Anti-melanoma differentiation-associated gene 5 antibody detection kit and application thereof

Abstract

This invention provides a detection kit for anti-melanoma differentiation-associated gene 5 (AAA5) antibodies and its application, relating to the field of biotechnology. The kit includes a working solution for AAA5-coupled magnetic microparticles and a tracer-labeled antibody working solution. The AAA5-coupled magnetic microparticle working solution includes AAA5-coupled magnetic microparticles and magnetic bead buffer. The tracer-labeled antibody working solution includes a tracer-labeled first antibody and a diluent. The AAA5-coupled magnetic microparticles include AAA5 peptide A1-coupled magnetic microparticles, AAA5 peptide A2-coupled magnetic microparticles, and AAA5 peptide A3-coupled magnetic microparticles. This configuration significantly improves the sensitivity of anti-melanoma differentiation-associated gene 5 antibody detection.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an antibody detection kit for anti-melanoma differentiation-associated gene 5 and its application. Background Technology

[0002] Anti-melanoma differentiation-associated gene 5 (MDA5) antibody is a relatively common myositis-specific antibody (MSA) in the clinical amyopathic derma-tomyositis (CADM) subtype of idiopathic inflammatory myopathy (IIM). MDA5 antibody is a myositis-specific autoantibody and can serve as a biomarker for patients with dermatomyositis (DM). It has been confirmed that this antibody is closely related to the development of interstitial lung disease (ILD) in DM patients. The presence of anti-MDA5 antibody in human serum is an important biomarker for diagnosing and predicting the prognosis of patients with idiopathic inflammatory myopathy. Anti-MDA5 antibody is associated with DM and can serve as a biomarker for the clinical diagnosis of CADM. The presence of anti-MDA5 antibody is also associated with poor overall survival prognosis in DM-ILD patients.

[0003] As a novel rare disease and a novel indicator, there are currently no commercially available detection kits for anti-melanoma differentiation-associated gene 5 antibodies. The research kits made by a few medical institutions are complicated in their preparation process, difficult to control, and have low sensitivity, which cannot meet the current detection needs for anti-melanoma differentiation-associated gene 5 antibodies.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a detection kit for anti-melanoma differentiation-associated gene 5 antibodies and its application, thereby addressing the problem of low sensitivity in existing in vitro detection kits for anti-melanoma differentiation-associated gene 5 antibodies. This invention's kit uses melanoma differentiation-associated gene 5 peptides A1, A2, and A3, respectively conjugated to magnetic microparticles, further improving the sensitivity of the detection results.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In one aspect, the present invention provides an anti-melanoma differentiation-associated gene 5 antibody detection kit, comprising: a melanoma differentiation-associated gene 5 coupled magnetic microparticle working solution, wherein the melanoma differentiation-associated gene 5 coupled magnetic microparticle working solution comprises melanoma differentiation-associated gene 5 coupled magnetic microparticles and magnetic bead buffer; a tracer-labeled antibody working solution, wherein the tracer-labeled antibody working solution comprises a tracer-labeled first antibody and a diluent; wherein the melanoma differentiation-associated gene 5 coupled magnetic microparticles comprise melanoma differentiation-associated gene 5 polypeptide A1 coupled magnetic microparticles, melanoma differentiation-associated gene 5 polypeptide A2 coupled magnetic microparticles, and melanoma differentiation-associated gene 5 polypeptide A3 coupled magnetic microparticles.

[0008] The melanoma differentiation-associated gene 5 (MEA5) coupled magnetic microparticles in the kit of this invention include MEA5 polypeptide A1 coupled magnetic microparticles, MEA5 polypeptide A2 coupled magnetic microparticles, and MEA5 polypeptide A3 coupled magnetic microparticles. This configuration allows for more complete exposure of each antigenic site in MEA5. Validated by immunoserological detection technology, compared with existing MEA5 antibody detection kits, this kit demonstrates higher sensitivity in detecting MEA5 antibodies.

[0009] Furthermore, the melanoma differentiation-associated gene 5 polypeptide A1 has the amino acid sequence shown in SEQ ID No. 1; the melanoma differentiation-associated gene 5 polypeptide A2 has the amino acid sequence shown in SEQ ID No. 2; and the melanoma differentiation-associated gene 5 polypeptide A3 has the amino acid sequence shown in SEQ ID No. 3.

[0010] Further, the magnetic microparticles are toluenesulfonyl magnetic beads; preferably, the coating ratio of melanoma differentiation-associated gene 5 peptide A1 to the magnetic microparticles in the melanoma differentiation-associated gene 5 peptide A1-conjugated magnetic microparticles is 5 μg / mg; the coating ratio of melanoma differentiation-associated gene 5 peptide A2 to the magnetic microparticles in the melanoma differentiation-associated gene 5 peptide A2-conjugated magnetic microparticles is 2.5 μg / mg; and the coating ratio of melanoma differentiation-associated gene 5 peptide A3 to the magnetic microparticles in the melanoma differentiation-associated gene 5 peptide A3-conjugated magnetic microparticles is 3 μg / mg.

[0011] The melanoma differentiation-associated gene 5 (MEA5) coupled magnetic microparticles in the kit of this invention include MEA5 peptide A1 coupled magnetic microparticles, MEA5 peptide A2 coupled magnetic microparticles, and MEA5 peptide A3 coupled magnetic microparticles. The coating ratio of MEA5 peptide A1 to the magnetic microparticles in the MEA5 peptide A1 coupled magnetic microparticles is 5 μg / mg; the coating ratio of MEA5 peptide A2 to the magnetic microparticles in the MEA2 coupled magnetic microparticles is 2.5 μg / mg; and the coating ratio of MEA5 peptide A3 to the magnetic microparticles in the MEA3 coupled magnetic microparticles is 3 μg / mg. This ensures more complete exposure of each antigenic site in MEA5. Validated by immunoserological detection technology, compared with existing MEA5 antibody detection kits, this kit exhibits higher sensitivity in detecting anti-MEA5 antibodies.

