins, making it more suitable for cerebrospinal fluid samples with low immunoglobulin content. Additionally, selecting magnetic beads of this size results in higher efficiency and less loss during subsequent magnetic separation.

[0066] According to a specific embodiment of the present invention, the mass ratio of antibody to magnetic beads in the kit is 1:(20-200). For example, the mass ratio of antibody to magnetic beads in the kit can be 1:20, 1:30, 1:50, 1:100, 1:150, 1:200, etc. Preferably, the mass ratio of antibody to magnetic beads is 1:50, which results in the highest conjugation efficiency, reasonable antibody orientation, and best activity retention, while avoiding antibody waste and increased costs.

[0067] According to a specific embodiment of the present invention, the elution solution is not particularly limited, and all reagents capable of dissociating antigen-antibody complexes without affecting the antigen and antibody themselves are included within the scope of protection of the present invention. According to a specific embodiment of the present invention, the elution solution includes, but is not limited to, at least one of organic acid solutions, inorganic acid solutions, and alkaline solutions. The organic acid solutions include at least one selected from 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 solutions include at least one selected from hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution. The alkaline solutions include 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.

[0068] According to a specific embodiment of the present invention, the elution solution is a trifluoroacetic acid solution. The mass concentration of the trifluoroacetic acid solution is 0.05-5%. Preferably, the present invention uses a 0.1% trifluoroacetic acid solution, thereby achieving higher antigen-antibody dissociation efficiency.

[0069] According to a specific embodiment of the present invention, the reaction buffer includes at least one selected from PBS, PBST, and TBST solutions, preferably, the reaction buffer is a PBST solution.

[0070] According to a specific embodiment of the present invention, the kit further includes a washing solution; the washing solution includes at least one selected from PBS, PBST, and TBST solutions. Preferably, the washing solution is a PBST solution.

[0071] It should be noted that when the reaction buffer and washing solution in the kit are the same reagent, such as PBST solution, the reagent in the kit can be used as both the reaction buffer and the washing solution.

[0072] According to a specific embodiment of the present invention, the reducing agent includes at least one selected from TCEP, DTT, β-mercaptoethanol, and glutathione. Preferably, the reducing agent is TCEP.

[0073] According to a specific embodiment of the present invention, the matrix in the matrix solution may be selected from at least one of sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid.

[0074] According to a specific embodiment of the present invention, the matrix solution is preferably sinapic acid, and more preferably a 20 mg / mL sinapic acid solution. This allows for better protein ionization and higher dissociation efficiency. Furthermore, using this matrix can significantly improve the protein detection signal, thereby enhancing detection sensitivity.

[0075] According to a specific embodiment of the present invention, the present invention provides a method for detecting endogenous immunoglobulins expressed in the central nervous system, comprising using the aforementioned kit to detect a sample to be tested in order to determine whether the central nervous system expresses endogenous immunoglobulins and the expressed endogenous immunoglobulin subtypes, wherein the sample to be tested is a cerebrospinal fluid sample.

[0076] According to a specific embodiment of the present invention, the present invention provides a method for detecting endogenous immunoglobulins expressed in the central nervous system, comprising: S1: The sample to be tested is divided into three parts, and each part is contacted with magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibody, magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibody, and magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibody in a reaction buffer, so that the IgG, IgA and IgM contained in the sample to be tested can specifically bind to the antibodies bonded to the magnetic beads, and three antigen-antibody complex magnetic beads are obtained. S2: The three portions of antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched IgG, IgA and IgM from the antibodies, and obtain a test solution containing IgG, IgA and IgM. S3: The test solution is reduced using a reducing agent in order to break down the immunoglobulins; S4: The test solution after reduction treatment is mixed with the matrix solution and spotted onto the sample chip for crystallization. MALDI-TOF MS is then used for detection to determine whether the central nervous system abnormally expresses the endogenous immunoglobulin and the subtype of the expressed endogenous immunoglobulin. In the antibody-bonded magnetic beads, the mass ratio of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads is 1:(20-200); The magnetic beads have a particle size of 0.2-5 μm; The sample to be tested is a cerebrospinal fluid sample.

