Anti-Mi-2 antibody detection reagent and detection kit

Abstract

The invention relates to an anti-Mi-2 antibody detection reagent and a detection kit.The anti-Mi-2 antibody detection reagent comprises a first antigen fragment and a second antigen fragment, the first antigen fragment is a biotin-labeled recombinant Mi-2 antigen protein fragment with the amino acid sequence shown as SEQ ID NO.1, and the second antigen fragment is a biotin-labeled recombinant Mi-2 antigen protein fragment with the amino acid sequence shown as SEQ ID NO.2. And the second antigen fragment is a biotin-labeled recombinant Mi-2 antigen protein fragment with an amino acid sequence as shown in SEQ ID NO.2. The anti-Mi-2 antibody detection reagent can effectively reduce the false positive of the anti-Mi-2 antibody in a serum sample detected by a magnetic particle chemiluminescence method, can improve the positive detection rate, and provides a more economical and reliable choice for clinical diagnosis and research of dermatomyositis.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagents, specifically relating to an anti-Mi-2 antibody detection reagent and detection kit. Background Technology

[0002] Anti-Mi-2 antibody is a specific autoantibody for dermatomyositis. Its positive rate in dermatomyositis patients is about 4%-18%, and its specificity is as high as 98%-100%. Anti-Mi-2 antibody detection is one of the important serological markers for the diagnosis of dermatomyositis.

[0003] Patients with dermatomyositis who are positive for anti-Mi-2 antibodies typically present with typical clinical features of dermatomyositis, such as skin symptoms (e.g., V-shaped or scarf-shaped rashes on the chest, neck, and shoulders) and muscle involvement. Furthermore, these patients usually respond well to glucocorticoid therapy and have a good prognosis. Anti-Mi-2 antibodies are primarily associated with dermatomyositis and are rarely found in other autoimmune diseases (such as systemic lupus erythematosus and polymyositis). Therefore, the detection of anti-Mi-2 antibodies can help differentiate dermatomyositis from other types of myositis or connective tissue diseases.

[0004] The high specificity of anti-Mi-2 antibodies and their correlation with typical dermatomyositis symptoms make them an important tool for diagnosing dermatomyositis. The detection results of anti-Mi-2 antibodies can help clinicians differentiate dermatomyositis from other similar diseases and provide patients with personalized treatment plans.

[0005] Common antibody detection methods include enzyme-linked immunosorbent assay (ELISA), Western blotting, solid-phase radioimmunoassay, and magnetic particle chemiluminescence immunoassay. ELISA is the most widely used, but it is complex, requiring coating and washing processes, which are time-consuming and labor-intensive. Magnetic particle chemiluminescence immunoassay utilizes the free energy released from a chemical reaction to excite intermediates, causing them to release photons as they return to their ground state. Quantitative analysis by measuring these photons offers rapid, automated, and highly sensitive results, along with the specificity of immunoassay.

[0006] The detection of anti-Mi-2 antibodies using magnetic microparticle chemiluminescence assay requires the specific binding of the target antigen of biotinylated anti-Mi-2 antibody to the anti-Mi-2 antibody in the sample to be tested. The resulting fluorescence signal, generated by the reaction of streptavidin magnetic beads and alkaline phosphatase-labeled human IgG antibody, indicates the content of anti-Mi-2 antibody.

[0007] The target antigen of anti-Mi-2 antibodies is the Mi-2 antigen protein, located in the nucleus and nucleoplasm of cells, with a relative molecular mass of 218 kDa. It is an alias for CHD4 (Chromodomain-Helicase-DNA-binding protein 4) and is a core component of the Mi-2 / NuRD (Nucleosome Remodeling and Deacetylase) complex. Most existing anti-Mi-2 antibody detection kits use the full-length Mi-2 protein, which consists of 1912 amino acids. This protein has a relatively large molecular weight, and recombinant expression using HEK293T yields low protein levels. Furthermore, it is prone to false positives due to non-specific binding (such as cross-reactivity with other CHD family proteins), failing to meet the performance requirements of diagnostic reagents. Summary of the Invention

