Anti-mutant citrullinated vimentin antibody detection kit and its application

Abstract

The present invention provides an anti-mutant citrullinated vimentin antibody detection kit and its application, which relates to the field of biotechnology. The kit includes a mutant citrullinated vimentin-coupled magnetic particle working solution, which is mixed with a final feed ratio of 0.6 ng / (unit × mL) mutant citrullinated vimentin polypeptide E1-coupled magnetic particles, 0.3 ng / (unit × mL) mutant citrullinated vimentin polypeptide H1-coupled magnetic particles, 0.3 ng / (unit × mL) mutant citrullinated vimentin polypeptide G5-coupled magnetic particles and a buffer; a tracer-labeled antibody working solution, which is composed of a tracer-labeled anti-human IgM antibody and a diluent. The kit of the present invention uses a mixture of mutant citrullinated vimentin polypeptide E1-coupled magnetic particles, mutant citrullinated vimentin polypeptide H1-coupled magnetic particles, and mutant citrullinated vimentin polypeptide G5-coupled magnetic particles to greatly improve sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an anti-mutant citrullinated vimentin antibody detection kit and applications thereof. Background Art

[0002] The glycine residue in the natural vimentin protein was mutated to arginine. After the arginine was citrullinated by peptidylarginine iminase, the potential protein determinant cluster of citrulline was added, the structure of vimentin was changed, and the mutant citrullinated vimentin protein was produced.

[0003] Compared with anti-cyclic citrullinated peptide antibodies, anti-mutant citrullinated vimentin antibodies have higher sensitivity and specificity in the diagnosis of rheumatoid arthritis (RA), particularly in the early stages of RA. Anti-mutant citrullinated vimentin antibodies appear in the serum of RA patients at an earlier stage than other markers. Detecting changes in anti-mutant citrullinated vimentin antibody levels can reflect the severity of RA and assist in monitoring the treatment process of RA patients, thus being of great significance for the early diagnosis of RA.

[0004] Currently, using mutant citrullinated vimentin to detect anti-mutant anti-citrullinated vimentin antibodies in blood samples to aid in the diagnosis of early-stage rheumatoid arthritis is a common medical practice. However, existing anti-mutant citrullinated vimentin antibody kits often use commercially available or homemade mutant citrullinated vimentin as the antigen. The preparation process is complex and difficult to control, resulting in low sensitivity and specificity for the mutant citrullinated vimentin produced, making it unable to meet the current needs for detecting anti-mutant citrullinated vimentin antibodies.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a detection kit for mutant citrullinated vimentin antibodies and its use, addressing the low sensitivity and specificity of existing in vitro detection reagents for mutant citrullinated vimentin antibodies. The kit utilizes three mutant citrullinated vimentin antigens with different specific sequences coupled to magnetic microparticles, further enhancing the sensitivity and specificity of the detection results.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides an anti-mutant citrullinated vimentin antibody detection kit, comprising: a mutant citrullinated vimentin-coupled magnetic particle working solution, which is composed of mutant citrullinated vimentin-coupled magnetic particles and a buffer; a tracer-labeled antibody working solution, which is composed of a tracer-labeled anti-human IgM antibody and a diluent; wherein the mutant citrullinated vimentin-coupled magnetic particles are prepared by mixing mutant citrullinated vimentin polypeptide E1-coupled magnetic particles, mutant citrullinated vimentin polypeptide H1-coupled magnetic particles, and mutant citrullinated vimentin polypeptide G5-coupled magnetic particles; the final feed ratio of mutant citrullinated vimentin polypeptide E1 to magnetic particles in the mutant citrullinated vimentin polypeptide E1-coupled magnetic particles is 0.6 ng / (piece × mL); the final feed ratio of mutant citrullinated vimentin polypeptide H1 to magnetic particles in the mutant citrullinated vimentin polypeptide H1-coupled magnetic particles is 0.3 ng / (piece × mL).

[0009] The final feed ratio of the mutant citrullinated vimentin polypeptide G5 to the magnetic particles in the mutant citrullinated vimentin polypeptide G5-coupled magnetic particles is 0.3 ng / (piece×mL).

[0010] Unlike commonly used commercially available mutant citrullinated vimentin antigens, the kit of the present invention uses a final feed ratio of 0.6 ng / (unit × mL) mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles, 0.3 ng / (unit × mL) mutant citrullinated vimentin polypeptide H1-coupled magnetic microparticles, 0.3 ng / (unit × mL) mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles, and a buffer solution to more fully expose each antigenic site. The final feed ratios of the mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles, the mutant citrullinated vimentin polypeptide H1-coupled magnetic microparticles, and the mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles were screened. As verified by immunoserological detection technology, the kit has higher sensitivity and specificity for detecting anti-mutant citrullinated vimentin antibodies than a kit prepared using the existing commercially available mutant citrullinated vimentin antigen.

[0011] In one embodiment, in the kit of the present invention, the mutant citrullinated vimentin polypeptide E1 has the amino acid sequence shown as SEQ ID No. 1; the mutant citrullinated vimentin polypeptide H1 has the amino acid sequence shown as SEQ ID No. 2; and the mutant citrullinated vimentin polypeptide G5 has the amino acid sequence shown as SEQ ID No. 3.

[0012] In a preferred embodiment, the mutant citrullinated vimentin polypeptide E1, the mutant citrullinated vimentin polypeptide H1, and the mutant citrullinated vimentin polypeptide G5 are all modified with biotin.

[0013] Because the mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 used in the present application are all 17 amino acids in length, after modification with biotin, the spatial structure of biotin is smaller, which does not affect the exposure of the antigenic site on the polypeptide, and is more conducive to the connection of the polypeptide to the magnetic barcode particles, thereby improving the utilization rate of the polypeptide and the magnetic barcode particles. If other methods are used to connect the polypeptide to the magnetic barcode particles, the exposure of the antigenic epitope will be affected, the polypeptide will not be able to be connected to the magnetic barcode particles, the utilization rate of the polypeptide or the magnetic barcode particles will be low, and the sensitivity and specificity of the kit for detecting antibodies against mutant citrullinated vimentin will be seriously affected.

[0014] In one embodiment, the magnetic particles are magnetic barcode particles, and the magnetic barcode particles are modified with streptavidin.