[0077] According to a specific embodiment of the present invention, in step S1, during contact, the volume ratio of the sample to be tested to the reaction buffer is 1:(20-100). This can improve the binding efficiency of the target protein and the antibody in the sample, thereby improving the enrichment effect, detection efficiency, and detection sensitivity.

[0078] According to a specific embodiment of the present invention, in the reaction buffer, the mass concentrations of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 0.003-0.01 mg / mL. This mass concentration is the optimal range obtained by the inventors for cerebrospinal fluid samples. By adding antibodies at the above-mentioned mass concentrations, the present invention can ensure sufficient enrichment of immunoglobulins in the cerebrospinal fluid without wasting antibodies, thereby minimizing antibody costs. According to a specific embodiment of the present invention, the final concentration of the above three antibodies in the reaction buffer is preferably 0.005 mg / mL, thereby minimizing antibody costs while ensuring sufficient enrichment of immunoglobulins in the cerebrospinal fluid.

[0079] According to a specific embodiment of the present invention, the reaction time during contact is 10-40 min and the reaction temperature is 20-30℃.

[0080] According to a specific embodiment of the present invention, in step S2, the elution process 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, the type of which is as described above.

[0081] According to a specific embodiment of the present invention, the method further includes washing the antigen-antibody complex magnetic beads before step S2. According to a specific embodiment of the present invention, the washing process is performed using a washing solution, the type of which is described above.

[0082] According to a specific embodiment of the present invention, the volume ratio of the test solution after reduction treatment to the matrix solution is 1:(1-10). According to a specific embodiment of the present invention, the matrix solution is preferably sinapic acid, and more preferably a 20 mg / mL sinapic acid solution. This allows for better protein ionization and higher dissociation efficiency. Simultaneously, using this matrix can significantly improve the protein detection signal, thereby increasing detection sensitivity.

[0083] According to a preferred embodiment of the present invention, the volume ratio of the sample to the sinapic acid matrix is ​​1:2, and the mass ratio of the antibody to the sinapic acid matrix is ​​1:100. Using this amount of matrix can fully disperse the immunoglobulins and make them fully ionized, thereby improving the ionization efficiency and detection sensitivity.

[0084] According to a specific embodiment of the present invention, the method further includes: (1) Immunoglobulins from serum and cerebrospinal fluid samples from the same individual were detected simultaneously using the above detection method to obtain mass spectra of the serum and cerebrospinal fluid samples. (2) Compare the mass spectra of the serum sample and the cerebrospinal fluid sample. If the mass spectra peaks of the two samples in the range of 20,000-30,000 m / z are inconsistent and there is a "sharp peak" in the cerebrospinal fluid sample, it is determined that the cerebrospinal fluid sample has endogenous immunoglobulin synthesis. Based on the different detection antibodies, the subtype of the synthesized endogenous immunoglobulin can be determined.

[0085] Therefore, if the mass spectrum of a cerebrospinal fluid (CSF) sample does not contain any "peaks" in the 20,000-30,000 m / z range, it is determined that the CSF sample does not have abnormal endogenous immunoglobulin synthesis. If the CSF sample's mass spectrum does contain "peaks" in the 20,000-30,000 m / z range, it is necessary to compare the mass spectrum of a serum sample to see if there are any "peaks" at the same positions. If they are present, it indicates that the monoclonal immunoglobulins in the CSF originate from plasma ultrafiltration, and it is determined that the CSF sample does not have abnormal endogenous immunoglobulin synthesis. If they are not present, it is determined that the CSF sample has abnormal endogenous immunoglobulin synthesis.

[0086] According to a specific embodiment of the present invention, the immunoglobulins in the sample are detected using a MALDI-TOF mass spectrometer. It should be noted that there are no particular restrictions on the model of the mass spectrometer. Preferably, the QuanTOF mass spectrometer from Rongzhi Biotechnology is used.

[0087] According to a more specific embodiment of the present invention, the main detection parameters of the QuanTOF mass spectrometer can be: spectrum storage range of m / z 5000~100000, focusing mass of 23000 Da, detection voltage of -0.59 kV, and laser energy of 9.4 μJ.