[0008] The technical problem solved by the present invention is to overcome the shortcomings of the prior art and provide an anti-Mi-2 antibody detection reagent that can effectively reduce false positives when detecting anti-Mi-2 antibodies in serum samples by magnetic particle chemiluminescence method.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides an anti-Mi-2 antibody detection reagent, which includes a first antigen fragment and a second antigen fragment, wherein the first antigen fragment is a recombinant Mi-2 antigen protein fragment with a biotin-labeled amino acid sequence as shown in SEQ ID NO.1, and the second antigen fragment is a recombinant Mi-2 antigen protein fragment with a biotin-labeled amino acid sequence as shown in SEQ ID NO.2.

[0011] In some embodiments, the mass ratio of the first antigen fragment to the second antigen fragment is (0.8 to 1.2):1, for example, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1 or any mass ratio between any two of the above.

[0012] In some embodiments, the solvent of the detection reagent is a 0.01-0.05M phosphate buffer solution with a pH of 7.2-8.0 containing 0.5wt%-1.5wt% bovine serum albumin.

[0013] In some embodiments, the sum of the concentrations of the first antigen fragment and the second antigen fragment in the detection reagent is 1 to 10 μg / mL, for example, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, or any concentration between any two of the above.

[0014] A second aspect of the present invention provides an anti-Mi-2 antibody detection kit, the detection kit comprising the above-mentioned detection reagents for the anti-Mi-2 antibody.

[0015] In some embodiments, the test kit further includes magnetic microparticle reagent, alkaline phosphatase-labeled anti-human IgG antibody solution, and chemiluminescent substrate solution.

[0016] In some embodiments, the magnetic microparticle reagent is a streptavidin-labeled magnetic microparticle solution.

[0017] In some embodiments, the concentration of the streptavidin-labeled magnetic microparticle solution is 0.1–1 mg / mL.

[0018] In some embodiments, the magnetic microparticles have a particle size of 0.1 to 0.5 μm.

[0019] In some embodiments, the solvent for the streptavidin-labeled magnetic microparticle solution is a phosphate buffer solution with a pH of 7.2 to 7.5 containing 1–2 wt% BSA, 1–3 wt% glycerol and 2–5 wt% mannitol.

[0020] In some embodiments, the concentration of the alkaline phosphatase-labeled anti-human IgG antibody solution is 0.1–1 μg / mL.

[0021] In some embodiments, the solvent for the alkaline phosphatase-labeled anti-human IgG antibody solution is a 0.01-0.05M phosphate buffer with a pH of 7.2-8.0 containing 0.5wt%-1.5wt% bovine serum albumin.

[0022] In some embodiments, the luminescent substrate solution is an AMPPD solution.

[0023] In some embodiments, the test kit further includes one or more of a sample diluent, a calibrator, and a quality control, wherein the sample diluent is a 0.01–0.05 M phosphate buffer with a pH of 7.2–8.0.

[0024] According to some implementation methods, the concentrations of the calibrators are 0, 5 RU / mL, 20 RU / mL, 100 RU / mL, 200 RU / mL, 400 RU / mL, and 1600 RU / mL.

[0025] According to some implementation methods, the concentration of the quality control sample is 20 RU / mL or 400 RU / mL.

[0026] A third aspect of the present invention also provides an anti-Mi-2 antibody detection system, which includes the above-mentioned anti-Mi-2 antibody detection kit and a fully automated chemiluminescence analyzer.

[0027] In some embodiments, the fully automated chemiluminescence analyzer is the SMART 6500 fully automated chemiluminescence analyzer from Jiangsu Haooubo Biomedical Co., Ltd. (HOB).