[0012] Because the melanoma differentiation-associated gene 5 polypeptides A1, A2, and A3 used in this invention application, compared to the complete melanoma differentiation-associated gene 5 antigen, allow for full exposure of the melanoma differentiation-associated gene 5 antigen epitopes, thus improving detection sensitivity, and because the three polypeptides have relatively large molecular weights, they can be directly coated onto toluenesulfonyl magnetic beads without being affected by steric hindrance, simplifying the process, reducing costs, and avoiding the need for coupling agent treatment during the coupling process, thereby avoiding adverse effects on polypeptide activity and structure after treatment and improving polypeptide stability.

[0013] Furthermore, in the melanoma differentiation-associated gene 5 coupled magnetic microparticle working solution, the final concentration of the melanoma differentiation-associated gene 5 peptide A1 coupled magnetic microparticle is 0.05 mg / mL, the final concentration of the melanoma differentiation-associated gene 5 peptide A2 coupled magnetic microparticle is 0.03 mg / mL, and the final concentration of the melanoma differentiation-associated gene 5 peptide A3 coupled magnetic microparticle is 0.02 mg / mL.

[0014] This application employs a method where three different melanoma differentiation-associated gene 5 peptides are first coupled separately to toluenesulfonyl magnetic beads to form different melanoma differentiation-associated gene 5 coupled magnetic microparticles. These different melanoma differentiation-associated gene 5 coupled magnetic microparticles are then mixed at a specific final magnetic bead concentration. Compared to simultaneously coupling different melanoma differentiation-associated gene 5 peptides to magnetic beads in the same liquid, the melanoma differentiation-associated gene 5 peptides coupled to the magnetic beads are more uniform. This avoids the significant steric hindrance and poor uniformity that can occur when multiple melanoma differentiation-associated gene 5 peptides are coupled to the same magnetic bead, further improving the sensitivity of the kit's detection results.

[0015] Furthermore, the magnetic bead buffer is a phosphate buffer with a pH of 7.4 and a concentration of 0.01 M, consisting of 0.01 mg / mL bovine serum albumin, 0.9 mg / mL sodium chloride, 0.1 mL / L Triton-X1000, 0.01 mL / mL Procline-300.

[0016] Furthermore, the tracer is alkaline phosphatase.

[0017] Furthermore, the first antibody is an anti-human IgG antibody.

[0018] Furthermore, the diluent is a MES buffer solution with a concentration of 0.1 mol / L, consisting of 0.1 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.01 mL / mL proclin 300, pH 6.0.

[0019] Furthermore, the kit also includes a sample diluent, which is a 0.01M pH 7.4 phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.001 mL / mL Procline-300, and 0.001 mL / mL Triton X-100.

[0020] In another aspect, the present invention provides the application of the kit in the in vitro detection of anti-melanoma differentiation-associated gene 5 antibody for non-disease diagnostic purposes.

[0021] Furthermore, the samples that the kit of the present invention is used to detect include whole blood, serum or plasma.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] Anti-melanoma differentiation-associated gene 5 antibodies produced in the blood of patients with autoimmune myositis and dermatomyositis are mixtures of antibodies targeting different antigenic epitopes (which can be understood as polyclonal antibodies). The types and proportions of antibodies produced vary among individuals. Artificially synthesized melanoma differentiation-associated gene 5 antigens are genetically engineered antigens, with only some antigenic determinants fully exposed. Using a single antigen for immunological testing can easily lead to false negatives, resulting in low sensitivity. The combined use of melanoma differentiation-associated gene 5 peptide A1, A2, and A3 covers as many specific and effective antigenic epitopes as possible.

[0024] The melanoma differentiation-associated gene 5 peptides A1, A2, and A3 selected in this application each contain different antigenic determinants. The binding rate constants of the antigenic determinants of the three antigens and their corresponding antibodies are constant but different. The signal value of a single antigen concentration is positively correlated with the signal value level. The immunoassay methodology determines the positive or negative result by comparing the signal value with a pre-set threshold. The three antigens have a complementary relationship in antibody detection in the population. The final magnetic bead concentration when the three antigens are used simultaneously is particularly important. Only when the signal value levels of the mixed antigens are adjusted to be similar will the positive judgment values ​​of the three antigens be similar, resulting in the highest sensitivity and specificity. If the concentration of a certain antigen is relatively low, the signal value of the sample that can only be detected by that antigen will be below the threshold, leading to a false negative result and decreased sensitivity. Similarly, if the concentration of a certain antigen is relatively high, the overall signal value of the sample will be increased, the threshold will be shifted upward, and a false positive result will be obtained, resulting in a false positive and decreased specificity.

[0025] The melanoma differentiation-associated gene 5 antibody detection kit provided in this invention includes melanoma differentiation-associated gene 5 peptide A1-coupled magnetic microparticles, melanoma differentiation-associated gene 5 peptide A2-coupled magnetic microparticles, and melanoma differentiation-associated gene 5 peptide A3-coupled magnetic microparticles. The coating ratio of melanoma differentiation-associated gene 5 peptide A1 to the magnetic microparticles in the melanoma differentiation-associated gene 5 peptide A1-coupled magnetic microparticles is 5 μg / mg; the coating ratio of melanoma differentiation-associated gene 5 peptide A2 to the magnetic microparticles in the melanoma differentiation-associated gene 5 peptide A2-coupled magnetic microparticles is 2.5 μg / mg; and the coating ratio of melanoma differentiation-associated gene 5 peptide A3 to the magnetic microparticles in the melanoma differentiation-associated gene 5-coupled magnetic microparticles is 3 μg / mg. With this configuration, the detection sensitivity of the anti-melanoma differentiation-associated gene 5 antibody is 98.00%, the specificity is 100.00%, the false positive rate is 0.00%, and the false negative rate is 2.00%. Existing kits using melanoma differentiation-associated gene 5 (MAG5) monoantigen have a sensitivity of 90.00%, a specificity of 98.00%, a false positive rate of 2.00%, and a false negative rate of 10.00%. The anti-melanoma MAG5 antibody detection kit provided in this invention reduces false negatives by 80%, demonstrating significant effectiveness. It further improves the sensitivity of detecting anti-melanoma MAG5 antibodies in the serum of different patients, contributing to accurate clinical diagnosis and treatment. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] The "melanoma differentiation-associated gene 5 coupled magnetic microparticles" mentioned in this application refers to the coupling of the melanoma differentiation-associated gene 5 antigen to magnetic microparticles. The melanoma differentiation-associated gene 5 antigen refers to a substance that can specifically recognize and bind to anti-melanoma differentiation-associated gene 5 antibodies, including but not limited to melanoma differentiation-associated gene 5 protein, melanoma differentiation-associated gene 5 polypeptide, and aptamers that can specifically recognize anti-melanoma differentiation-associated gene 5 antibodies prepared according to the methods described in the background art. The magnetic microparticles refer to a magnetic solid-phase carrier, including but not limited to magnetic beads, magnetic particles, and magnetic microparticles. In this embodiment, the magnetic microparticles used are toluenesulfonyl nanomagnetic beads.