[0015] In a preferred embodiment, the structure of the mutant citrullinated vimentin polypeptide E1 coupled to the magnetic microparticles is mutant citrullinated vimentin polypeptide E1-biotin-streptavidin-magnetic barcode microparticles; the structure of the mutant citrullinated vimentin polypeptide H1 coupled to the magnetic microparticles is mutant citrullinated vimentin polypeptide H1-biotin-streptavidin-magnetic barcode microparticles; the structure of the mutant citrullinated vimentin polypeptide G5 coupled to the magnetic microparticles is mutant citrullinated vimentin polypeptide G5-biotin-streptavidin-magnetic barcode microparticles.

[0016] In this application, different mutant citrullinated vimentin polypeptides are first coupled to different magnetic barcode microparticles to form different mutant citrullinated vimentin-coupled magnetic microparticles, and then the different mutant citrullinated vimentin-coupled magnetic microparticles are mixed at a specific final feed ratio. Compared to simultaneously coupling different mutant citrullinated vimentin polypeptides to magnetic barcode microparticles in the same liquid, the mutant citrullinated vimentin polypeptides coupled to the magnetic barcode microparticles are more uniform, avoiding the large steric hindrance and poor uniformity that occurs when multiple mutant citrullinated vimentin polypeptides are coupled to the same magnetic barcode microparticle, further improving the sensitivity and specificity of the test kit detection results.

[0017] In a preferred embodiment, in the phosphate buffer, the ratio of the mutant citrullinated vimentin-coupled magnetic particles to the buffer in the mutant citrullinated vimentin-coupled magnetic particle working solution is 1000 magnetic barcode particles / mL buffer.

[0018] In a preferred embodiment, the buffer is 1 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.05 mol / L phosphate buffer.

[0019] In one embodiment, the tracer is phycoerythrin, and the diluent is 0.2 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.1 mg / mL proclin 300, and a phosphate buffer solution with a pH of 6.5 and a concentration of 0.1 mol / L.

[0020] In one embodiment, the kit further comprises a zero-value control solution and a sample diluent, wherein the zero-value control solution and the sample diluent are phosphate buffer containing 0.05 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.01 mol / L.

[0021] In one embodiment, the sample suitable for detection in the kit of the present invention includes whole blood, serum or plasma.

[0022] In another aspect, the present invention provides use of the kit in detecting anti-mutant citrullinated vimentin antibodies in vitro for non-disease diagnosis purposes.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The mutant citrullinated vimentin produced in the blood of rheumatoid arthritis patients is a mixture of antibodies (polyclonal antibodies) targeting different antigenic epitopes. The types and proportions of antibodies produced by different individuals vary. Artificially synthesized mutant citrullinated vimentin antigens / genetically engineered antigens only have partially exposed antigenic determinants, making it very easy to miss detections when using a single antigen for immunological testing, resulting in low sensitivity. By combining mutant citrullinated vimentin peptide E1, mutant citrullinated vimentin peptide H1, and mutant citrullinated vimentin peptide G5, as many specific and effective antigenic epitopes as possible are covered.

[0025] The present invention provides an anti-mutant citrullinated vimentin antibody detection kit. The kit uses mutant citrullinated vimentin polypeptide E1 and magnetic barcode microparticles at a final feeding ratio of 0.6 ng / (piece × mL), mutant citrullinated vimentin polypeptide H1 and magnetic barcode microparticles at a final feeding ratio of 0.3 ng / (piece × mL), and mutant citrullinated vimentin polypeptide G5 and magnetic barcode microparticles at a final feeding ratio of 0.3 ng / (piece × mL) to replace commercially available mutant citrullinated vimentin-coupled magnetic microparticles. The kit has a sensitivity of 90.00%, a specificity of 90.20%, a false positive rate of 9.80%, and a false negative rate of 10.00%. A kit prepared with commercially available mutant citrullinated vimentin-coupled magnetic microparticles at a final feeding ratio of 0.3 ng / (piece × mL) has a false positive rate of 29.41% and a false negative rate of 30.00%. The anti-mutant citrullinated vimentin antibody detection kit provided by the present invention reduces both false positives and false negatives by 66.67% compared with the commercially available mutant citrullinated vimentin-coupled magnetic microparticles, demonstrating significant results. The sensitivity and specificity of anti-mutant citrullinated vimentin antibody detection in sera from different patients were further improved.

[0026] The mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 selected in this application all contain different antigenic determinants. The binding rate constants of the antigenic determinants of the three antigens and their corresponding antibodies are constant and different. The concentration level signal value of a single antigen is positively correlated with the signal value level. The immunoassay methodology judges the positive and negative nature of the test result by comparing the signal value with a pre-set critical value. The three antigens have a complementary relationship in the detection of antibodies in the population. The final feed ratio of the three antigens when mixed and used at the same time is particularly important. Only when the mixed antigen test results are adjusted to a signal value level close to the three antigen positive judgment values ​​are close, the sensitivity and specificity are the highest at this time. When the concentration of a certain antigen is relatively low, the signal value of the sample that can only be detected by this antigen will be lower than the critical point, mistakenly judged as negative, resulting in missed detection and decreased sensitivity. Similarly, when the concentration of a certain antigen is relatively high, the overall signal value of the entire sample will be raised, the critical point will be moved up, and it will be mistakenly judged as positive, resulting in wrong detection and decreased specificity.

[0027] The kit of the present invention has wide applicability, especially when used in a fully automated multiplex liquid chip immunoassay system. Steps such as sample addition, incubation, washing, and detection can all be automated, thus avoiding result deviations caused by manual operation and improving work efficiency. By incorporating a calibration curve into the test software, only the test sample needs to be tested to qualitatively or quantitatively detect anti-mutant citrullinated vimentin antibodies in the sample, making the detection faster, more reliable, and more stable. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] The term "mutant citrullinated vimentin coupled to magnetic microparticles" as used herein refers to coupling a mutant citrullinated vimentin antigen to magnetic microparticles. The mutant citrullinated vimentin antigen refers to a substance that can specifically recognize and bind to an anti-mutant citrullinated vimentin antibody, including but not limited to mutant citrullinated vimentin prepared according to the method described in the background art, mutant citrullinated vimentin polypeptides, and commercially available mutant citrullinated vimentin. The magnetic microparticles refer to solid-phase carriers having magnetic properties, including but not limited to magnetic beads, magnetic particles, and magnetic barcode microparticles. The magnetic microparticles used in this embodiment are magnetic barcode microparticles.