[0028] A fourth aspect of the present invention also provides a method for detecting anti-Mi-2 antibodies in serum samples using the above-described anti-Mi-2 antibody detection reagent, the above-described anti-Mi-2 antibody detection kit, or the above-described anti-Mi-2 antibody detection system.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] This invention improves upon the low immunogenicity and cross-reactivity of the full-length Mi-2 antigen protein by using a reactive Mi-2 antigen fragment instead of the full-length Mi-2 antigen protein for biotin labeling. This effectively avoids false positives caused by cross-reactivity and increases the positive detection rate, thus simultaneously improving the sensitivity and specificity of anti-Mi-2 antibody detection in clinical serum samples, providing strong support for the rapid diagnosis of dermatomyositis. Furthermore, compared to the full-length Mi-2 antigen protein, the reactive Mi-2 antigen fragment is easier to produce and yields higher quantities, significantly reducing production costs and providing a more economical and reliable option for the production and application of anti-Mi-2 antibody detection reagents and kits. Attached Figure Description

[0031] Figure 1 The image shows the biotinylation result of the recombinant Mi-2 antigen protein fragment (amino acid sequence as shown in SEQ ID NO.1);

[0032] Figure 2 The image shows the biotinylation results of the recombinant Mi-2 antigen protein fragment (amino acid sequence as shown in SEQ ID NO.2);

[0033] Figure 3 The image shows the biotinylation results of the recombinant Mi-2 antigen protein fragment (amino acid sequence as shown in SEQ ID NO.3);

[0034] Figure 4 This is the standard curve of the detection kit in Example 1;

[0035] Figure 5 The standard curve for the test kit in Comparative Example 1;

[0036] Figure 6 The standard curve for the detection kit in Comparative Example 2;

[0037] Figure 7 The standard curve for the detection kit used in Comparative Example 3;

[0038] Figure 8The standard curve for the detection kit in Comparative Example 4;

[0039] Figure 9 The standard curve for the detection kit used in Comparative Example 5;

[0040] Figure 10 This is the standard curve for the test kit used in Comparative Example 6. Detailed Implementation

[0041] To improve the sensitivity and specificity of detecting anti-Mi-2 antibodies in clinical serum samples, the inventors of this application conducted bioinformatics analysis on the antigenic epitopes of the Mi-2 antigen protein (UniProtKB-Q14839(CHD4_HUMAN)), designed several Mi-2 antigen protein fragments, produced recombinant Mi-2 antigen protein fragments using an E. coli system, and initially screened the binding efficiency of the recombinant Mi-2 antigen protein fragments to antibodies using ELISA. The obtained sequences were then biotinylated and used to develop antigen reagents for chemiluminescent detection methods of anti-Mi-2 antibodies. After extensive research and experimental screening and verification, the technical solution of this application was obtained.

[0042] The present invention will be further described below with reference to embodiments and comparative examples. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0043] In the following examples and comparative examples, the implementation conditions not specified are standard conditions in the industry, and the reagents not specified are all commercially available products. The detection instrument was a SMART6500 fully automated chemiluminescence analyzer from Jiangsu Haooubo Biomedical Co., Ltd. (HOB).

[0044] The three biotinylated Mi-2 antigen protein fragments (the amino acid sequences of the Mi-2 antigen protein fragments are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively) involved in the following examples and comparative examples were prepared by the following methods.

[0045] (1) Nanjing Genscript Biotech Co., Ltd. optimized the codons of the Mi-2 antigen protein fragment provided by the inventors of this application, and attached an Avi Tag tag (amino acid sequence GLNDIFEAQKIEWHE) to the N-terminus (nitrogen end) of the Mi-2 antigen protein fragment, synthesized the gene, and inserted it before the NdeⅠ restriction site of the pET-28a(+)-TEV vector to obtain a recombinant Mi-2 antigen protein fragment expression plasmid with the AviTag tag.

[0046] (2) The recombinant Mi-2 antigen protein fragment expression plasmid with Avi Tag tag was transformed into the expression strain BL21(DE3). The transformation steps are as follows: Take 50 μL of competent Escherichia coli cells, add an appropriate amount of plasmid, incubate on ice for 30 min, then heat shock at 42℃ for 90 s, immediately put back on ice, and incubate on ice for 2 min; add 500 μL of LB medium, and culture on a shaker at 37℃ for 60 min. Take 100 μL and spread it on LB solid medium containing kanamycin (50 μg / mL), and incubate upside down at 37℃ overnight.