[0028] The “melanoma differentiation-associated gene 5 polypeptide A1 coupled to magnetic microparticles” described in this application refers to the coupling of the melanoma differentiation-associated gene 5 polypeptide A1 with the amino acid sequence as described in SEQ ID No. 1 to magnetic microparticles.

[0029] The “melanoma differentiation-associated gene 5 polypeptide A2 coupled to magnetic microparticles” described in this application refers to the coupling of the melanoma differentiation-associated gene 5 polypeptide A2 with the amino acid sequence as described in SEQ ID No. 2 to magnetic microparticles.

[0030] The “melanoma differentiation-associated gene 5 polypeptide A3 coupled to magnetic microparticles” described in this application refers to the coupling of the melanoma differentiation-associated gene 5 polypeptide A3 with the amino acid sequence as described in SEQ ID No. 3 to magnetic microparticles.

[0031] The "magnetic bead coating ratio" mentioned in this application refers to the relative coating amount formed during the coupling process of melanoma differentiation-associated gene 5 peptide and magnetic microparticles. For example, a magnetic bead coating ratio of 5ug / mg for melanoma differentiation-associated gene 5 peptide A1 and magnetic microparticles indicates that when preparing melanoma differentiation-associated gene 5 peptide A1 coupled with magnetic microparticles alone, the actual reaction ratio of the two after the addition of magnetic microparticles and melanoma differentiation-associated gene 5 peptide A1 is 5ug of melanoma differentiation-associated gene 5 peptide in the solution containing 1mg of magnetic microparticles.

[0032] The "final concentration of magnetic microparticles" mentioned in this application refers to the final concentration of each melanoma differentiation-related gene 5 peptide-coupled magnetic microparticle in the magnetic microparticle working solution after it has been prepared. For example, "the final concentration of the melanoma differentiation-related gene 5 peptide A1-coupled magnetic microparticle is 0.05 mg / mL, the final concentration of the melanoma differentiation-related gene 5 peptide A2-coupled magnetic microparticle is 0.03 mg / mL, and the final concentration of the melanoma differentiation-related gene 5 peptide A3-coupled magnetic microparticle is 0.02 mg / mL" can be understood as the absolute content of melanoma differentiation-related gene 5 peptide A1-coupled magnetic microparticles in 1 mL of the mixed magnetic microparticle working solution being 0.05 mg, 0.03 mg, and 0.02 mg.

[0033] 1. Raw material: Biotinylated melanoma differentiation-associated gene 5 peptide

[0034] The melanoma differentiation-associated gene 5 antigen used in this invention is melanoma differentiation-associated gene 5 polypeptide A1, melanoma differentiation-associated gene 5 polypeptide A2, and melanoma differentiation-associated gene 5 polypeptide A3. The amino acid sequence of melanoma differentiation-associated gene 5 polypeptide A1 is SEQ ID No. 1, the amino acid sequence of melanoma differentiation-associated gene 5 polypeptide A2 is SEQ ID No. 2, and the amino acid sequence of melanoma differentiation-associated gene 5 polypeptide A3 is SEQ ID No. 3.

[0035]

[0036] (SEQ ID No.1).

[0037]

[0038] (SEQ ID No.2).

[0039]

[0040] (SEQ ID No.3).

[0041] 2. Preparation of magnetic microparticles coupled to melanoma differentiation-related gene 5:

[0042] Melanoma differentiation-associated gene 5 peptide A1, peptide A2, and peptide A3 were coated with toluenesulfonyl magnetic nanobeads at ratios of 5 μg / mg, 2.5 μg / mg, and 3 μg / mg, respectively. After shaking at 37 ± 1.5 °C for 18 h, the supernatant was removed by magnetic separation and adsorption. The nanobeads were washed three times with the same volume of phosphate buffer (pH 7.4, concentration 0.01 mol / L) to obtain melanoma differentiation-associated gene 5 coupled magnetic microparticles.

[0043] The prepared melanoma differentiation-associated gene 5 coupled magnetic microparticles were blocked by shaking and resuspending at 37±1.5℃ for 6h using a phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.005 mg / mL proclin 300, pH 7.4, and a concentration of 0.01 mol / L.

[0044] After blocking, melanoma differentiation-associated gene 5 coupled magnetic microparticles were resuspended in phosphate buffer at pH 7.4 and a concentration of 0.01 mol / L to a concentration of 10 mg / mL.

[0045] The working solution was prepared with the following final concentrations of magnetic microparticles: 0.05 mg / ml for melanoma differentiation-associated gene 5 peptide A1 coupled with magnetic microparticles, 0.03 mg / ml for melanoma differentiation-associated gene 5 peptide A2 coupled with magnetic microparticles, and 0.02 mg / ml for melanoma differentiation-associated gene 5 peptide A3 coupled with magnetic microparticles. The buffer solution was a phosphate buffer solution with a pH of 7.4 and a concentration of 0.01 M, consisting of 0.01 mg / mL bovine serum albumin, 0.9 mg / mL sodium chloride, 0.1 mL / L Triton-X1000, 0.01 mL / mL Procline-300, bovine serum albumin, bovine serum albumin, and phosphate buffer solution.

