Anti-sperm antibody IgM chemiluminescence kit and preparation method thereof

Abstract

The invention relates to the technical field of chemiluminiscence detection, in particular to an anti-sperm antibody IgM chemiluminiscence kit and a preparation method thereof.The anti-sperm antibody IgM chemiluminiscence kit comprises a reagent M, a reagent R2 and a reagent R1, and the test repeatability CV is smaller than 3%; a biotin interference sample with the addition concentration of 50ng / mL is tested, and the result has no obvious interference phenomenon; 50 clinical samples are tested and compared with the result of a comparison manufacturer, the total coincidence rate is 98%, meanwhile, a Guilin superior chemiluminescence immunoassay analyzer IA260 is matched to form a detection system, the testing amount per hour reaches 130 tests, and compared with a traditional colloidal gold immunochromatography method and an enzyme-linked immunosorbent assay method, the test speed is greatly increased, and the result optimization accuracy is greatly improved. Therefore, the problems that a colloidal gold immunochromatography method and an enzyme-linked immunosorbent assay method used for existing anti-sperm antibody detection are low in test flux, and other similar reagents cannot solve biotin interference are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of chemiluminescence detection, and in particular to an anti-sperm antibody IgM chemiluminescence kit and a preparation method thereof. Background Art

[0002] Sperm antigen is a hidden antigen. After contacting with the body's immune system, it will produce autoimmune or alloimmune reactions, leading to the production of antisperm antibodies. In infertile patients, the detection rate of antisperm antibodies is usually 10%-30%, and the positive rate in patients with obstructive azoospermia is as high as 60%. Clinically, about 10% of male infertility is related to immune factors. The blood-testis barrier is damaged and antisperm antibodies are produced. The production of antisperm antibodies has an inhibitory effect on the vitality and production of sperm. When the immunosuppressive substances in sperm are missing and the female reproductive tract mucosa is damaged, sperm enters the female body and produces an immune response, prompting women to produce antisperm antibodies, dissolving and damaging sperm, resulting in decreased female conception ability, miscarriage, and infertility. Antisperm antibodies are divided into IgG, IgM, and IgA3 subtypes. Antisperm antibody IgM (ASA-IgM) is mainly used clinically for auxiliary diagnosis of decreased conception ability or infertility.

[0003] At present, the detection kits for anti-sperm antibody IgM (ASA-IgM) on the market are mainly divided into colloidal gold immunochromatography, enzyme-linked immunosorbent assay (ELISA), and chemiluminescent immunoassay (CLIA). Traditional colloidal gold immunoassay cannot achieve accurate quantitative analysis, while ELISA is greatly affected by individual factors and has a low test throughput, which is not conducive to fully automated detection. Chemiluminescent immunoassay has the advantages of high degree of automation, large test throughput, high detection sensitivity, and accurate results.

[0004] Biotin, also known as vitamin B7, is a sulfur-containing water-soluble vitamin in the B vitamins. Biotin is an important factor in the fixation of carbon dioxide in organisms and a necessary substance for the synthesis of vitamin C. It plays an important role in biochemical reaction pathways such as fat synthesis and gluconeogenesis. It is a necessary nutrient for maintaining the natural growth and development of the human body and the normal health of human functions, and has multiple biological effects. Currently, the main source of biotin interference is the widespread use of various nutritional supplements and the use of ultra-high doses of biotin to treat multiple sclerosis and biotin-responsive neurobasal ganglia diseases.

[0005] The biotin in the sample will compete with the biotin component in the chemiluminescent reagent to bind to the streptavidin magnetic particles, so that the luminescent substance in the reagent is not captured enough by the streptavidin magnetic particles, resulting in a weakening of the measurable light signal, thereby causing a false decrease in the sandwich method results and a false increase in the competition method results. Summary of the invention

[0006] The purpose of the present invention is to provide an anti-sperm antibody IgM chemiluminescent kit and a preparation method thereof, aiming to solve the problem that the colloidal gold immunochromatography and enzyme-linked immunosorbent assay used in the existing anti-sperm antibody detection have low test throughput and other similar reagents cannot solve the problem of biotin interference.

[0007] To achieve the above-mentioned purpose, in the first aspect, the present invention provides an anti-sperm antibody IgM chemiluminescent kit, comprising reagent M, reagent R2 and reagent R1, wherein the reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibodies labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer containing a blocking agent and streptavidin.

[0008] The reagent M includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 5-25 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and biotin-labeled sperm antigen 0.5 μg / mL-2 μg / mL.