[0047] (3) Select a single clone of the strain and add it to 100 mL of LB medium containing kanamycin. Incubate overnight at 37°C and 220 rpm. Take 10 mL of the bacterial culture and add it to 1000 mL of LB medium. Add the corresponding antibiotic at a ratio of 1:1000. Incubate at 37°C and 220 rpm with shaking until the OD600 is 0.6-0.8 (about 3 h). Then add IPTG to make the final concentration 1 mmol / L. Incubate at 16°C and 180 rpm for about 20 h. Centrifuge at 4°C and 8000 rpm for 10 min to collect the bacterial pellet.

[0048] (4) After resuspending the bacterial pellet with bacterial lysis buffer (20mM Na2HPO4, 500mM NaCl, 5mM imidazole, 1% Triton X-100, pH=7.5), the cells were lysed using an ultrasonic cell disruptor. The lysed cells were centrifuged for 30 minutes and the supernatant was collected.

[0049] (5) Obtain the target protein with high purity by metal ion affinity chromatography and size exclusion chromatography. Specifically, follow the instructions provided by the chromatographic column manufacturer or adjust the operation method according to conventional methods in this field:

[0050] Prepare the affinity chromatography column: Select a suitable metal ion affinity chromatography column according to experimental requirements, such as a Ni-NTA or Co-NTA column. If using Ni-NTA resin, ensure that it has been equilibrated with a buffer solution (e.g., 20 mM Na2HPO4, 500 mM NaCl, 20 mM imidazole, pH = 7.5).

[0051] Sample loading: The supernatant (containing the target protein) after ultrasonic disruption and centrifugation is added to the affinity chromatography column. The sample is slowly loaded onto the column using a peristaltic pump or gravity flow, ensuring full contact between the sample and the resin.

[0052] Washing impurities: Wash the chromatography column with an appropriate amount of washing buffer (e.g., 10 column volumes of equilibration buffer). The purpose of this step is to remove impurity proteins that are weakly bound to the target protein, while retaining the target protein that is tightly bound to metal ions.

[0053] Elution of the target protein: The target protein is eluted with an elution buffer (e.g., 20 mM Na2HPO4, 500 mM NaCl, 250 mM imidazole, pH = 7.5). Increasing the imidazole concentration will competitively bind metal ions, thereby eluting the target protein from the resin.

[0054] Collect elution peaks: Monitor the eluent with a UV detector (e.g., 280 nm) and collect the elution peaks containing the target protein.

[0055] Prepare a size-exclusion column: Select a suitable size-exclusion column, such as Sephadex G-25 or Sephacryl S-200. Choose a suitable gel medium based on the molecular weight of the target protein to ensure effective separation.

[0056] Equilibrate the column: Equilibrate the column with an appropriate equilibration buffer (e.g., 20 mM Hepes, 500 mM NaCl, 10% glycerol, pH 7.5). Ensure the column is free of air bubbles and the flow rate is stable.

[0057] Sample loading: The target protein sample, purified by metal ion affinity chromatography, is loaded onto a size exclusion column. The sample volume should be as small as possible to avoid overload.

[0058] Separation and Collection: The sample is eluted with equilibration buffer at a constant flow rate. Based on the molecular weight of the target protein, it will be separated within a specific elution volume range. The eluent is monitored using a UV detector, and the elution peak containing the target protein is collected.

[0059] (6) Based on the ratio of TEV enzyme mass to target protein mass = 1:20, add TEV enzyme to the target protein and digest overnight at 4°C.

[0060] (7) The target protein digested overnight was processed by size exclusion chromatography to obtain a high-purity target fragment that retains the AviTag, i.e. a high-purity recombinant Mi-2 antigen protein fragment with the Avi Tag tag. The method is the same as the size exclusion chromatography in step (5).