[0030] The “mutant citrullinated vimentin polypeptide E1 coupled to magnetic microparticles” mentioned in the present application refers to coupling the mutant citrullinated vimentin polypeptide E1 with the amino acid sequence as described in SEQ ID No. 1 to magnetic microparticles.

[0031] The “mutant citrullinated vimentin polypeptide H1 coupled to magnetic microparticles” mentioned in the present application refers to coupling the mutant citrullinated vimentin polypeptide H1 with the amino acid sequence as described in SEQ ID No. 2 to magnetic microparticles.

[0032] The “mutant citrullinated vimentin polypeptide G5 coupled to magnetic microparticles” mentioned in the present application refers to coupling the mutant citrullinated vimentin polypeptide G5 with the amino acid sequence as described in SEQ ID No. 3 to magnetic microparticles.

[0033] The "final feed ratio" mentioned in this application refers to the feed ratio formed during the coupling process of mutant citrullinated vimentin polypeptide and magnetic microparticles. For example, the final feed ratio of mutant citrullinated vimentin polypeptide E1 to magnetic microparticles is 0.6 ng / (unit × mL), which means that when the mutant citrullinated vimentin polypeptide E1 coupled to magnetic microparticles is prepared separately, the actual reaction ratio of the magnetic microparticles and the mutant citrullinated vimentin polypeptide E1 after feeding is 0.6 ng mutant citrullinated vimentin polypeptide and 1 magnetic microparticle in 1 mL of liquid. When the mutant citrullinated vimentin polypeptide E1 is coupled to magnetic microparticles, the mutant citrullinated vimentin polypeptide H1 is coupled to magnetic microparticles, and the mutant citrullinated vimentin polypeptide E1 is coupled to magnetic microparticles. After the peptide G5-coupled magnetic particles were mixed, the feed ratio, amount of magnetic particles added, and amount of buffer for preparing the mutant citrullinated vimentin polypeptide E1 polypeptide-coupled magnetic particles were adjusted according to the ratio, so that after the mutant citrullinated vimentin polypeptide E1-coupled magnetic particles, the mutant citrullinated vimentin polypeptide H1-coupled magnetic particles, and the mutant citrullinated vimentin polypeptide G5-coupled magnetic particles were mixed, the feed ratio of the mutant citrullinated vimentin polypeptide E1-coupled magnetic particles in the mutant citrullinated vimentin polypeptide-coupled magnetic particle working solution was still maintained at 0.6 ng / (piece × mL).

[0034] In the present application, "mutant citrullinated vimentin polypeptide E1-biotin-streptavidin-magnetic barcode microparticles" refers to a complex formed by the reaction of mutant citrullinated vimentin polypeptide E1 modified with biotin and magnetic barcode microparticles modified with streptavidin, with biotin and streptavidin linked.

[0035] 1. Raw material: biotinylated mutant citrullinated vimentin peptide.

[0036] The mutant citrullinated vimentin antigens used in the present invention are mutant citrullinated vimentin polypeptides E1, H1, and G5. The amino acid sequence of E1 is SEQ ID No. 1, the amino acid sequence of H1 is SEQ ID No. 2, and the amino acid sequence of G5 is SEQ ID No. 3, wherein X in the amino acid sequence represents citrulline Cit. Mutant citrullinated vimentin polypeptides E1, H1, and G5 are all modified with biotin.

[0037] SXPSSSXSYVTTSTXTY(SEQ ID No. 1).

[0038] TYSLGSALXPSTSXHLY(SEQ ID No. 2).

[0039] NASLAXLDLEXKVESLQ(SEQ ID No. 3).

[0040] 2. Prepare Streptavidin Magnetic Particle Working Solution:

[0041] The magnetic barcode particles used in this example are magnetic barcode particles coupled with streptavidin. One magnetic barcode particle has multiple barcode regions. The magnetic barcode particles in the suspension of magnetic barcode particles coupled with streptavidin are subjected to magnetic separation to obtain magnetic barcode particles. The magnetic barcode particles are resuspended in phosphate buffer with a pH of 7.0 and a concentration of 0.05 mol / L to a concentration of 2×10 5 The magnetic barcode microparticle solution was washed three times with phosphate buffer at pH 7.0 and a concentration of 0.05 mol / L and resuspended to 2×10 5 / mL magnetic barcode microparticle solution.

[0042] 3. Preparation of mutant citrullinated vimentin-coupled magnetic microparticles:

[0043] Biotinylated mutant citrullinated vimentin polypeptides E1, H1, and G5 were coated with streptavidin magnetic particle working solution at final feed ratios of 1.25 ng / (piece × mL), 0.8 ng / (piece × mL), 0.6 ng / (piece × mL), 0.4 ng / (piece × mL), 0.3 ng / (piece × mL), 0.2 ng / (piece × mL), and 0.125 ng / (piece × mL), respectively. After shaking at room temperature for 2 hours, the supernatant was removed by magnetic separation and adsorption, and the supernatant was washed three times with the same volume of the above-mentioned phosphate buffer with a pH of 7.0 and a concentration of 0.05 mol / L to obtain mutant citrullinated vimentin-coupled magnetic particles.

[0044] Here, 1.25 ng / (unit × mL) means that on average 1 mL of liquid contains 1.25 ng of biotinylated mutant citrullinated vimentin polypeptide and 1 magnetic barcode microparticle of streptavidin, or that on average 1 mL of liquid contains 1 magnetic barcode microparticle, on which 1.25 ng of biotinylated mutant citrullinated vimentin polypeptide is coupled via streptavidin.

[0045] The prepared mutant citrullinated vimentin-coupled magnetic microparticles were resuspended and blocked for 1 hour using phosphate buffer containing 2 mg / mL bovine serum albumin, 0.005 mg / mL proclin 300, pH 0.7, and a concentration of 0.05 mol / L.