[0009] The reagent R2 includes 0.05-0.1 mol / L MES buffer, pH value in the range of 6.0-8.0, zinc chloride, magnesium chloride, 0.1 g / L-10 g / L protein stabilizer, 1-5 g / L carbohydrate molecules, 0.5 g / L-5 g / L nonionic surfactant, 0.5 g / L-1 g / L preservative, and 0.5 μg / mL-2 μg / mL alkaline phosphatase-labeled anti-human IgM antibody.

[0010] Among them, the reagent R1 includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1g / L-10g / L, carbohydrate molecules 5-10g / L, non-ionic surfactant 0.5g / L-5g / L, preservative 0.5g / L-1g / L, blocking agent 50μg / mL-100μg / mL, and streptavidin 0.5μg / mL-1μg / mL.

[0011] In a second aspect, a method for preparing an anti-sperm antibody IgM chemiluminescent kit is provided, which is used to prepare the anti-sperm antibody IgM chemiluminescent kit described in the first aspect, comprising the following steps:

[0012] The biotin-labeled sperm antigen is coated with magnetic beads to prepare reagent M;

[0013] The alkaline phosphatase-labeled anti-human IgM antibody is added at a concentration of 0.5 μg / mL-2 μg / mL to 0.05-0.1 mol / L MES buffer, zinc chloride, magnesium chloride, 0.1 g / L-10 g / L protein stabilizer, 1-5 g / L carbohydrate molecule, 0.5 g / L-5 g / L nonionic surfactant and 0.5 g / L-1 g / L preservative to prepare reagent R2;

[0014] Adding blocking agent BR0110-100 μg / mL, HBR-PLUS10-100 μg / mL, and streptavidin 0.5-1 μg / mL to phosphate buffer 0.05-0.1 mol / L, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, nonionic surfactant 0.5 g / L-5 g / L, and preservative 0.5 g / L-1 g / L to prepare reagent R1;

[0015] The ASA-IgM chemiluminescence kit is composed based on the reagent M, the reagent R2 and the reagent R1.

[0016] The present invention discloses an anti-sperm antibody IgM chemiluminescence kit, comprising reagent M, reagent R2 and reagent R1, wherein the reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibody labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer containing a blocking agent and streptavidin. The test repeatability CV of the present invention is less than 3%; the test adds a biotin interference sample with a concentration of 50 ng / mL, and the result shows no obvious interference phenomenon; 50 clinical samples are tested and compared with the results of the comparison manufacturer, and the total compliance rate is 98%. At the same time, the detection system is matched with Guilin Youlite chemiluminescence immunoassay instrument IA260, and the single-hour test volume reaches 130 tests. Compared with the traditional colloidal gold immunochromatography and enzyme-linked immunosorbent assay, the test speed is greatly improved and the result accuracy is optimized. Thus, the problem that the colloidal gold immunochromatography and enzyme-linked immunosorbent assay used in the existing anti-sperm antibody detection have low test throughput and other similar reagents cannot solve the problem of biotin interference. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 The present invention provides a flow chart of a method for preparing an anti-sperm antibody IgM chemiluminescence kit. DETAILED DESCRIPTION

[0019] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0020] In the first aspect, the present invention provides an anti-sperm antibody IgM chemiluminescent kit, comprising reagent M, reagent R2 and reagent R1, wherein the reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibodies labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer containing a blocking agent and streptavidin.

[0021] The reagent M includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, sugar molecule 5-25 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and biotin-labeled sperm antigen 0.5 μg / mL-2 μg / mL.

[0022] The reagent R2 includes MES buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and alkaline phosphatase-labeled anti-human IgM antibody 0.5 μg / mL-2 μg / mL.

[0023] The reagent R1 includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 5-10 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, blocking agent 50 μg / mL-100 μg / mL, and streptavidin 0.5 μg / mL-1 μg / mL.

[0024] In this embodiment, reagent M, reagent R2 and reagent R1 are included. The reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibodies labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer containing a blocking agent and streptavidin. The test repeatability CV of the present invention is less than 3%; the test adds a biotin interference sample with a concentration of 50ng / mL, and the result has no obvious interference phenomenon; 50 clinical samples are tested and compared with the results of the comparison manufacturer, and the total compliance rate is 98%. At the same time, the detection system is matched with Guilin Youlite chemiluminescence immunoassay instrument IA260, and the single-hour test volume reaches 130 tests. Compared with the traditional colloidal gold immunochromatography and enzyme-linked immunosorbent assay, the test speed is greatly improved and the result accuracy is optimized. This solves the problem that the colloidal gold immunochromatography and enzyme-linked immunosorbent assay used in the existing anti-sperm antibody detection have low test throughput, and other similar reagents cannot solve the problem of biotin interference.