[0061] (8) Calculate the required amount of BirA enzyme based on the ratio of 2.5 μg BirA enzyme added per 40 μM of recombinant Mi-2 antigen protein fragment tagged with Avi Tag. Add the corresponding amount of BirA enzyme and 1 / 10 volume of Biomix B (100 mM ATP, 100 mM MgOAc, 500 μM D-biotin) to the target protein. Incubate overnight at 4°C to complete biotinylation. Dialyze to remove unreacted Biomix B. The dialysate consisted of 20 mM Hepes, 500 mM NaCl, 10% glycerol, and pH = 7.5, to obtain a biotinylated recombinant Mi-2 antigen protein fragment with a concentration of 0.5 mg / mL. Label the fragment as the first biotinylated Mi-2 antigen fragment (amino acid sequence as shown in SEQ ID NO.1), the second biotinylated Mi-2 antigen fragment (amino acid sequence as shown in SEQ ID NO.2), and the third biotinylated Mi-2 antigen fragment (amino acid sequence as shown in SEQ ID NO.3).

[0062] (9) To assess the biotinylation effect, SA4 was added to the biotinylated Mi-2 antigen fragments at the above molar ratio and reacted for 5 min. The capture of the three biotinylated Mi-2 antigen protein fragments by streptavidin (SA4) was then detected by SDS-PAGE protein gel electrophoresis. The results are shown in […]. Figures 1-3 The results showed that all three Mi-2 antigen protein fragments were successfully biotinylated.

[0063] Example 1

[0064] This embodiment provides a detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a cleaning solution.

[0065] The first reagent contains a biotinylated Mi-2 antigen fragment. In this embodiment, equal masses of the first biotinylated Mi-2 antigen fragment and the second biotinylated Mi-2 antigen fragment are mixed, and the mixed antigen is diluted to a total antigen concentration of 4 μg / mL using a 0.02M phosphate buffer containing 1 wt% bovine serum albumin at pH 7.5.

[0066] The second reagent is an alkaline phosphatase-labeled anti-human IgG antibody solution with a concentration of 0.1 μg / mL, and the solvent is a 0.02 M phosphate buffer with a pH of 7.5 containing 1 wt% bovine serum albumin.

[0067] The magnetic microparticle reagent was a streptavidin-labeled magnetic microparticle solution with a concentration of 0.5 mg / mL and a particle size of 0.1–0.5 μm. The solvent was a 0.02 M phosphate buffer solution with a pH of 7.5 containing 1.5 wt% BSA, 2 wt% glycerol and 3 wt% mannitol.

[0068] The chemiluminescent substrate is an alkaline phosphatase enzyme-catalyzed luminescent substrate based on AMPPD luminescence, which is produced in-house. For details, please refer to the invention patent "An Enzymatic Chemiluminescent Substrate for Alkaline Phosphatase", authorized announcement number CN 104990912 B.

[0069] Calibrators: Dilute the anti-Mi-2 antibody with 0.02M phosphate buffer at pH 7.5 to concentrations of 0, 5 RU / mL, 20 RU / mL, 100 RU / mL, 200 RU / mL, 400 RU / mL, and 1600 RU / mL.

[0070] Quality control: Dilute the anti-Mi-2 antibody with 0.02M phosphate buffer at pH 7.5 to concentrations of 20RU / mL and 400RU / mL.

[0071] Sample dilution solution: 0.02M phosphate buffer at pH 7.5.

[0072] The cleaning solution is a product of Jiangsu Haooubo Biomedical Co., Ltd., product number: MY00071.

[0073] In this embodiment, all reagents except the first reagent can be directly used from the corresponding reagents in other existing antibody detection kits, preferably products of Jiangsu Haooubo Biomedical Co., Ltd., but can also be prepared in-house according to conventional methods in the art, and are not limited in this embodiment. The anti-Mi-2 antibody used to prepare the calibrators and quality control samples is produced by a heterologous hybridoma cell line according to conventional methods in the art (this cell line is constructed by fusing peripheral B lymphocytes of GammaPrim mice immunized with Mi-2 antigen with an artificially constructed heterologous hybridoma fusion cell line (possessing unlimited proliferative capacity)).

[0074] Comparative Example 1

[0075] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is a mixture of a first biotinylated Mi-2 antigen fragment and a third biotinylated Mi-2 antigen fragment.

[0076] Comparative Example 2

[0077] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is a mixture of a second biotinylated Mi-2 antigen fragment and a third biotinylated Mi-2 antigen fragment.