[0046] After blocking, the mutant citrullinated vimentin-coupled magnetic particles were resuspended in phosphate buffer with a pH of 0.7 and a concentration of 0.05 mol / L to 10 5 magnetic barcoded particles / mL.

[0047] Use 10 for each test 3The working solution was prepared at a ratio of 100 magnetic barcode particles / mL buffer solution, wherein the buffer solution was a phosphate buffer solution containing 1 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.05 mol / L.

[0048] 4. Prepare phycoerythrin-labeled anti-human IgM antibody working solution:

[0049] The phycoerythrin-labeled anti-human IgM antibody was diluted to 300 ng / mL with phosphate buffer containing 0.2 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, and 0.1 mg / mL proclin 300, at a pH of 6.5 and a concentration of 0.1 mol / L.

[0050] 5. Prepare zero-value control solution and sample dilution solution:

[0051] The zero-value control solution and sample diluent are phosphate buffer containing 0.05 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, pH 7.0, and a concentration of 0.01 mol / L.

[0052] 6. Preparation of anti-mutant citrullinated vimentin antibody detection kit.

[0053] The anti-mutant citrullinated vimentin antibody detection kit includes: mutant citrullinated vimentin-coupled magnetic particles 6.5 mL / bottle, phycoerythrin-labeled anti-human IgM antibody 3.5 mL / bottle, and zero-value control working solution 1.0 mL / tube. After packaging, assemble together and store at 2-8°C.

[0054] In one embodiment, a sample to be tested and mutant citrullinated vimentin-coupled magnetic microparticles can be mixed and incubated, followed by the addition of a phycoerythrin-labeled anti-human IgM antibody working solution. When anti-mutant citrullinated vimentin antibodies are present in the sample, a magnetic barcode microparticle-streptavidin-biotin-mutant citrullinated vimentin polypeptide-anti-mutant citrullinated vimentin antibody-anti-human IgM antibody complex is formed. Qualitative or quantitative analysis can then be performed using the phycoerythrin on the anti-human IgM antibody and the barcode on the magnetic barcode microparticles to detect the anti-mutant citrullinated vimentin antibodies in the sample.

[0055] 1. Detection of the feed ratio of magnetic microparticles coated with mutant citrullinated vimentin peptides E1, H1, and G5.

[0056] Experimental Example 1.1

[0057] The mutant citrullinated vimentin antigen used in this example is mutant citrullinated vimentin polypeptide E1. The amino acid sequence of E1 is SEQ ID No. 1, wherein X in the amino acid sequence represents citrulline Cit. The mutant citrullinated vimentin polypeptide E1 is modified with biotin.

[0058] The magnetic barcode particles used in this example are magnetic barcode particles coupled with streptavidin. One magnetic barcode particle has multiple barcode regions. The magnetic barcode particles in the suspension of magnetic barcode particles coupled with streptavidin are subjected to magnetic separation to obtain magnetic barcode particles. The magnetic barcode particles are resuspended in phosphate buffer with a pH of 7.0 and a concentration of 0.05 mol / L to a concentration of 2×10 5 The magnetic barcode microparticle solution was washed three times with phosphate buffer at pH 7.0 and a concentration of 0.05 mol / L and resuspended to 2×10 5 / mL magnetic barcode microparticle solution.

[0059] The biotinylated mutant citrullinated vimentin polypeptide E1 was coated with streptavidin magnetic particle working solution at a feed ratio of 1.25 ng / (piece × mL), 0.8 ng / (piece × mL), 0.6 ng / (piece × mL), 0.4 ng / (piece × mL), 0.3 ng / (piece × mL), 0.2 ng / (piece × mL), and 0.125 ng / (piece × mL), respectively. After shaking at room temperature for 2 hours, the supernatant was removed by magnetic separation and adsorption, and the supernatant was washed three times with the same volume of the above-mentioned phosphate buffer with a pH of 7.0 and a concentration of 0.05 mol / L to obtain mutant citrullinated vimentin-coupled magnetic particles.

[0060] Here, 1.25 ng / (microparticle × mL) means that on average, 1 mL of liquid contains 1.25 ng of biotinylated mutant citrullinated vimentin polypeptide and 1 magnetic barcode microparticle of streptavidin, or that on average, 1 mL of liquid contains 1 magnetic barcode microparticle to which 1.25 ng of biotinylated mutant citrullinated vimentin polypeptide is coupled via streptavidin.

[0061] The prepared mutant citrullinated vimentin-coupled magnetic microparticles were resuspended and blocked for 1 hour using phosphate buffer containing 2 mg / mL bovine serum albumin, 0.005 mg / mL proclin 300, pH 0.7, and a concentration of 0.05 mol / L.

[0062] After blocking, the mutant citrullinated vimentin-coupled magnetic particles were resuspended in phosphate buffer with a pH of 0.7 and a concentration of 0.05 mol / L to 10 5 magnetic barcoded particles / mL.

[0063] Use 10 for each test3 The working solution was prepared at a ratio of 100 magnetic barcode particles / mL buffer solution, wherein the buffer solution was a phosphate buffer solution containing 1 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.05 mol / L.

[0064] The phycoerythrin-labeled anti-human IgM antibody was diluted to 300 ng / mL with phosphate buffer containing 0.2 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, and 0.1 mg / mL proclin 300, at a pH of 6.5 and a concentration of 0.1 mol / L.

[0065] A zero-value control solution and a sample diluent were prepared, which were phosphate buffer containing 0.05 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, pH 7.0, and a concentration of 0.01 mol / L.

[0066] The anti-mutant citrullinated vimentin antibody detection kit includes: mutant citrullinated vimentin-coupled magnetic particles 6.5 mL / bottle, phycoerythrin-labeled anti-human IgM antibody 3.5 mL / bottle, and zero-value control working solution 1.0 mL / tube. After packaging, assemble together and store at 2-8°C.

[0067] The anti-mutant citrullinated vimentin antibody detection kit prepared above was used to detect a sample with a value of 0, 10 positive samples from patients with rheumatoid arthritis who had anti-mutant citrullinated vimentin antibodies in their blood, and 10 negative samples from patients without rheumatoid arthritis. The assay was performed using the MCLIA-800 fully automated multiplex liquid phase chip immunoassay system of Zhuhai Livzon Diagnostics Co., Ltd. The assay process is as follows:

[0068] Step 1: The MCLIA-800 fully automatic multiplex liquid chip immunoassay system draws 15 μL of the sample and automatically dilutes it 13 times.