[0025] See also Figure 1 In the second aspect, a method for preparing an anti-sperm antibody IgM chemiluminescent kit is used to prepare the anti-sperm antibody IgM chemiluminescent kit described in the first aspect, comprising the following steps:

[0026] S1 prepares reagent M by coating biotin-labeled sperm antigen with magnetic beads;

[0027] Specifically, according to the working concentration of biotin-labeled sperm antigen 0.5μg / mL-2μg / mL, the magnetic bead stock solution was taken out according to the coating ratio of 1mg:5μg-1mg:20μg; the magnetic microparticle stock solution was mixed on a blood mixer for 10 minutes until it was suspended without obvious precipitation and aggregated particles; the mixed magnetic bead stock solution was placed on a magnetic separator for 10 minutes, and when the supernatant was completely clear and transparent, the supernatant was discarded, and 3 times the volume of the magnetic bead stock solution, the coating solution Buffer E, was added, and the above operation was repeated 3 times; 3 times the volume of the coating solution Buffer E. Mix and resuspend the magnetic beads, add the biotin-labeled sperm antigen according to the calculated amount determined above, place on a blood mixer, and mix at room temperature for 30min-2h; add the blocking agent 1 according to the calculated amount, place on a blood mixer, and mix at room temperature for 1h-4h; place on a magnetic separator until the liquid becomes clear and transparent, discard the supernatant, take 3 times the volume of the magnetic bead stock solution BufferA to wash 3 times; add the above solution to the magnetic particle working solution BufferA to make up to the target concentration, and the result is reagent M.

[0028] S2: adding alkaline phosphatase-labeled anti-human IgM antibody at a concentration of 0.5 μg / mL-2 μg / mL to 0.05-0.1 mol / L MES buffer, zinc chloride, magnesium chloride, 0.1 g / L-10 g / L protein stabilizer, 1-5 g / L carbohydrate molecule, 0.5 g / L-5 g / L nonionic surfactant and 0.5 g / L-1 g / L preservative to prepare reagent R2;

[0029] Specifically, alkaline phosphatase-labeled anti-human IgM antibody is added at a concentration of 0.5 μg / mL-2 μg / mL to MES buffer 0.05-0.1 mol / L with a pH value in the range of 6.0-8.0 (containing 0.1-0.2 mol / L sodium chloride), zinc chloride, magnesium chloride, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecules 1-5 g / L, nonionic surfactant 0.5 g / L-5 g / L, and preservative 0.5 g / L-1 g / L, and the result is reagent R2.

[0030] S3: adding blocking agent BR01 10-100 μg / mL, HBR-PLUS 10-100 μg / mL, and streptavidin 0.5-1 μg / mL to phosphate buffer 0.05-0.1 mol / L, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, nonionic surfactant 0.5 g / L-5 g / L, and preservative 0.5 g / L-1 g / L to prepare reagent R1;

[0031] Specifically, blocker BR0110-100 μg / mL, HBR-PLUS10-100 μg / mL, streptavidin 0.5-1 μg / mL are added to phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and the result is reagent R1.

[0032] S4 forms an ASA-IgM chemiluminescent kit based on the reagent M, the reagent R2 and the reagent R1.

[0033] Specifically, reagent M and reagents R2 and R1 together constitute the ASA-IgM chemiluminescent kit. Wherein, the magnetic particle working solution Buffer A is a phosphate buffer solution of pH 8.0±0.05, with the addition of 0.2mol NaCl, 5g / L bovine serum albumin, 10g / L mannitol, 0.1g / L 4-aminopyrine, 1g / L Tween-20, and 1g / L PC-300. Wherein, the coating solution Buffer E is a PBS buffer solution of pH 7.40±0.05, with the addition of 0.2mol NaCl, 5g / L casein, 1g / L bovine serum albumin, 10g / L sucrose, 1g / L Tween-20, and 0.5g / L PC-300. Wherein, the blocking agent 1 is a Tris buffer with a pH of 7.40±0.05, to which 0.2 mol of NaCl, 10 g / L of bovine serum albumin, 5 g / L of deep-sea fish skin gelatin, 5 g / L of sucrose, 1 g / L of TX-100, and 0.5 g / L of PC-300 are added.