[0088] According to a more specific embodiment of the present invention, the present invention provides a method for detecting endogenous immunoglobulins expressed in the central nervous system based on MALDI-TOF MS, comprising the following steps: (1) Add an appropriate amount of buffer to the serum or cerebrospinal fluid sample, vortex mix, add magnetic beads-antibody, and vortex mix again. (2) Place the sample on a vortex mixer and mix at room temperature. After the mixture is mixed, centrifuge briefly, place it on a magnetic rack and magnetically absorb it for 1 min, then discard the supernatant. (3) Add cleaning solution A, vortex mix, magnetically aspirate for 1 min and discard the supernatant. Repeat the same steps to wash once with cleaning solution A and once with cleaning solution B. (4) After cleaning, magnetically aspirate and discard the supernatant. After the sample is centrifuged briefly, magnetically aspirate again and use a 10 μL pipette tip to aspirate the remaining liquid in the tube. (5) Add elution buffer and vortex to mix well, then elute the sample; (6) Take the eluent into a new EP tube, add the reducing agent, vortex to mix, and shake at room temperature for 15 min; (7) Add the matrix directly, vortex mix, and then take 2 μL of the elution solution to spot the target plate and detect it by MALDI-TOF mass spectrometry.

[0089] (8) Results analysis: After specific enrichment of a certain type (IgG, IgA or IgM) of immunoglobulin antibody in the cerebrospinal fluid and serum samples of the same individual, the peak shapes of the two in the 20,000~30,000 m / z range in the mass spectrometry were compared to determine the presence of endogenously synthesized Ig in the central nervous system, regardless of whether the peak shapes of the two samples are consistent. If there is at least one "peak" in the cerebrospinal fluid that is different from that in the serum, it indicates that the central nervous system contains endogenously synthesized Ig of this type (IgG, IgA or IgM).

[0090] According to a more specific embodiment of the present invention, the target plate type is a hydrophobic target plate.

[0091] According to a more specific embodiment of the present invention, the sample volume is 5 μL to 50 μL, the reaction buffer volume is 150 μL to 500 μL, and the amount of magnetic beads and antibody added is 5 μL to 20 μL.

[0092] The present disclosure will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0093] Example 1: Comparison of Endogenous Immunoglobulin Synthesis Detection and Isoelectric Focusing Electrophoresis Results The antibody bonding method in this embodiment is as follows: (1) Take 10 mg (100 μL, 1:50) of 3 μm carboxyl magnetic beads into a centrifuge tube, wash 3 times with 1 mL of 0.1 M MES (pH=4), then resuspend the magnetic beads with 200 μL of MES (pH=4), add 400 μL each of freshly prepared EDC and NHS (10 mg / mL, prepared and used immediately with pre-cooled MES stored at 4℃ pH=6), and invert for 30 min to activate the carboxyl groups; (2) Take 200 μg of rabbit polyclonal antibody specific to human IgG (affinity constant KD value is 2.9×10). -10 Add 200 μL of MES (pH=5) buffer solution to a 30 kDa ultrafiltration tube, wash 4 times to remove antibody protectant, and then set aside for use. (3) Wash 4 times with 1 mL MES (pH=5), transfer all the antibody (about 40 μL) in the centrifuge tube to the activated carboxyl magnetic bead solution, then rinse the centrifuge tube 3 times with 360 μL MES (pH=5) and combine it into the magnetic bead solution, and invert and couple at room temperature for 3 h. (4) Add 600 μL of CE510 and invert at room temperature for 3 h; (5) Wash 3 times with 1 mL TBS-T, then wash 3 times with 1 mL PBST; (6) Resuspend in 1 mL of PBST and store at 4°C (10 μL is equivalent to 2 μg of antibody).

[0094] This embodiment specifically includes the following experimental steps: Three pairs of paired clinical serum (S) and cerebrospinal fluid (C) samples (from the same individual) were tested, designated S / C-1, S / C-2, and S / C-3, using IgG antibody. The results were compared with isoelectric focusing electrophoresis results to determine the feasibility and accuracy of this detection method.