[0069] Step 2: Take 50 μL of the diluted sample, add 100 μL of mutant citrullinated vimentin-coupled magnetic particles, incubate at 37°C for 15 minutes, perform magnetic separation after incubation, and wash three times.

[0070] Step 3: Add 50 μL of phycoerythrin-labeled anti-human IgM antibody, incubate at 37°C for 15 min, perform magnetic separation after incubation, and wash three times.

[0071] Step 4: Add 150 μL of signal enhancement solution, read the signal value, and calculate the signal-to-noise ratio. The anti-mutant citrullinated vimentin antibody test results are shown in Table 1.

[0072] Experimental Example 1.2

[0073] The mutant citrullinated vimentin antigen was mutant citrullinated vimentin polypeptide H1. The amino acid sequence of H1 is SEQ ID No. 2, where X in the amino acid sequence represents citrulline (Cit). The remaining conditions were the same as those in Experimental Example 1. The results of the anti-mutant citrullinated vimentin antibody test are shown in Table 2.

[0074] Experimental Example 1.3

[0075] The mutant citrullinated vimentin antigen was the mutant citrullinated vimentin polypeptide G5. The amino acid sequence of G5 is SEQ ID No. 3, where X in the amino acid sequence represents citrulline (Cit). The remaining conditions were the same as those in Experimental Example 1. The results of the anti-mutant citrullinated vimentin antibody test are shown in Table 3.

[0076] Comparative Example 1

[0077] The mutant citrullinated vimentin antigen was a commercially available mutant citrullinated vimentin. The remaining conditions were the same as those in Experimental Example 1. The results of the anti-mutant citrullinated vimentin antibody test are shown in Table 4.

[0078] Table 1. Experimental Example 1.1 Detection of the final feed ratio of magnetic microparticles coated with mutant citrullinated vimentin polypeptide E1

[0079]

[0080]

[0081] Table 2. Experimental Example 1.2 Detection of the final feed ratio of the magnetic microparticles coated with mutant citrullinated vimentin polypeptide H1

[0082]

[0083] Table 3. Experimental Example 1.3 Detection of the final feed ratio of mutant citrullinated vimentin polypeptide G5 coated magnetic microparticles

[0084]

[0085] Table 4. Comparative Example 1: Testing of the feed ratio of commercially available mutant citrullinated vimentin-coated magnetic microparticles

[0086]

[0087]

[0088] Result analysis:

[0089] Because a dissociation equilibrium exists between the biotin-modified mutant citrullinated vimentin peptide and the streptavidin-modified magnetic barcode microparticles, and because the magnetic barcode microparticles and the mutant citrullinated vimentin peptide cannot always maintain sufficient contact, the coupled mutant citrullinated vimentin peptide on the magnetic barcode microparticles may be uneven and vary greatly in number. Therefore, the amount of amino acids present in the coupling buffer can only be calculated after dissociation during the coupling process, thereby calculating the amount of mutant citrullinated vimentin peptide bound to the magnetic barcode microparticles. Ignoring the fact that a small number of magnetic barcode microparticles have less coupled mutant citrullinated vimentin peptide, the calculated average ratio of mutant citrullinated vimentin peptide, magnetic barcode microparticles, and buffer is the sole criterion for representing the amount of antigen coupled to the mutant citrullinated vimentin peptide-coupled magnetic microparticles.

[0090] Based on the comprehensive judgment of the signal-to-noise ratio and signal value, the higher the signal-to-noise ratio of the positive sample and the lower the signal-to-noise ratio of the negative sample, the better the effect.

[0091] Table 1 shows that the optimal antigen-to-magnetic-particle ratio for magnetic microparticles coated with mutant citrullinated vimentin peptide E1 is 0.6 ng / (particle × mL). The signal-to-noise ratio for positive samples is 6.84, while that for negative samples is 1.67. The upper limit of the antigen-to-magnetic-particle ratio is 0.8 ng / (particle × mL), and the lower limit is 0.4 ng / (particle × mL).

[0092] Table 2 shows that the optimal antigen-to-magnetic-particle ratio for magnetic microparticles coated with mutant citrullinated vimentin peptide H1 is 0.3 ng / (particle × mL). The signal-to-noise ratio for positive samples is 7.01, and for negative samples is 1.65. The upper limit of the antigen-to-magnetic-particle ratio is 0.4 ng / (particle × mL), and the lower limit is 0.2 ng / (particle × mL).

[0093] Table 3 shows that the optimal antigen-to-magnetic-particle ratio for magnetic microparticles coated with mutant citrullinated vimentin peptide G5 is 0.3 ng / (particle × mL). The signal-to-noise ratio for positive samples is 6.90, while that for negative samples is 1.64. The upper limit of the antigen-to-magnetic-particle ratio is 0.4 ng / (particle × mL), and the lower limit is 0.2 ng / (particle × mL).

[0094] Table 4 shows that the optimal antigen-to-magnetic-particle ratio for commercially available mutant citrullinated vimentin-coated magnetic microparticles was 0.3 ng / (particle × mL), resulting in a signal-to-noise ratio of 4.07 for positive samples and 1.83 for negative samples. The upper limit of the antigen-to-magnetic-particle ratio was 0.4 ng / (particle × mL), and the lower limit was 0.2 ng / (particle × mL). The signal-to-noise ratio of the best positive sample for commercially available mutant citrullinated vimentin was lower than the signal-to-noise ratios of the worst positive samples for mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5, indicating that the detection performance of the anti-mutant citrullinated vimentin antibody for commercially available mutant citrullinated vimentin was inferior to that of mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, or mutant citrullinated vimentin polypeptide G5.

[0095] 2. Detection of the mixing ratio of mutant citrullinated vimentin peptide-coupled magnetic microparticles

[0096] Experimental Example 2.1

[0097] The mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles prepared in Experimental Example 1.1 were used to prepare an anti-mutant citrullinated vimentin antibody detection kit at a feed ratio of 0.6 ng / (microparticle × mL). The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 5.