[0046] 3. Preparation of alkaline phosphatase-labeled anti-human IgG antibody working solution:

[0047] The alkaline phosphatase-labeled anti-human IgG antibody was diluted to 0.1 μg / mL using MES buffer containing 0.1 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.1 mg / mL proclin 300, pH 6.0, and a concentration of 0.1 mol / L.

[0048] 4. Prepare sample diluent:

[0049] The sample diluent was a 0.01M pH 7.4 phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.001 mL / mL Procline-300, and 0.001 mL / mL Triton X-100.

[0050] 5. Preparation of the anti-melanoma differentiation-associated gene 5 antibody detection kit:

[0051] The anti-melanoma differentiation-associated gene 5 antibody detection kit includes: 6.5 mL / vial of melanoma differentiation-associated gene 5 coupled magnetic microparticles, 6.5 mL / vial of alkaline phosphatase-labeled anti-human IgG antibody, and 1.0 mL / vial of sample diluent. After aliquoting, the kit is assembled together and stored at 2-8℃.

[0052] When testing the sample, the sample and melanoma differentiation-associated gene 5 (MEA5) coupled magnetic microparticles can be mixed and incubated, followed by incubation with alkaline phosphatase-labeled anti-human IgG antibody working solution. When anti-MEA5 antibody is present in the sample, a magnetic microparticle-MEA5 polypeptide-anti-MEA5 antibody-anti-human IgG antibody complex is formed. The alkaline phosphatase on the anti-human IgG antibody promotes substrate luminescence. Qualitative or quantitative analysis is performed by detecting the luminescence signal to detect the anti-MEA5 antibody in the sample. I. Detection of the coating amount of MEA5 polypeptides A1, A2, and A3.

[0053] Experimental Example 1.1

[0054] The melanoma differentiation-associated gene 5 antigen used in this embodiment is melanoma differentiation-associated gene 5 polypeptide A1. The amino acid sequence of A1 is SEQ ID No. 1.

[0055] The magnetic microparticles used in this embodiment are toluenesulfonyl magnetic beads. The toluenesulfonyl magnetic bead suspension was magnetically separated to obtain the magnetic microparticles. The magnetic microparticles were resuspended in a 10 mg / mL magnetic microparticle solution using a 0.01 mol / L phosphate buffer solution (pH 7.4). The microparticles were washed three times with a 0.01 mol / L phosphate buffer solution (pH 7.4) and resuspended in a 10 mg / mL magnetic microparticle working solution.

[0056] Melanoma differentiation-associated gene 5 peptide A1 was coated with toluenesulfonyl magnetic beads at coating ratios of 1.25 μg / mg, 2.5 μg / mg, 5 μg / mg, 7.5 μg / mg, and 10 μg / mg, respectively. After shaking at 37 ± 1.5 °C for 18 h, the supernatant was removed by magnetic separation and adsorption. The particles were washed three times with the same volume of the above-mentioned phosphate buffer (pH 7.4, concentration 0.01 mol / L) to obtain melanoma differentiation-associated gene 5 coupled magnetic microparticles.

[0057] The ratio of melanoma differentiation-associated gene 5 peptide A1 to magnetic bead coating of magnetic microparticles is 5ug / mg, indicating that when preparing melanoma differentiation-associated gene 5 peptide A1 coupled with magnetic microparticles alone, the actual reaction ratio of the two after the addition of magnetic microparticles and melanoma differentiation-associated gene 5 peptide A1 is 5ug of melanoma differentiation-associated gene 5 peptide in 1mg magnetic bead solution.

[0058] The prepared melanoma differentiation-associated gene 5 coupled magnetic microparticles were resuspended and blocked at 37±1.5℃ for 6h using phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.005 mg / mL proclin 300, pH 7.4, and a concentration of 0.01 mol / L.

[0059] After blocking, melanoma differentiation-associated gene 5 coupled magnetic microparticles were resuspended in phosphate buffer at pH 7.4 and a concentration of 0.01 mol / L to a concentration of 10 mg / mL.

[0060] The working solution was prepared according to a ratio of 0.05 mg of coupled magnetic microparticles / mL of magnetic bead buffer. The buffer solution was a phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.9 mg / mL sodium chloride, 0.1 mL / L Triton-X1000, 0.01 mL / mL Procline-300, pH 7.4, and a concentration of 0.01 M.

[0061] The alkaline phosphatase-labeled anti-human IgG antibody was diluted to 0.1 μg / mL using MES buffer containing 0.1 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.01 mL / mL proclin 300, pH 6.0, and a concentration of 0.1 mol / L.

[0062] Prepare a sample diluent, which is a 0.01M pH 7.4 phosphate buffer containing 0.1 mg / mL bovine serum albumin, 0.001 mL / mL Procline-300, and 0.001 mL / mL Triton X-100.

[0063] The anti-melanoma differentiation-associated gene 5 antibody detection kit includes: 6.5 mL / vial of melanoma differentiation-associated gene 5 coupled magnetic microparticles, 6.5 mL / vial of alkaline phosphatase-labeled anti-human IgG antibody, and 1.0 mL / vial of sample diluent. After aliquoting, the kit is assembled together and stored at 2-8℃.

[0064] The above-prepared anti-melanoma differentiation-associated gene 5 antibody detection kit was used to detect samples with a value of 0, 10 positive samples from patients with dermatomyositis and containing anti-melanoma differentiation-associated gene 5 antibodies in their blood, and 10 negative samples from patients without dermatomyositis. The detection process was performed using a fully automated chemiluminescence immunoassay system from Zhuhai Lizhu Reagent Co., Ltd. The detection procedure is as follows:

[0065] Step 1: The fully automated chemiluminescence immunoassay system aspirates 20 μL of sample and automatically dilutes it 15 times.

[0066] Step 2: Take 20 μL of the diluted sample, add 100 μL of melanoma differentiation-related gene 5 coupled magnetic microparticles, incubate at 37°C for 10 min, then perform magnetic separation and wash 3 times.

[0067] Step 3: Add 100 μL of alkaline phosphatase-labeled anti-human IgG antibody, incubate at 37°C for 10 min, then perform magnetic separation and wash 3 times.