[0095] (1) Add 395 μL PBST to 5 μL serum / cerebrospinal fluid, vortex mix, then add 10 μL of carboxyl magnetic beads bonded with antibody IgG, and vortex mix again. (2) Place the sample on a vortex mixer and mix at room temperature for 10 min. After the mixture is finished, centrifuge briefly, place it on a magnetic rack and magnetically aspirate for 1 min, then discard the supernatant. (3) Add 600 μL PBST, vortex to mix, magnetically aspirate for 1 min and discard the supernatant. Repeat the same steps to wash once with 400 μL PBST and once with 200 μL ddH2O. Each vortexing time is 15~20 s. (4) After washing with ddH2O, discard the supernatant, centrifuge the sample briefly and magnetically aspirate it, and use a 10 μL pipette tip to aspirate the remaining liquid in the tube; (5) Add 10 μL of 0.1% TFA, vortex and mix well, then elute the sample; (6) Take 9 μL into a new EP tube, add 1 μL of TCEP, vortex to mix, and react at room temperature for 15 min. (7) Add 10 μL of matrix (20 mg / mL sinapic acid), vortex for about 15 s, take 2 μL of elution buffer and spot it on a single layer plate. The target plate type is 2.4 mm hydrophobic target plate.

[0096] Mass spectrometry detection: Mass spectrometry was performed using Rongzhi Bio's QuanTOF type MALDI-TOF MS.

[0097] Main detection parameters: spectrum storage range is m / z 5000~100000, focusing quality is 23000 Da, detection voltage is -0.59 kV, and laser energy is 9.4 μJ.

[0098] Analysis of experimental results: (1) Determine whether IgG is detected in serum (S) and cerebrospinal fluid (C) samples. Check the mass spectrum in the range of 20,000~30,000 m / z. If the Y value of the mass spectrum peak is less than 0.00015, it is considered undetectable. The rest are considered detected.

[0099] (2) Compare the mass spectra of paired serum and cerebrospinal fluid samples. If the mass spectra peaks in the range of 20,000 to 30,000 m / z are inconsistent and there is a “sharp peak” in the cerebrospinal fluid sample, then it is considered that there is endogenous IgG synthesis.

[0100] (3) In this test, the peak shapes of the S / C-1 serum and cerebrospinal fluid spectra of the three paired samples were consistent. Figure 1The presence of oligoclonal "peaks" and isoelectric focusing electrophoresis results indicated type IV cerebrospinal fluid (CSF) samples, consistent with both methods, suggest the absence of abnormally synthesized endogenous immunoglobulins. The peak shapes in the serum and CSF spectra of samples S / C-2 and S / C-3 were inconsistent. Figure 2 and Figure 3 The serum sample showed a normal negative spectrum, while the cerebrospinal fluid sample showed monoclonal spikes, indicating the presence of abnormally synthesized endogenous immunoglobulins in the cerebrospinal fluid. The isoelectric focusing electrophoresis results for samples S / C-2 and S / C-3 were type II, consistent with the MALDI-TOF detection results.

[0101] (4) Comparison of the results of the two detection methods showed that MALDI-TOF MS mass spectrometry and isoelectric focusing electrophoresis had high consistency in determining the synthesis of endogenous immunoglobulins in the central nervous system, confirming that the results of the MALDI-TOF MS mass spectrometry detection method are reliable and can be used for related detection and evaluation.

[0102] Table 1. Comparison of detection results and isostatic focusing electrophoresis results for three pairs of clinically paired serum and cerebrospinal fluid samples.

[0103] Note 1: To determine whether the peak shapes of serum and cerebrospinal fluid in paired samples are consistent, "√" indicates consistent peak shapes; "×" indicates inconsistent peak shapes.

[0104] Note 2: For S (serum) sample test results, whether there are "peaks" in the mass spectrometry peaks in the range of 20000~30000 m / z, "√" indicates the presence of a peak, indicating abnormal Ig proliferation; "×" indicates the absence of a peak, indicating normal Ig synthesis in the sample.