[0034] Example 1

[0035] The invention provides an ASA-IgM chemiluminescence kit and a preparation method thereof, comprising a reagent M and reagents R2 and R1; the reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibodies labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer containing a blocking agent and streptavidin.

[0036] The reagent M includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 5-25 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and biotin-labeled sperm antigen 0.5 μg / mL-2 μg / mL.

[0037] The reagent R2 includes 0.05-0.1 mol / L MES buffer, pH value in the range of 6.0-8.0, zinc chloride, magnesium chloride, 0.1 g / L-10 g / L protein stabilizer, 1-5 g / L carbohydrate molecules, 0.5 g / L-5 g / L nonionic surfactant, 0.5 g / L-1 g / L preservative, and 0.5 μg / mL-2 μg / mL alkaline phosphatase-labeled anti-human IgM antibody.

[0038] Wherein, the reagent R1 includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1g / L-10g / L, carbohydrate molecule 5-10g / L, non-ionic surfactant 0.5g / L-5g / L, preservative 0.5g / L-1g / L, blocking agent 50μg / mL-100μg / mL, and streptavidin 0.5μg / mL-1μg / mL.

[0039] Example 2

[0040] A method for preparing an ASA-IgM chemiluminescent kit comprises the following steps:

[0041] Prepare Reagent M:

[0042] According to the working concentration of biotin-labeled sperm antigen 0.5μg / mL-2μg / mL, take out the magnetic bead stock solution according to the coating ratio of 1mg:5μg-1mg:20μg;

[0043] Mix the magnetic microparticle stock solution on a blood mixer for 10 minutes until it is suspended and there is no obvious precipitation or aggregated particles.

[0044] Place the mixed magnetic bead stock solution on a magnetic separator for 10 minutes. When the supernatant is completely clear and transparent, discard the supernatant and add 3 times the volume of Buffer E of the magnetic bead stock solution. Repeat the above operation 3 times.

[0045] Add 3 times the volume of coating buffer E, mix and resuspend the magnetic beads, add biotin-labeled sperm antigen according to the above-determined ratio, place on a blood mixer, and mix at room temperature for 30 minutes to 2 hours;

[0046] Add the calculated amount of sealant 1, place on a blood mixer, and mix at room temperature for 2h-4h;

[0047] Place on a magnetic separator until the liquid becomes clear and transparent, discard the supernatant, and wash three times with Buffer A, a working solution 3 times the volume of the magnetic bead stock solution.

[0048] The above solution was added to the magnetic particle working solution Buffer A to make up to the target concentration, and the result was reagent M.

[0049] Prepare reagent R2:

[0050] The alkaline phosphatase-labeled anti-human IgM antibody is added at a concentration of 0.5 μg / mL-2 μg / mL to MES buffer 0.05-0.1 mol / L pH 6.0-8.0 (containing 0.1-0.2 mol / L sodium chloride), zinc chloride, magnesium chloride, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecules 1-5 g / L, nonionic surfactant 0.5 g / L-5 g / L, and preservative 0.5 g / L-1 g / L to obtain reagent R2.

[0051] Prepare reagent R1:

[0052] Add blocking agent BR0110-100μg / mL, HBR-PLUS10-100μg / mL, and streptavidin 0.5-1μg / mL to phosphate buffer 0.05-0.1mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1g / L-10g / L, carbohydrate molecules 1-5g / L, non-ionic surfactant 0.5g / L-5g / L, and preservative 0.5g / L-1g / L to obtain reagent R1.

[0053] Reagent M and reagents R2 and R1 together constitute the ASA-IgM chemiluminescence kit.

[0054] The magnetic particle working solution Buffer A is a phosphate buffer solution with a pH of 8.0±0.05, to which 0.2 mol NaCl, 5 g / L bovine serum albumin, 10 g / L mannitol, 0.1 g / L 4-aminopyrine, 1 g / L Tween-20, and 1 g / L PC-300 are added.

[0055] The coating solution Buffer E is a PBS buffer with a pH of 7.40±0.05, to which 0.2 mol NaCl, 5 g / L casein, 1 g / L bovine serum albumin, 10 g / L sucrose, 1 g / L Tween-20, and 0.5 g / L PC-300 are added.