[0098] Experimental Example 2.2

[0099] The mutant citrullinated vimentin polypeptide H1-coupled magnetic microparticles prepared in Experimental Example 1.2 were used to prepare an anti-mutant citrullinated vimentin antibody detection kit at a feed ratio of 0.3 ng / (microparticle × mL). The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 6.

[0100] Experimental Example 2.3

[0101] The mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles prepared in Experimental Example 1.3 were used to prepare an anti-mutant citrullinated vimentin antibody detection kit at a feed ratio of 0.3 ng / (particle × mL). The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 7.

[0102] Experimental Example 2.4

[0103] The mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles prepared in Experimental Example 1.1 were mixed at a final feed ratio of 0.6 ng / (unit × mL) and the mutant citrullinated vimentin polypeptide H1-coupled magnetic microparticles prepared in Experimental Example 1.2 were mixed at a final feed ratio of 0.3 ng / (unit × mL) to prepare an anti-mutant citrullinated vimentin antibody detection kit. The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody detection kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 8.

[0104] Experimental Example 2.5

[0105] The mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles prepared in Experimental Example 1.1 were mixed at a final feed ratio of 0.6 ng / (unit × mL) and the mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles prepared in Experimental Example 1.3 were mixed at a final feed ratio of 0.3 ng / (unit × mL) to prepare an anti-mutant citrullinated vimentin antibody detection kit. The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody detection kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 9.

[0106] Experimental Example 2.6

[0107] The mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles prepared in Experimental Example 1.2 and the mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles prepared in Experimental Example 1.3 were mixed at a final ratio of 0.3 ng / (unit × mL) to prepare an anti-mutant citrullinated vimentin antibody detection kit. The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody detection kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 10.

[0108] Example 1

[0109] The mutant citrullinated vimentin polypeptide E1-coupled magnetic microparticles prepared in Experimental Example 1.1 were mixed at a final feed ratio of 0.6 ng / (unit × mL), the mutant citrullinated vimentin polypeptide H1-coupled magnetic microparticles prepared in Experimental Example 1.2 were mixed at a final feed ratio of 0.3 ng / (unit × mL), and the mutant citrullinated vimentin polypeptide G5-coupled magnetic microparticles prepared in Experimental Example 1.3 were mixed at a final feed ratio of 0.3 ng / (unit × mL) to prepare an anti-mutant citrullinated vimentin antibody detection kit. The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody detection kit was used to test 50 positive samples and 51 negative samples, and the sensitivity and specificity were calculated based on the test results. The test results are shown in Table 11.

[0110] Comparative Example 2

[0111] The commercially available mutant citrullinated vimentin-coupled magnetic microparticles prepared in Experimental Example 1.4 were used at a feed ratio of 0.3 ng / (microparticle × mL) to prepare an anti-mutant citrullinated vimentin antibody detection kit. The remaining conditions were the same as those in Experimental Example 1.1. The prepared anti-mutant citrullinated vimentin antibody kit was used to test 50 positive samples and 51 negative samples. The sensitivity and specificity were calculated based on the test results. The test results are shown in Table 12.

[0112] Among them, the test results of the eight kits for 50 positive samples are shown in Table 13, the test results of the eight kits for 50 negative samples are shown in Table 14, and the sensitivity, specificity, false positive and false negative results of each kit are shown in Table 15.

[0113] Table 5. Sensitivity and specificity test of mutant citrullinated vimentin peptide E1

[0114]

[0115] Table 6. Sensitivity and specificity test of mutant citrullinated vimentin peptide H1

[0116]

[0117] Table 7. Sensitivity and specificity test of mutant citrullinated vimentin peptide G5

[0118]

[0119] Table 8. Sensitivity and specificity test of mutant citrullinated vimentin peptide E1+H1

[0120]

[0121] Table 9. Sensitivity and specificity test of mutant citrullinated vimentin peptide E1+G5

[0122]

[0123] Table 10. Sensitivity and specificity test of mutant citrullinated vimentin peptide H1+G5

[0124]

[0125]

[0126] Table 11. Sensitivity and specificity test of mutant citrullinated vimentin peptide E1+H1+G5

[0127]

[0128] Table 12. Sensitivity and specificity test of mutant citrullinated vimentin

[0129]

[0130] Table 13. Positive test results of eight kits

[0131]

[0132]

[0133] Table 14. Negative test results of eight kits

[0134]

[0135]

[0136]

[0137] Table 15. Sensitivity, specificity, false positive and false negative test results of eight kits

[0138]

[0139] Result analysis:

[0140] From the data in Table 15, it can be seen that the sensitivity and specificity of the anti-mutant citrullinated vimentin antibody detection kit prepared by mixing mutant citrullinated vimentin polypeptide E1 coupled to magnetic microparticles at a final feed ratio of 0.6 ng / (piece × mL), mutant citrullinated vimentin polypeptide H1 coupled to magnetic microparticles at a final feed ratio of 0.3 ng / (piece × mL), and mutant citrullinated vimentin polypeptide G5 coupled to magnetic microparticles at a final feed ratio of 0.3 ng / (piece × mL) are better than those of the commercially available mutant citrullinated vimentin coupled to magnetic microparticles at a feed ratio of 0.3 ng / (piece × mL) for preparing the anti-mutant citrullinated vimentin antibody detection kit. The false negative rate was reduced from 30.00% to 10.00%, a decrease of 66.7%; the false positive rate was reduced from 29.41% to 10.00%, a decrease of 66.0%, and the sensitivity and specificity were greatly improved.

[0141] The inventors speculate that the mutant citrullinated vimentin produced in the blood of rheumatoid arthritis patients is a mixture of antibodies (polyclonal antibodies) targeting different antigenic epitopes. The types and proportions of antibodies produced by different individuals are different. Artificially synthesized mutant citrullinated vimentin antigens / genetically engineered antigens only have some antigenic determinants fully exposed, and it is very easy to miss detection when using a single antigen for immunological testing, resulting in low sensitivity. By using mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 in combination, as many specific and effective antigenic epitopes as possible are covered.