[0068] Step 4: Add 200 μL of substrate solution, read the signal value, calculate the signal-to-noise ratio, and the results of the antibody test for anti-melanoma differentiation-related gene 5 are shown in Table 1.

[0069] Experimental Example 1.2

[0070] The melanoma differentiation-associated gene 5 antigen is melanoma differentiation-associated gene 5 polypeptide A2. The amino acid sequence of A2 is SEQ ID No. 2. Melanoma differentiation-associated gene 5 polypeptide A2 was coated with toluenesulfonyl magnetic beads at coating ratios of 0.625 ug / mg, 1.25 ug / mg, 2.5 ug / mg, 5 ug / mg, and 10 ug / mg, respectively, with other conditions the same as in Experiment 1. The results of the anti-melanoma differentiation-associated gene 5 antibody test are shown in Table 2.

[0071] Experimental Example 1.3

[0072] The melanoma differentiation-associated gene 5 antigen is melanoma differentiation-associated gene 5 polypeptide A3. The amino acid sequence of A3 is SEQ ID No. 3. Melanoma differentiation-associated gene 5 polypeptide A3 was coated with toluenesulfonyl magnetic beads at coating ratios of 1.25 μg / mg, 1.5 μg / mg, 3 μg / mg, 6 μg / mg, and 12 μg / mg, respectively, with other conditions the same as in Experiment 1. The results of the anti-melanoma differentiation-associated gene 5 antibody test are shown in Table 3.

[0073] Comparative Example 1

[0074] The melanoma differentiation-associated gene 5 antigen was a commercially available melanoma differentiation-associated gene 5. The commercially available melanoma differentiation-associated gene 5 was coated with toluenesulfonyl magnetic beads at concentrations of 1.25 μg / mg, 2.5 μg / mg, 5 μg / mg, 7 μg / mg, and 10 μg / mg, respectively. All other conditions were the same as in Experiment 1. The results of the anti-melanoma differentiation-associated gene 5 antibody test are shown in Table 4.

[0075] Table 1. Detection of the proportion of magnetic microparticles coated with the 5-peptide A1 of melanoma differentiation-related genes in Experiment 1.1.

[0076]

[0077] Table 2. Detection of the proportion of magnetic microparticles coated with the 5-peptide A2 of the melanoma differentiation-related gene in Experiment 1.2.

[0078]

[0079] Table 3. Detection of the proportion of magnetic microparticles coated with the 5-peptide A3 of the melanoma differentiation-related gene in Experiment 1.3.

[0080]

[0081] Table 4. Comparative Example 1: Detection of the coating ratio of 5 commercially available melanoma differentiation-related genes with magnetic microparticles.

[0082]

[0083] Results Analysis:

[0084] Although there is a dissociation equilibrium between the melanoma differentiation-associated gene 5 peptide and the toluenesulfonyl magnetic microparticles, and because the magnetic microparticles and the melanoma differentiation-associated gene 5 peptide cannot always maintain sufficient contact, there will be uneven coupling of the melanoma differentiation-associated gene 5 peptide on the magnetic microparticles and large differences in the number of peptides. However, the peptide is usually in a state of relative excess. Ignoring the cases where a few magnetic microparticles have fewer melanoma differentiation-associated gene 5 peptides coupled to them, the coating ratio of the magnetic microparticles with the melanoma differentiation-associated gene 5 peptide is the only standard to indicate the amount of antigen coupling between the melanoma differentiation-associated gene 5 peptide and the magnetic microparticles.

[0085] Based on a comprehensive judgment of signal-to-noise ratio and signal value, the higher the signal-to-noise ratio of positive samples and the lower the signal-to-noise ratio of negative samples, the better the effect.

[0086] Table 1 shows that the optimal antigen-magnetic microparticle coating ratio for melanoma differentiation-associated gene 5 peptide A1 is 5 μg / mg, with a signal-to-noise ratio of 703.85 for positive samples and 18.37 for negative samples. The upper limit for the antigen-magnetic microparticle coating ratio is 7.5 μg / mg, and the lower limit is 2.5 μg / mg.

[0087] Table 2 shows that the optimal antigen-to-magnetic-microparticle coating ratio for melanoma differentiation-associated gene 5 peptide A2 is 2.5 ug / mg, with a signal-to-noise ratio of 1652.51 for positive samples and 43.59 for negative samples. The upper limit for the antigen-to-magnetic-microparticle coating ratio is 5 ug / mg, and the lower limit is 1.25 ug / mg.

[0088] Table 3 shows that the optimal antigen-magnetic microparticle coating ratio for melanoma differentiation-associated gene 5 peptide A3 is 3 μg / mg, with a signal-to-noise ratio of 729.08 for positive samples and 72.35 for negative samples. The upper limit for the antigen-magnetic microparticle coating ratio is 6 μg / mg, and the lower limit is 1.5 μg / mg.

[0089] Table 4 shows that the optimal antigen-magnetic microparticle coating ratio for commercially available melanoma differentiation-associated gene 5 (MEA5) is 5 μg / mg, with a signal-to-noise ratio (SNR) of 470.23 for positive samples and 129.57 for negative samples. The upper limit for the antigen-magnetic microparticle coating ratio is 7.5 μg / mg, and the lower limit is 2.5 μg / mg. The SNR of the best positive sample for commercially available MEA5 is lower than that of the worst positive samples for MEA5 peptides A1, A2, and A3, indicating that the detection performance of commercially available MEA5 antibodies against MEA5 is lower than that of MEA5 peptides A1, A2, or A3.

[0090] II. Detection of the concentration of melanoma differentiation-associated gene 5 peptide-coupled magnetic microparticles and mixed magnetic beads.

[0091] Experimental Example 2.1

[0092] The melanoma differentiation-associated gene 5 polypeptide A1 prepared in Experiment 1.1 was coupled with magnetic microparticles at a magnetic bead concentration of 0.05 mg / mL to prepare an anti-melanoma differentiation-associated gene 5 antibody detection kit. All other conditions were the same as in Experiment 1.1. The prepared anti-melanoma differentiation-associated gene 5 antibody kit was used to detect 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the detection results, and the results are shown in Table 5.