[0105] Note 3: For C (cerebrospinal fluid) sample test results, whether there are "peaks" in the mass spectrometry peaks in the range of 20000~30000 m / z, "√" indicates the presence of a peak, indicating abnormal Ig proliferation; "×" indicates the absence of a peak, indicating normal Ig synthesis in the sample.

[0106] Example 2: Detection of endogenous immunoglobulin IgG, IgA and IgM synthesis The antibody bonding method in this embodiment is as follows: (1) Take 10 mg (100 μL, 1:50) of 3 μm carboxyl magnetic beads into a centrifuge tube, wash 3 times with 1 mL of 0.1 M MES (pH=4), then resuspend the magnetic beads with 200 μL of MES (pH=4), add 400 μL each of freshly prepared EDC and NHS (10 mg / mL, prepared and used immediately with pre-cooled MES stored at 4℃ pH=6), and invert for 30 min to activate the carboxyl groups; (2) Take 200 μg of rabbit polyclonal antibody specific to human IgG (affinity constant KD value is 2.9×10).-10 (or IgA-specific rabbit polyclonal antibody (affinity constant KD value of 9.1 × 10⁻⁶)) -11 (or IgM-specific rabbit polyclonal antibody (affinity constant KD value is 3.9 × 10⁻⁶)) -11 Add 200 μL of MES (pH=5) buffer solution to a 30 kDa ultrafiltration tube, wash 4 times to remove antibody protectant, and then set aside for use. (3) Wash 4 times with 1 mL MES (pH=5), transfer all the antibody (about 40 μL) in the centrifuge tube to the activated carboxyl magnetic bead solution, then rinse the centrifuge tube 3 times with 360 μL MES (pH=5) and combine it into the magnetic bead solution, and invert and couple at room temperature for 3 h. (4) Add 600 μL of CE510 and invert at room temperature for 3 h; (5) Wash 3 times with 1 mL TBS-T, then wash 3 times with 1 mL PBST; (6) Resuspend in 1 mL of PBST and store at 4°C (10 μL is equivalent to 2 μg of antibody).

[0107] The above steps are used to prepare magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibodies (hereinafter referred to as IgG magnetic beads), magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibodies (hereinafter referred to as IgA magnetic beads), and magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibodies (hereinafter referred to as IgM magnetic beads) for later use.

[0108] This embodiment specifically includes the following experimental steps: A total of 43 pairs of paired clinical serum (S) and cerebrospinal fluid (C) samples, numbered 01 to 43, were tested to determine whether there was abnormal synthesis of endogenous immunoglobulins. The antibodies detected included IgG, IgA, and IgM, and the antibody magnetic beads were prepared using the antibody bonding method described above.

[0109] (1) Prepare three 5 μL serum samples and three 5 μL cerebrospinal fluid samples for each number. Add 395 μL PBST to each sample, vortex to mix, and then add 10 μL of IgG magnetic beads, IgA magnetic beads and IgM magnetic beads to the three serum samples in sequence. Add 10 μL of IgG magnetic beads, IgA magnetic beads and IgM magnetic beads to the three cerebrospinal fluid samples in sequence, and vortex to mix again.

[0110] (2) Place the sample on a vortex mixer and mix at room temperature for 10 min. After the mixture is finished, centrifuge briefly, place it on a magnetic rack and magnetically aspirate for 1 min, then discard the supernatant. (3) Add 600 μL PBST, vortex to mix, magnetically aspirate for 1 min and discard the supernatant. Repeat the same steps to wash once with 400 μL PBST and once with 200 μL ddH2O. Each vortexing time is 15~20 s. (4) After washing with ddH2O, discard the supernatant, centrifuge the sample briefly and magnetically aspirate it, and use a 10 μL pipette tip to aspirate the remaining liquid in the tube; (5) Add 10 μL of 0.1% TFA, vortex and mix well, then elute the sample; (6) Take 9 μL into a new EP tube, add 1 μL of TCEP, vortex to mix, and react at room temperature for 15 min. (7) Add 10 μL of matrix (20 mg / mL sinapic acid), vortex for about 15 s, take 2 μL of monolayer spot plate, the target plate type is 2.4 mm hydrophobic target plate.