[0142] The combined use of mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 had the highest sensitivity and strongest specificity, and maintained specificity above 90%. At this time, the final feed ratios of the three antigens were 0.6 ng / (piece × mL) of mutant citrullinated vimentin polypeptide E1 and magnetic barcode microparticles, 0.3 ng / (piece × mL) of mutant citrullinated vimentin polypeptide H1 and magnetic barcode microparticles, and 0.3 ng / (piece × mL) of mutant citrullinated vimentin polypeptide G5 and magnetic barcode microparticles.

[0143] Because the mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 used in the present application are all 17 amino acids in length, after modification with biotin, the spatial structure of biotin is smaller, which does not affect the exposure of the antigenic site on the polypeptide, and is more conducive to the connection of the polypeptide to the magnetic barcode particles, thereby improving the utilization rate of the polypeptide and the magnetic barcode particles. If other methods are used to connect the polypeptide to the magnetic barcode particles, the exposure of the antigenic epitope will be affected, the polypeptide will not be able to be connected to the magnetic barcode particles, the utilization rate of the polypeptide or the magnetic barcode particles will be low, and the sensitivity and specificity of the kit for detecting antibodies against mutant citrullinated vimentin will be seriously affected.

[0144] In this application, different mutant citrullinated vimentin polypeptides are first coupled to different magnetic barcode microparticles to form different mutant citrullinated vimentin-coupled magnetic microparticles, and then the different mutant citrullinated vimentin-coupled magnetic microparticles are mixed at a specific final feed ratio. Compared to simultaneously coupling different mutant citrullinated vimentin polypeptides to magnetic barcode microparticles in the same liquid, the mutant citrullinated vimentin polypeptides coupled to the magnetic barcode microparticles are more uniform, avoiding the large steric hindrance and poor uniformity that occurs when multiple mutant citrullinated vimentin polypeptides are coupled to the same magnetic barcode microparticle, further improving the sensitivity and specificity of the test kit detection results.

[0145] 3. Screening of the final feed ratio of mutant citrullinated vimentin peptides E1, H1, and G5 to magnetic particles

[0146] Comparative Example 3

[0147] Orthogonal experiments were conducted using the final feed ratios of mutant citrullinated vimentin polypeptide E1 to magnetic barcode microparticles, mutant citrullinated vimentin polypeptide H1 to magnetic barcode microparticles, and mutant citrullinated vimentin polypeptide G5 to magnetic barcode microparticles according to the combinations 1-9 shown in Table 16. The remaining procedures were the same as in Example 1. The sensitivity and specificity test results corresponding to the combinations 1-9 are shown in Tables 17-26.

[0148] Table 16. Experimental table of final feed ratios of mutant citrullinated vimentin peptides E1, H1, G5 and magnetic barcode microparticles

[0149]

[0150] Table 17. Sensitivity and specificity test of group 1 in comparative example 3

[0151]

[0152] Table 18. Comparative Example 3 Group 2 Sensitivity and Specificity Test

[0153]

[0154] Table 19. Comparative Example 3 Group 3 Sensitivity and specificity test

[0155]

[0156] Table 20. Comparative Example 3 Group 4 Sensitivity and Specificity Test

[0157]

[0158] Table 21. Comparative Example 3 Group 5 Sensitivity and Specificity Test

[0159]

[0160] Table 22. Sensitivity and specificity test of group 6 in comparative example 3

[0161]

[0162]

[0163] Table 23. Sensitivity and specificity test of group 7 in comparative example 3

[0164]

[0165] Table 24. Comparative Example 3 Group 8 Sensitivity and Specificity Test

[0166]

[0167] Table 25. Comparative Example 3 Group 9 Sensitivity and Specificity Test

[0168]

[0169] Table 26. Sensitivity and specificity test results of mutant citrullinated vimentin peptide E1+H1+G5 mixed antigen at different final feed ratios

[0170]

[0171]

[0172] Result analysis:

[0173] In Table 26, in Group 1, Group 3, and Group 4, false positive results were superimposed due to excessively high feed ratios of one or more polypeptides in mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5, resulting in the specificity of the final test result being less than 90%, and false positives increased to varying degrees.

[0174] In Groups 5, 7, and 9, false negative results were superimposed due to the low feed ratio of one or more polypeptides in the mutant citrullinated vimentin polypeptide E1, the mutant citrullinated vimentin polypeptide H1, and the mutant citrullinated vimentin polypeptide G5, resulting in the sensitivity of the final test results being lower than 90%, and the false negative results increased to varying degrees.

[0175] In Groups 2, 6, and 8, the specificity was lower than 90%, and the false positive rate increased by nearly 100%, due to the high or low feed rate of one or more polypeptides in the mutant citrullinated vimentin polypeptide E1, the mutant citrullinated vimentin polypeptide H1, and the mutant citrullinated vimentin polypeptide G5.

[0176] The inventors speculate that the mutant citrullinated vimentin polypeptide E1, mutant citrullinated vimentin polypeptide H1, and mutant citrullinated vimentin polypeptide G5 selected in this application all contain different antigenic determinants. The binding rate constants of the antigenic determinants of the three antigens to their corresponding antibodies are constant and distinct. The signal value of the concentration level of each antigen is positively correlated with the signal value level. Immunoassay methodology determines the positive or negative nature of the test result by comparing the signal value with a pre-set critical value. The three antigens have a complementary relationship in the detection of antibodies in the human population. The final feed ratio of the three antigens is particularly important when the three antigens are mixed together. Sensitivity and specificity are highest only when the mixed antigen test results are adjusted to similar signal value levels, and the positive judgment values ​​of the three antigens are close. When the concentration of a particular antigen is relatively low, the signal value of the sample that can only be detected by that antigen will fall below the critical value, resulting in a false negative, missed detection, and reduced sensitivity. Similarly, when the concentration of a particular antigen is relatively high, the overall signal value of the sample is increased, the critical point is shifted upward, and false positives are misclassified, resulting in false detections and reduced specificity.