[0093] Experimental Example 2.2

[0094] The melanoma differentiation-associated gene 5 polypeptide A2 prepared in Experiment 1.2 was coupled with magnetic microparticles to prepare an anti-melanoma differentiation-associated gene 5 antibody detection kit with a final magnetic microparticle concentration of 0.03 mg / mL. All other conditions were the same as in Experiment 1.1. The prepared anti-melanoma differentiation-associated gene 5 antibody kit was used to detect 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the detection results, and the results are shown in Table 6.

[0095] Experimental Example 2.3

[0096] The melanoma differentiation-associated gene 5 polypeptide A3 prepared in Experiment 1.3 was coupled with magnetic microparticles to a final magnetic microparticle concentration of 0.02 mg / mL to prepare an anti-melanoma differentiation-associated gene 5 antibody detection kit. All other conditions were the same as in Experiment 1.1. The prepared anti-melanoma differentiation-associated gene 5 antibody kit was used to detect 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the detection results, and the results are shown in Table 7.

[0097] Experimental Example 2.4

[0098] A melanoma differentiation-associated gene 5 (MAG5) antibody detection kit was prepared by mixing the melanoma MAG5 peptide A1 prepared in Experiment 1.1 with magnetic microparticles at a final magnetic microparticle concentration of 0.05 mg / mL, and the melanoma MAG5 peptide A2 prepared in Experiment 1.2 with magnetic microparticles at a final magnetic microparticle concentration of 0.03 mg / mL. All other conditions were the same as in Experiment 1.1. The prepared anti-melanoma MAG5 antibody detection kit was used to test 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the test results, and the results are shown in Table 8.

[0099] Experimental Example 2.5

[0100] A melanoma differentiation-associated gene 5 (MEA5) antibody detection kit was prepared by mixing the melanoma differentiation-associated gene 5 peptide A1 prepared in Experiment 1.1 with magnetic microparticles at a final magnetic microparticle concentration of 0.05 mg / mL, and the melanoma differentiation-associated gene 5 peptide A3 prepared in Experiment 1.3 with magnetic microparticles at a final magnetic microparticle concentration of 0.02 mg / mL. All other conditions were the same as in Experiment 1.1. The prepared anti-melanoma differentiation-associated gene 5 antibody detection kit was used to test 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the test results, and the results are shown in Table 9.

[0101] Experimental Example 2.6

[0102] A melanoma differentiation-associated gene 5 (MEA5) antibody detection kit was prepared by mixing the melanoma differentiation-associated gene 5 peptide A2 prepared in Experiment 1.2 with magnetic microparticles at a final concentration of 0.03 mg / mL, and the melanoma differentiation-associated gene 5 peptide A3 prepared in Experiment 1.3 with magnetic microparticles at a final concentration of 0.02 mg / mL. All other conditions were the same as in Experiment 1.1. The prepared MEA5 antibody detection kit was used to test 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the test results, and the results are shown in Table 10.

[0103] Example 1

[0104] A melanoma differentiation-associated gene 5 (MEA5) antibody detection kit was prepared by mixing the following: MEA5 peptide A1 prepared in Example 1.1 with magnetic microparticles at a final concentration of 0.05 mg / mL; MEA2 prepared in Example 1.2 with magnetic microparticles at a final concentration of 0.03 mg / mL; and MEA3 prepared in Example 1.3 with magnetic microparticles at a final concentration of 0.02 mg / mL. All other conditions were the same as in Example 1.1. The prepared MEA5 antibody detection kit was used to test 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the test results, which are shown in Table 11.

[0105] Comparative Example 2

[0106] The commercially available melanoma differentiation-associated gene 5 (MEA5)-coupled magnetic microparticles prepared in Example 1.4 were used to prepare an anti-MEA5 antibody detection kit at a final concentration of 0.1 mg / mL. All other conditions were the same as in Example 1.1. The prepared anti-MEA5 antibody kit was used to detect 50 positive samples and 50 negative samples. Sensitivity and specificity were calculated based on the results, and the results are shown in Table 12.

[0107] The detection results of eight reagent kits on 50 positive samples are shown in Table 13, the detection results of eight reagent kits on 50 negative samples are shown in Table 14, and the sensitivity, specificity, false positive and false negative results of each reagent kit are shown in Table 15.

[0108] Table 5. Sensitivity and specificity tests of melanoma differentiation-associated gene 5 peptide A1.

[0109]

[0110] Table 6. Sensitivity and specificity tests for melanoma differentiation-associated gene 5 peptide A2

[0111]

[0112] Table 7. Sensitivity and specificity tests of melanoma differentiation-associated gene 5 peptide A3.

[0113]

[0114] Table 8. Sensitivity and specificity tests of the five melanoma differentiation-associated gene peptides A1+A2

[0115]

[0116]

[0117] Table 9. Sensitivity and specificity tests of melanoma differentiation-associated gene 5 peptides A1+A3

[0118]

[0119] Table 10. Sensitivity and specificity tests of melanoma differentiation-associated gene 5 peptides A2+A3

[0120]

[0121] Table 11. Sensitivity and specificity tests of the five melanoma differentiation-associated gene peptides A1+A2+A3

[0122]

[0123] Table 12. Sensitivity and specificity tests for five melanoma differentiation-related genes.

[0124]

[0125] Table 13. Positive test results from eight test kits

[0126]

[0127]

[0128] Table 14. Negative test results from eight test kits

[0129]

[0130]

[0131] Table 15. Sensitivity, specificity, false positive, and false negative test results for eight reagent kits

[0132]

[0133]

[0134] Results analysis:

[0135] As shown in Table 15, the anti-melanoma differentiation-associated gene 5 antibody detection kit prepared by mixing melanoma differentiation-associated gene 5 peptide A1 coupled with magnetic microparticles at a final magnetic microparticle concentration of 0.05 mg / ml, melanoma differentiation-associated gene 5 peptide A2 coupled with magnetic microparticles at a final magnetic microparticle concentration of 0.03 mg / ml, and melanoma differentiation-associated gene 5 peptide A3 coupled with magnetic microparticles at a final magnetic microparticle concentration of 0.02 mg / ml, exhibits superior sensitivity and specificity compared to commercially available anti-melanoma differentiation-associated gene 5 antibody detection kits prepared using melanoma differentiation-associated gene 5 coupled with magnetic microparticles at a final magnetic microparticle concentration of 0.1 mg / ml. False negatives decreased from 10.00% to 2.00%, a reduction of 80.00%; false positives decreased from 2.00% to 0.00%, a reduction of 100%, demonstrating significant improvements in both sensitivity and specificity.