[0111] (8) Place the prepared spot plate target plate in the mass spectrometer for data acquisition. The mass spectrometer used is QuanTOF type MALDI-TOF MS of Rongzhi Biotechnology. The main detection parameters are set as follows: spectrum storage range is m / z 5000~100000, focusing mass is 23000Da, detection voltage is -0.59 kV, and laser energy is 9.4 μJ.

[0112] Analysis of experimental results: (1) Determine whether Ig is detected in serum (S) and cerebrospinal fluid (C) samples. Check the mass spectra of different types of antibodies in 43 pairs of samples in the range of 20,000~30,000 m / z. If the Y value of the mass spectra peak is less than 0.00015, it is considered that the immunoglobulin subtype is not detected. The rest are detected.

[0113] (2) By comparing the mass spectra of paired serum and cerebrospinal fluid samples, if the mass spectra peaks in the range of 20,000 to 30,000 m / z are inconsistent and "sharp peaks" are present in the cerebrospinal fluid sample, it is considered that there is abnormal synthesis of endogenous immunoglobulins. Based on the different antibodies detected, the subtype of the synthesized endogenous immunoglobulin can be determined. Among the 43 pairs of paired samples tested in this study, 11 cerebrospinal fluid samples were found to have abnormally synthesized immunoglobulins, and the immunoglobulin subtype was IgG. Specifically, these are the cerebrospinal fluid samples numbered 03, 04, 06, 08, 12, 15, 17, 22, 25, 30, and 43 in Table 2.

[0114] (3) Appendix Figure 4-12 The comparison of mass spectra of several paired samples is shown. Figure 4-9 The test results are for samples enriched with IgG antibodies. Figure 10-11 The results of the test for samples enriched with IgA antibodies. Figure 12 The results of the test for samples enriched with IgM antibodies.

[0115] Figure 4 In the paired sample No. 09, no "peak" was detected in either the cerebrospinal fluid or serum sample, indicating that there was no abnormally synthesized IgG in the cerebrospinal fluid sample.

[0116] Figure 5 In the 33rd paired sample, both the cerebrospinal fluid and serum samples showed "peaks", and the positions of the "peaks" were consistent, indicating that the abnormal IgG in the cerebrospinal fluid sample came from plasma ultrafiltration, and the central nervous system itself did not synthesize IgG abnormally.

[0117] Figure 6 In the 30 paired samples, both cerebrospinal fluid and serum samples showed "peaks", but the positions of the "peaks" were inconsistent, indicating that there was abnormally synthesized IgG in the cerebrospinal fluid sample.

[0118] Figure 7 In the 17th paired sample, a "peak" was detected in the cerebrospinal fluid sample, while no "peak" was detected in the serum sample at the same location, indicating that there was abnormally synthesized IgG in the cerebrospinal fluid sample.

[0119] Figure 8-9 In the paired samples 10 and 16, no "peak" was detected in the cerebrospinal fluid sample, while a "peak" was detected in the serum sample, indicating that there was no abnormally synthesized IgG in the cerebrospinal fluid sample.

[0120] Figure 10 In the 20th paired sample, no "peak" was detected in either the cerebrospinal fluid or serum sample, indicating that there was no abnormally synthesized IgA in the cerebrospinal fluid sample.

[0121] Figure 11 In the paired sample No. 02, both the cerebrospinal fluid and serum samples showed "peaks", and the positions of the "peaks" were consistent, indicating that the abnormal IgA in the cerebrospinal fluid sample came from plasma ultrafiltration, and the central nervous system itself did not synthesize IgA abnormally.

[0122] Figure 12 In the paired sample No. 03, no "peak" was detected in the cerebrospinal fluid sample, but a "peak" was detected in the serum sample, indicating that there was no abnormally synthesized IgM in the cerebrospinal fluid sample, but there was abnormal IgM in the serum.

[0123] Table 2. Detection results of 43 pairs of clinically paired serum and cerebrospinal fluid samples.