[0177] From Table 15 and Table 26, it can be seen that the optimal combination is the final feed ratio of mutant citrullinated vimentin polypeptide E1 to magnetic barcode microparticles of 0.6 ng / (piece × mL), the final feed ratio of mutant citrullinated vimentin polypeptide H1 to magnetic barcode microparticles of 0.3 ng / (piece × mL), and the final feed ratio of mutant citrullinated vimentin polypeptide G5 to magnetic barcode microparticles of 0.3 ng / (piece × mL). At this time, the sensitivity and specificity of anti-mutant citrullinated vimentin antibody detection are the highest, with a sensitivity of 90.00%, a specificity of 90.20%, a false positive rate of 9.80%, and a false negative rate of 10.00%. Compared with the commercially available mutant citrullinated vimentin-coupled magnetic microparticles with a final feed ratio of 0.3 ng / (piece × mL), the false positive rate is 29.41%, the false negative rate is 30.00%, and both false positives and false negatives are reduced by 66.67%, which is a significant effect.

[0178] The present invention uses a final feeding ratio of mutant citrullinated vimentin polypeptide E1 to magnetic barcode microparticles of 0.6 ng / (piece × mL), a final feeding ratio of mutant citrullinated vimentin polypeptide H1 to magnetic barcode microparticles of 0.3 ng / (piece × mL), and a final feeding ratio of mutant citrullinated vimentin polypeptide G5 to magnetic barcode microparticles of 0.3 ng / (piece × mL) to replace commercially available mutant citrullinated vimentin-coupled magnetic microparticles, thereby further improving the sensitivity and specificity of anti-mutant citrullinated vimentin antibody detection in sera from different patients.

[0179] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. SEQUENCE LISTING <110> Zhuhai Livzon Diagnostics Co., Ltd. <120> Anti-mutant citrullinated vimentin antibody detection kit and its application <130> 20220427 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 17 <212> PRT <213> artificial synthesis <220> <221> misc_feature <222> (2)..(2) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (7)..(7) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (15)..(15) <223> Xaa can be any naturally occurring amino acid <400> 1 Ser Xaa Pro Ser Ser Ser Xaa Ser Tyr Val Thr Thr Ser Thr Xaa Thr 1 5 10 15 Tyr <210> 2 <211> 17 <212> PRT <213> Synthetic <220> <221> misc_feature <222> (9)..(9) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (14)..(14) <223> Xaa can be any naturally occurring amino acid <400> 2 Thr Tyr Ser Leu Gly Ser Ala Leu Xaa Pro Ser Thr Ser Xaa His Leu 1 5 10 15 Tyr <210> 3 <211> 17 <212> PRT <213> Synthetic <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (11)..(11) <223> It can be any naturally occurring amino acid. <400> 3 Asn Only Be Leu Only Be Leu Asp Leu Glu Only Lys Val Glu Ser Leu 1 5 10 15 Gln

Claims

1. A kit for detecting mutant citrullinated vimentin antibodies, characterized in that: include: A mutant citrullinated vimentin-coupled magnetic particle working solution, which consists of mutant citrullinated vimentin-coupled magnetic particles and a buffer solution; a tracer-labeled antibody working solution, which is composed of a tracer-labeled anti-human IgM antibody and a diluent; The mutant citrullinated vimentin-coupled magnetic particles are prepared by mixing mutant citrullinated vimentin polypeptide E1-coupled magnetic particles, mutant citrullinated vimentin polypeptide H1-coupled magnetic particles, and mutant citrullinated vimentin polypeptide G5-coupled magnetic particles; the mutant citrullinated vimentin polypeptide E1 has the amino acid sequence shown in SEQ ID No. 1; the mutant citrullinated vimentin polypeptide H1 has the amino acid sequence shown in SEQ ID No. 2; and the mutant citrullinated vimentin polypeptide G5 has the amino acid sequence shown in SEQ ID No.

3. The final feed ratio of the mutant citrullinated vimentin polypeptide E1 to the magnetic particles in the mutant citrullinated vimentin polypeptide E1 coupled magnetic particles is 0.6 ng / (piece × mL); The final feed ratio of the mutant citrullinated vimentin polypeptide H1 to the magnetic particles in the mutant citrullinated vimentin polypeptide H1-coupled magnetic particles is 0.3 ng / (piece × mL); The final feeding ratio of the mutant citrullinated vimentin polypeptide G5 to the magnetic particles in the mutant citrullinated vimentin polypeptide G5-coupled magnetic particles is 0.3 ng / (piece×mL).

2. The kit according to claim 1, wherein The mutant citrullinated vimentin polypeptide E1, the mutant citrullinated vimentin polypeptide H1, and the mutant citrullinated vimentin polypeptide G5 are all modified with biotin.

3. The kit according to claim 1, wherein The magnetic particles are magnetic barcode particles, and the magnetic barcode particles are modified with streptavidin.

4. The kit according to claim 3, wherein The structure of the mutant citrullinated vimentin polypeptide E1 coupled to the magnetic microparticles is mutant citrullinated vimentin polypeptide E1-biotin-streptavidin-magnetic barcode microparticles; The structure of the mutant citrullinated vimentin polypeptide H1 coupled to the magnetic particles is mutant citrullinated vimentin polypeptide H1-biotin-streptavidin-magnetic barcode particles; The structure of the mutant citrullinated vimentin polypeptide G5 coupled to the magnetic particles is mutant citrullinated vimentin polypeptide G5-biotin-streptavidin-magnetic barcode particles.

5. The kit according to claim 4, characterized in that The ratio of the mutant citrullinated vimentin-coupled magnetic particles to the buffer in the mutant citrullinated vimentin-coupled magnetic particle working solution is 1000 magnetic barcode particles / mL buffer.

6. The kit according to claim 5, characterized in that The buffer solution is a phosphate buffer solution containing 1 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.05 mol / L.

7. The kit according to claim 1, wherein The tracer is phycoerythrin, and the diluent is 0.2 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.1 mg / mL proclin300, and a phosphate buffer solution with a pH of 6.5 and a concentration of 0.1 mol / L.

8. The kit according to claim 1, wherein The kit also includes a zero-value control solution and a sample diluent. The zero-value control solution and the sample diluent are phosphate buffer containing 0.05 mg / mL bovine serum albumin, 0.05 mg / mL sodium chloride, 0.01 mg / mL Tween 20, a pH of 7.0, and a concentration of 0.01 mol / L.

9. Use of the kit according to any one of claims 1 to 8 in detecting in vitro anti-mutant citrullinated vimentin antibodies for non-disease diagnosis purposes.

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