[0078] Comparative Example 3

[0079] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is only a first biotinylated Mi-2 antigen fragment.

[0080] Comparative Example 4

[0081] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is only the second biotinylated Mi-2 antigen fragment.

[0082] Comparative Example 5

[0083] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is only the third biotinylated Mi-2 antigen fragment.

[0084] Comparative Example 6

[0085] This comparative example provides another detection kit for anti-Mi-2 antibodies, which includes a first reagent, a second reagent, magnetic microparticle reagent, a chemiluminescent substrate, calibrators, quality control materials, and a washing solution. The kit in this comparative example is essentially the same as in Example 1, except that the antigen in the first reagent is a self-prepared recombinant Mi-2 antigen protein.

[0086] The test kits of the above examples and comparative examples were configured to match the SMART 6500 fully automated chemiluminescence analyzer (Haobo), and labels containing corresponding information were affixed to the test tubes of the kits and each reagent.

[0087] The usage method of the above-mentioned anti-Mi-2 antibody detection kit is as follows:

[0088] Step 1: Place the reagents from the kit into the corresponding positions in the reagent compartment of the SMART 6500 fully automated chemiluminescence analyzer (Haobo). The kit information can be entered into the instrument system via a barcode scanner or set through the instrument's software.

[0089] Step 2: Place the calibrators in the instrument's sample compartment. Identify the calibrator information using a barcode scanner and assign the calibrator location to the instrument system.

[0090] Step 3: Place the quality control sample / test sample into the instrument's sample compartment and edit the corresponding test information using the instrument's software. (Clinical samples should be diluted to the calibrator concentration range using sample diluent before testing.)

[0091] Step 4: Start the program and all calibrator / quality control / sample processing steps will be executed automatically.

[0092] Taking the equipment's detection of the sample as an example, the steps performed automatically by the equipment are as follows:

[0093] Step 4.1: Add 50 μL of magnetic microparticle reagent and 50 μL of the first reagent to the detection tube in sequence, then add 20 μL of the sample to be tested, mix well, and incubate at 37°C for 20 min;

[0094] Step 4.2: Add a magnetic field to allow the system after incubation in step 4.1 to settle in the magnetic field, remove the supernatant, add 500 μL of the instrument's cleaning solution and clean 3-5 times;

[0095] Step 4.3: Remove the magnetic field, then add 135 μL of the second reagent to the system cleaned in step 4.2, mix well, and incubate at 37°C for 15 min;

[0096] Step 4.4: Add a magnetic field to allow the system after incubation in step 4.3 to settle in the magnetic field, remove the supernatant, add 500 μL of washing solution to wash 3-5 times, remove the magnetic field, and shake to fully suspend the magnetic particles.

[0097] Step 4.4: Add a magnetic field to allow the suspended magnetic particles to settle. Remove the supernatant, remove the magnetic field, add 150 μL of chemiluminescent substrate, remove the magnetic field again, and after thorough resuscitation, incubate at 37°C for 5 min to detect the relative luminescence intensity. The instrument's software automatically fits a standard curve with the concentration of different calibrators as the x-axis and the luminescence value as the y-axis, and automatically outputs the test concentration value of the test sample. All steps are fully automated.

[0098] The standard curves corresponding to the detection kits of the above embodiments and comparative examples are shown in Table 1.

[0099] Table 1

[0100] Test kit Standard curve <![CDATA[R 2 ]]> Example 1 Y = 29322X + 666558 0.9956 Comparative Example 1 Y = 26823X + 598306 0.9958 Comparative Example 2 Y = 28107X + 539159 0.9972 Comparative Example 3 Y = 30804X + 512678 0.9982 Comparative Example 4 Y = 30389X + 549727 0.9976 Comparative Example 5 Y = 29771X + 703873 0.9951 Comparative Example 6 Y = 28603X + 414238 0.9988

[0101] Sensitivity and specificity detection

[0102] The above-mentioned anti-Mi-2 antibody detection kit and SMART 6500 fully automated chemiluminescence analyzer (Haobo) were used to detect 28 clinically positive serum samples and 32 clinically negative serum samples, respectively. The software automatically generated the corresponding luminescence value (RLU) and the calculated concentration value for each sample. A detection concentration value ≤20RU / mL was interpreted as negative, and a detection concentration value >20RU / mL was interpreted as positive. The number of positive samples detected in the clinically positive serum samples was counted and the positive detection rate was calculated. The number of negative samples detected in the clinically negative serum samples was counted and the negative detection rate was calculated. The positive detection rate is the sensitivity, and the negative detection rate is the specificity. The results are shown in Table 2.