[0124] Note 1: To determine whether the peak shapes of serum and cerebrospinal fluid in paired samples are consistent, "√" indicates consistent peak shapes; "×" indicates inconsistent peak shapes; " / " indicates that the peak shape was not detected in either serum or cerebrospinal fluid sample.

[0125] Note 2: For S (serum) sample test results, whether there are "peaks" in the mass spectrometry peaks in the range of 20000~30000 m / z, "√" indicates the presence of a peak, indicating abnormal Ig proliferation; "×" indicates the absence of a peak, indicating normal Ig synthesis in the sample; " / " indicates not detected.

[0126] Note 3: For C (cerebrospinal fluid) sample test results, whether there are "peaks" in the mass spectrometry peaks in the range of 20000~30000 m / z: "√" indicates the presence of a peak, indicating abnormal Ig proliferation; "×" indicates the absence of a peak, indicating normal Ig synthesis in the sample; " / " indicates not detected.

[0127] Note 4: In the results, "negative" means that there is no abnormally synthesized Ig in the cerebrospinal fluid sample, and "positive" means that there is abnormally synthesized Ig in the cerebrospinal fluid sample.

[0128] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," "some implementations," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0129] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A kit for detecting and typing endogenous immunoglobulins expressed in the central nervous system based on MALDI-TOF MS, characterized in that, include: Magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibodies; Magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibodies; Reaction buffer solution; Elution solution; reducing agent; Matrix solution; The mass ratios of the anti-human immunoglobulin heavy chain IgG antibody, anti-human immunoglobulin heavy chain IgA antibody, and anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads are each independently 1:(20-200). The magnetic beads have a particle size of 0.2-5 μm.

2. The reagent kit according to claim 1, characterized in that, The affinity constants (KD) of the anti-human immunoglobulin heavy chain IgG antibody, anti-human immunoglobulin heavy chain IgA antibody, and anti-human immunoglobulin heavy chain IgM antibody are each independently 9.9 × 10⁻⁶. -12 M-1×10 -9 M; Optionally, the surface of the magnetic beads is modified with chemical groups for binding to the antibody; Optionally, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups; Optionally, the elution solution includes at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions; The organic acid solution includes at least one selected from 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 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, tris(hydroxymethyl)aminomethane solution, tetraethylammonium bromide solution, sodium carbonate solution, sodium bicarbonate solution, ammonium bicarbonate solution, ammonia solution, and urea solution; Optionally, the elution solution is a trifluoroacetic acid solution; Optionally, the mass concentration of the trifluoroacetic acid solution is 0.05-5%; Optionally, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions; Preferably, the reaction buffer is a PBST solution; Optionally, the kit further includes a cleaning solution; Optionally, the washing solution includes at least one selected from PBS, PBST, and TBST solutions; Preferably, the cleaning solution is a PBST solution.

3. The reagent kit according to claim 1, characterized in that, The reducing agent includes at least one selected from TCEP, DTT, β-mercaptoethanol, and glutathione; Preferably, the reducing agent is TCEP.

4. The reagent kit according to claim 1, characterized in that, The matrix in the matrix solution includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid. Optionally, the matrix solution is sinapic acid; preferably, the matrix solution is a 20 mg / mL sinapic acid solution.

5. A method for detecting endogenous immunoglobulins expressed in the central nervous system, characterized in that, The method includes using the kit described in any one of claims 1-4 to detect and classify a sample to be tested in order to determine whether the central nervous system abnormally expresses endogenous immunoglobulins and the expressed endogenous immunoglobulin subtypes, wherein the sample to be tested is a cerebrospinal fluid sample.