[0136] The inventors hypothesize that melanoma differentiation-associated gene 5 (MEA5) produced in the blood of dermatomyositis patients is a mixture of antibodies (polyclonal antibodies) targeting different antigenic epitopes. The types and proportions of antibodies produced vary among individuals. Artificially synthesized MEA5 antigens / genetically engineered antigens only fully expose some antigenic determinants, making it highly susceptible to false negatives and resulting in low sensitivity when using a single antigen for immunological testing. By using MEA5 peptides A1, A2, and A3 in combination, as many specific and effective antigenic epitopes as possible can be covered.

[0137] The combined use of melanoma differentiation-associated gene 5 peptide A1, melanoma differentiation-associated gene 5 peptide A2, and melanoma differentiation-associated gene 5 peptide A3 showed the highest sensitivity and the strongest specificity, while maintaining 100% specificity. At this point, the final concentrations of the three magnetic microparticles were 0.05 mg / ml for melanoma differentiation-associated gene 5 peptide A1, 0.03 mg / ml for melanoma differentiation-associated gene 5 peptide A2, and 0.02 mg / ml for melanoma differentiation-associated gene 5 peptide A3.

[0138] Compared to the intact antigen, the melanoma differentiation-associated gene 5 polypeptide A1, A2, and A3 used in this invention application fully expose the antigenic epitopes, improving detection sensitivity and specificity. Furthermore, the relatively large molecular weight of the three polypeptides allows them to be directly coated onto toluenesulfonyl magnetic beads without steric hindrance, simplifying the process, reducing costs, and avoiding the need for coupling agent treatment during the coupling process. This avoids adverse effects on polypeptide activity and structure after treatment, thus improving polypeptide stability.

[0139] This application employs a method where different melanoma differentiation-associated gene 5 peptides are first coupled to different magnetic microparticles to form different melanoma differentiation-associated gene 5-coupled magnetic microparticles. These different melanoma differentiation-associated gene 5-coupled magnetic microparticles are then mixed at a specific final concentration. Compared to simultaneously coupling different melanoma differentiation-associated gene 5 peptides to magnetic microparticles in the same liquid, the melanoma differentiation-associated gene 5 peptides coupled to the magnetic microparticles are more uniform. This avoids the significant steric hindrance and poor uniformity that can occur when multiple melanoma differentiation-associated gene 5 peptides are coupled to the same magnetic microparticle, further improving the sensitivity and specificity of the kit's detection results.

[0140] III. Final concentration screening of melanoma differentiation-related gene 5 peptides A1, A2, A3 and magnetic microparticles

[0141] Comparative Example 3

[0142] Orthogonal experiments were conducted using the final concentrations of melanoma differentiation-associated gene 5 peptide A1 with magnetic microparticles, melanoma differentiation-associated gene 5 peptide A2 with magnetic microparticles, and melanoma differentiation-associated gene 5 peptide A3 with magnetic microparticles, according to combinations 1-9 as described in Table 16. The remaining procedures were the same as in Example 1. Combination 5 corresponds to Example 1. The sensitivity and specificity detection results for combinations 1-9 are shown in Tables 17-26.

[0143] Table 16. Experimental results on the final concentrations of melanoma differentiation-related gene 5 peptides A1, A2, and A3 with magnetic microparticles.

[0144]

[0145] Table 17. Sensitivity and specificity tests of Comparative Example Group 3-1

[0146]

[0147] Table 18. Sensitivity and specificity tests of Comparative Example Group 3

[0148]

[0149] Table 19. Sensitivity and specificity tests of Comparative Example Groups 3

[0150]

[0151] Table 20. Sensitivity and specificity tests for Comparative Example Groups 3 and 4

[0152]

[0153] Table 21. Sensitivity and Specificity Tests in Example 1

[0154]

[0155] Table 22. Sensitivity and specificity tests of Comparative Example Group 3 (Group 6)

[0156]

[0157] Table 23. Sensitivity and specificity tests of Comparative Example Group 3.7

[0158]

[0159] Table 24. Sensitivity and specificity tests of Comparative Example Group 3 (8 groups)

[0160]

[0161]

[0162] Table 25. Sensitivity and specificity tests of Comparative Example Group 3 (9 groups)

[0163]

[0164] Table 26. Sensitivity and specificity test results of mixed antigens containing different final magnetic bead concentrations of melanoma differentiation-associated gene 5 polypeptides A1+A2+A3.

[0165]

[0166] Results analysis:

[0167] In Table 26, combination 5 exhibits the best sensitivity and specificity.

[0168] The inventors hypothesize that the melanoma differentiation-associated gene 5 polypeptide A1, A2, and A3 selected in this application each contain different antigenic determinants. The binding rate constants of the antigenic determinants of the three antigens and their corresponding antibodies are constant but different. The concentration level signal value of a single antigen is positively correlated with the signal value level. The immunoassay methodology determines the positive or negative result by comparing the signal value with a pre-set threshold. The three antigens have a complementary relationship in antibody detection in the population. The final concentration of magnetic particles when the three antigens are used simultaneously is particularly important. Only when the signal value levels of the mixed antigens are adjusted to be similar will the positive judgment values ​​of the three antigens be similar, resulting in the highest sensitivity and specificity. If the concentration of a certain antigen is relatively low, the signal value of the sample that can only be detected by that antigen will be below the threshold, leading to a false negative result and decreased sensitivity. Similarly, if the concentration of a certain antigen is relatively high, the overall signal value of the sample will be increased, the threshold will shift upward, and a false positive result will be obtained, resulting in a false positive and decreased specificity.