[0103] Table 2

[0104]

[0105] Note: Clinically positive and clinically negative serum samples used for testing were analyzed and interpreted according to the instructions of the industry gold standard - EURODIAGNOSTICA Antimyositis Antibody Spectrum IgG Detection Kit.

[0106] According to the results in Table 1, the detection kit for anti-Mi-2 antibody in Example 1 has the best sensitivity and specificity. As a detection kit for dermatomyositis-specific autoantibodies—anti-Mi-2 antibodies, it is of great significance for the rapid differential diagnosis of dermatomyositis.

[0107] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the scope of protection of the present invention. Any equivalent changes or modifications made based on the spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A reagent for detecting anti-Mi-2 antibodies, characterized in that, It includes a first antigen fragment and a second antigen fragment, wherein the first antigen fragment is a recombinant Mi-2 antigen protein fragment with a biotin-labeled amino acid sequence as shown in SEQ ID NO.1, and the second antigen fragment is a recombinant Mi-2 antigen protein fragment with a biotin-labeled amino acid sequence as shown in SEQ ID NO.

2.

2. The anti-Mi-2 antibody detection reagent according to claim 1, characterized in that, The mass ratio of the first antigen fragment to the second antigen fragment is (0.8–1.2):

1.

3. The anti-Mi-2 antibody detection reagent according to claim 1, characterized in that, The solvent is a 0.01-0.05M phosphate buffer solution with a pH of 7.2-8.0 containing 0.5wt%-1.5wt% bovine serum albumin; And / or, the sum of the concentrations of the first antigen fragment and the second antigen fragment in the detection reagent is 1 to 10 μg / mL.

4. A kit for detecting anti-Mi-2 antibodies, characterized in that, It includes the anti-Mi-2 antibody detection reagent according to any one of claims 1 to 3.

5. The anti-Mi-2 antibody detection kit according to claim 4, characterized in that, It also includes magnetic microparticle reagents, alkaline phosphatase-labeled anti-human IgG antibody solution, and chemiluminescent substrate solution.

6. The anti-Mi-2 antibody detection kit according to claim 5, characterized in that, The magnetic microparticle reagent is a streptavidin-labeled magnetic microparticle solution.

7. The anti-Mi-2 antibody detection kit according to claim 6, characterized in that, The concentration of the streptavidin-labeled magnetic microparticle solution is 0.1–1 mg / mL. And / or, the particle size of the magnetic particles is 0.1–0.5 μm. And / or, the solvent for the streptavidin-labeled magnetic microparticle solution is a phosphate buffer solution with a pH of 7.2 to 7.5 containing 1–2 wt% BSA, 1–3 wt% glycerol and 2–5 wt% mannitol.

8. The anti-Mi-2 antibody detection kit according to claim 5, characterized in that, The concentration of the alkaline phosphatase-labeled anti-human IgG antibody solution is 0.1–1 μg / mL. And / or, the solvent for the alkaline phosphatase-labeled anti-human IgG antibody solution is a 0.01-0.05M phosphate buffer with a pH of 7.2-8.0 containing 0.5wt%-1.5wt% bovine serum albumin.

9. The anti-Mi-2 antibody detection kit according to claim 5, characterized in that, The luminescent substrate solution is an AMPPD solution.

10. The anti-Mi-2 antibody detection kit according to claim 5, characterized in that, The test kit also includes one or more of a sample diluent, calibrator, and quality control, wherein the sample diluent is a 0.01–0.05 M phosphate buffer with a pH of 7.2–8.0.

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