6. A method for detecting endogenous immunoglobulins expressed in the central nervous system, characterized in that, include: S1: The sample to be tested is divided into three parts, and each part is contacted with magnetic beads bonded to anti-human immunoglobulin heavy chain IgG antibody, magnetic beads bonded to anti-human immunoglobulin heavy chain IgA antibody, and magnetic beads bonded to anti-human immunoglobulin heavy chain IgM antibody in a reaction buffer, so that the IgG, IgA and IgM contained in the sample to be tested can specifically bind to the antibodies bonded to the magnetic beads, and three antigen-antibody complex magnetic beads are obtained. S2: The three portions of antigen-antibody complex magnetic beads are eluted and magnetically separated to elute and separate the enriched IgG, IgA and IgM from the antibodies, and obtain a test solution containing IgG, IgA and IgM. S3: The test solution is reduced using a reducing agent in order to break down the immunoglobulins; S4: The test solution after reduction treatment is mixed with the matrix solution and spotted onto the sample chip for crystallization. MALDI-TOF MS is then used for detection to determine whether the central nervous system abnormally expresses the endogenous immunoglobulin and the subtype of the expressed endogenous immunoglobulin. In the antibody-bonded magnetic beads, the mass ratio of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody to the magnetic beads is independently 1:(20-200); The magnetic beads have a particle size of 0.2-5 μm; The sample to be tested is a cerebrospinal fluid sample.

7. The method according to claim 6, characterized in that, The surface of the magnetic beads is modified with chemical groups, which are used to bind to the antibody. Optionally, the chemical group includes at least one selected from amino, carboxyl, epoxy, and hydroxyl groups; Optionally, the affinity constants (KD) of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 9.9 × 10⁻⁶. -12 M-1×10 -9 M; Optionally, the reaction buffer comprises at least one selected from PBS, PBST, and TBST solutions; Optionally, the reaction buffer is a PBST solution.

8. The method according to claim 6, characterized in that, In step S1, during contact, the volume ratio of the sample to be tested to the reaction buffer is 1:(20-100). Optionally, in the reaction buffer, the mass concentrations of the anti-human immunoglobulin heavy chain IgG antibody, the anti-human immunoglobulin heavy chain IgA antibody, and the anti-human immunoglobulin heavy chain IgM antibody are each independently 0.003-0.01 mg / mL; Optionally, when contact is carried out, the reaction time is 10-40 min and the reaction temperature is 20-30℃; Optionally, in step S2, the elution process is performed using an elution solution; Optionally, the elution solution contains an antigen-antibody complex dissociation reagent. Optionally, the antigen-antibody complex dissociation reagent includes at least one selected from organic acid solutions, inorganic acid solutions, and alkaline solutions; The organic acid solution includes at least one selected from 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 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, 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%, preferably 0.1%.

9. The method according to claim 8, characterized in that, The method further includes cleaning the antigen-antibody complex magnetic beads before step S2; Optionally, the cleaning process is performed using a cleaning solution; Optionally, the washing solution includes at least one selected from PBS, PBST, and TBST solutions; Preferably, the cleaning solution is a PBST solution; Optionally, the reducing agent includes at least one selected from TCEP, DTT, β-mercaptoethanol, and glutathione; Preferably, the reducing agent is TCEP; Optionally, the matrix in the matrix solution includes at least one selected from sinapic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxybenzoic acid, 2,4,6-trihydroxyacetophenone, anthraquinone, and 3-indoleacrylic acid; Optionally, the matrix solution is sinapic acid; preferably, the matrix solution is a 20 mg / mL sinapic acid solution. Optionally, the volume ratio of the test solution after the reduction treatment to the matrix solution is 1:(1-10). Optionally, the method further includes: (1) Using the method described above for detecting immunoglobulins in a sample, immunoglobulins from serum samples and cerebrospinal fluid samples from the same individual are detected simultaneously to obtain mass spectra of serum samples and cerebrospinal fluid samples. (2) Compare the mass spectra of the serum sample and the cerebrospinal fluid sample. If the mass spectra peaks of the two samples in the range of 20,000-30,000 m / z are inconsistent and there is a "sharp peak" in the cerebrospinal fluid sample, it is determined that the cerebrospinal fluid sample has endogenous immunoglobulin synthesis. The subtype of the synthesized endogenous immunoglobulin is determined according to the different detection antibodies.

10. The method according to claim 6, characterized in that, The MALDI-TOF MS is QuanTOF; Optionally, the detection parameters of the MALDI-TOF MS are: spectrum storage range of m / z 5000~100000, focusing quality of 23000 Da, detection voltage of -0.59 kV, and laser energy of 9.4 μJ.

Images