[0169] From Tables 15 and 26, it can be seen that the coating ratio of melanoma differentiation-related gene 5 polypeptide A1 to the magnetic microparticles in the melanoma differentiation-related gene 5 polypeptide A1-conjugated magnetic microparticles is 5 μg / mg; the coating ratio of melanoma differentiation-related gene 5 polypeptide A2 to the magnetic microparticles in the melanoma differentiation-related gene 5 polypeptide A2-conjugated magnetic microparticles is 2.5 μg / mg; and the coating ratio of melanoma differentiation-related gene 5 polypeptide A3 to the magnetic microparticles in the melanoma differentiation-related gene 5 polypeptide A3-conjugated magnetic microparticles is 3 μg / mg. The final concentration of the melanoma differentiation-related gene 5 polypeptide A1-conjugated magnetic microparticles is 0.05 mg / mL. The optimal combination is a final concentration of 0.03 mg / mL for peptide A2 coupled with magnetic microparticles, and a final concentration of 0.02 mg / mL for the melanoma differentiation-associated gene 5 peptide A3 coupled with magnetic microparticles. At this concentration, the sensitivity and specificity of the antibody detection against melanoma differentiation-associated gene 5 are highest, with a sensitivity of 98.00%, a specificity of 100%, a false positive rate of 0.00%, and a false negative rate of 2.00%. Compared with commercially available melanoma differentiation-associated gene 5 coupled magnetic microparticles with a final magnetic bead concentration of 0.1 mg / mL, the false positive rate is 2.00%, the false negative rate is 10.00%, the false negative rate is reduced by 80%, and the false positive rate is reduced by 100%. The sensitivity is significantly improved, the specificity is also improved, and the performance improvement effect is remarkable.

[0170] This invention utilizes magnetic microparticles coupled with melanoma differentiation-associated gene 5 (MEA5) peptide A1, where the coating ratio of MEA5 peptide A1 to the magnetic microparticles is 5 μg / mg; MEA2 coupled with magnetic microparticles, where the coating ratio of MEA2 to the magnetic microparticles is 2.5 μg / mg; and MEA3 coupled with magnetic microparticles, where the coating ratio of MEA3 to the magnetic microparticles is 3 μg / mg. Furthermore, the final concentrations of the MEA5 peptide A1-coupled magnetic microparticles are 0.05 mg / mL, 0.03 mg / mL, and 0.02 mg / mL, respectively. This further improves the sensitivity and specificity of detecting anti-MEA5 antibodies in the serum of different patients.

[0171] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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.

Claims

1. A detection kit for antibody against melanoma differentiation-associated gene 5, characterized in that, include: Melanoma differentiation-associated gene 5 coupled magnetic microparticle working solution, wherein the melanoma differentiation-associated gene 5 coupled magnetic microparticle working solution comprises melanoma differentiation-associated gene 5 coupled magnetic microparticles and magnetic bead buffer. A tracer-labeled antibody working solution, wherein the tracer-labeled antibody working solution comprises a tracer-labeled first antibody and a diluent; The melanoma differentiation-associated gene 5 coupled magnetic microparticles include melanoma differentiation-associated gene 5 polypeptide A1 coupled magnetic microparticles, melanoma differentiation-associated gene 5 polypeptide A2 coupled magnetic microparticles, and melanoma differentiation-associated gene 5 polypeptide A3 coupled magnetic microparticles. The amino acid sequence of the melanoma differentiation-associated gene 5 polypeptide A1 is shown in SEQ ID No. 1; The amino acid sequence of the melanoma differentiation-associated gene 5 polypeptide A2 is shown in SEQ ID No. 2; The amino acid sequence of the melanoma differentiation-associated gene 5 polypeptide A3 is shown in SEQ ID No. 3; The coating ratio of melanoma differentiation-associated gene 5 polypeptide A1 to the magnetic microparticles in the magnetic microparticles is 5 μg / mg. The coating ratio of melanoma differentiation-associated gene 5 peptide A2 to the magnetic microparticles in the magnetic microparticles is 2.5 ug / mg. The coating ratio of melanoma differentiation-associated gene 5 peptide A3 to the magnetic microparticles in the magnetic microparticles is 3 μg / mg. The final concentration of the melanoma differentiation-associated gene 5 peptide A1 coupled magnetic microparticles in the working solution is 0.05 mg / mL, the final concentration of the melanoma differentiation-associated gene 5 peptide A2 coupled magnetic microparticles is 0.03 mg / mL, and the final concentration of the melanoma differentiation-associated gene 5 peptide A3 coupled magnetic microparticles is 0.02 mg / mL.

2. The reagent kit according to claim 1, characterized in that, The magnetic particles are toluenesulfonyl magnetic beads.

3. The reagent kit according to claim 1, characterized in that, The magnetic bead buffer is a phosphate buffer with a pH of 7.4 and a concentration of 0.01 M, consisting of 0.01 mg / mL bovine serum albumin, 0.9 mg / mL sodium chloride, 0.1 mL / L Triton-X1000, 0.01 mL / mL Procline-300.

4. The reagent kit according to claim 1, characterized in that, The tracer is alkaline phosphatase.

5. The reagent kit according to claim 4, characterized in that, The first antibody is an anti-human IgG antibody.

6. The reagent kit according to claim 5, characterized in that, The diluent was a MES buffer solution containing 0.1 mg / mL bovine serum albumin, 0.1 mg / mL sodium chloride, 0.1 mg / mL sucrose, 0.01 mL / mL proclin 300, pH 6.0, and a concentration of 0.1 mol / L.

7. The kit according to claim 1, characterized in that, The kit also includes a sample diluent, which is a 0.01M pH 7.4 phosphate buffer containing 0.01 mg / mL bovine serum albumin, 0.001 mL / mL Procline-300, and 0.001 mL / mL Triton X-100.

8. The use of the kit according to any one of claims 1-7 in the in vitro detection of anti-melanoma differentiation-associated gene 5 antibody for non-disease diagnostic purposes.

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