[0056] Wherein, the blocking agent 1 is a Tris buffer with a pH of 7.40±0.05, to which 0.2 mol of NaCl, 10 g / L of bovine serum albumin, 5 g / L of deep-sea fish skin gelatin, 5 g / L of sucrose, 1 g / L of TX-100, and 0.5 g / L of PC-300 are added.

[0057] Among them, all the above buffers were filtered through a 0.22μm pore size nylon filter membrane after preparation.

[0058] Example 3

[0059] Anti-sperm antibody IgM chemiluminescent kit A

[0060] Preparation of Reagent M:

[0061] 1. The concentration of biotin-labeled sperm antigen working solution is 1 μg / mL. Take out the magnetic bead stock solution according to the coating ratio (magnetic beads: antibody) of 1 mg: 10 μg.

[0062] 2. Replace the magnetic bead stock solution (Agilent's streptavidin magnetic beads) by taking out 3 times the volume of the magnetic bead stock solution, add the coating solution Buffer C to the magnetic particles after discarding the supernatant in the magnetic separator, and mix on a mixer for 10 minutes.

[0063] 3. Take 3 times the volume of the original magnetic bead solution, add the coating solution Buffer C to the magnetic particles with the supernatant discarded, wash three times, place on a magnetic separator, and discard the supernatant after the liquid becomes clear and transparent.

[0064] 4. Add 3 times the volume of coating buffer C, mix and resuspend the magnetic beads, add biotin-labeled antibody according to the above-determined ratio, place on a blood mixer, and mix at room temperature for 30 minutes.

[0065] 5. Add the calculated amount of blocking agent 1 (add 50 μL of blocking solution to 1 mg of magnetic particles), place on a blood mixer, and mix at room temperature for 1 hour.

[0066] 6. Place the beads on a magnetic separator until the liquid becomes clear and transparent, then discard the supernatant. Take 3 times the volume of the original magnetic bead solution, Buffer A, and wash three times. Place the beads on a magnetic separator until the liquid becomes clear and transparent, then discard the supernatant.

[0067] 7. Add the above solution to the magnetic particle working solution Buffer A and make up to the target concentration. The result is reagent M.

[0068] Reagent R2 preparation:

[0069] 1. Preparation of enzyme labeling buffer: MES buffer 0.1 mol / L, adjust pH = 6.50 ± 0.05, zinc chloride prepared and used immediately (100 mM) 0.1 mL / L, magnesium chloride prepared and used immediately (500 mM) 10 mL / L, casein 5 g / L, sucrose 2 g / L, polyvinyl pyrrolidone 301 g / L, PEG-2000 1 g / L, PC-300 1 g / L.

[0070] 2. Calculate the amount of alkaline phosphatase-labeled anti-human IgM antibody at 1 μg / mL, add the calculated amount of anti-human IgM antibody to the enzyme-labeled buffer, and the result is reagent R2.

[0071] Reagent R1 preparation:

[0072] Add blocking agent BR01 50 μg / mL and HBR-PLUS 50 μg / mL to phosphate buffer 0.1 mol / L, adjust pH to 7.00±0.05, bovine serum albumin 5 g / L, mannitol 5 g / L, Tween-20 1 g / L, PC-300 1 g / L, and the result is reagent R1.

[0073] Example 4

[0074] Chemiluminescent kit B for anti-sperm antibody IgM

[0075] Preparation of Reagent M:

[0076] Add the calculated amount of blocking agent 1 (1 mg of magnetic particles added to 100 μL of blocking solution) in step 5 of Example 3, place on a blood mixer, and mix at room temperature for 2 h.

[0077] Reagents R2 and R1 and other operations were kept unchanged to obtain the chemiluminescent kit B for anti-sperm antibody IgM.

[0078] Example 5

[0079] Anti-sperm antibody IgM chemiluminescent kit C

[0080] Preparation of Reagent M:

[0081] Add the calculated amount of blocking agent 1 (1 mg of magnetic particles added to 100 μL of blocking solution) in step 5 of Example 3, place on a blood mixer, and mix at room temperature for 2 h.

[0082] Preparation of reagent R1:

[0083] Add blocking agent BR01 50 μg / mL, HBR-PLUS 50 μg / mL, and streptavidin 1 μg / mL to phosphate buffer 0.1 mol / L, adjust pH to 7.00±0.05, bovine serum albumin 5 g / L, mannitol 5 g / L, Tween-20 1 g / L, and PC-300 1 g / L to obtain reagent R1.

[0084] Reagent R2 and other operations were not changed to obtain the chemiluminescent kit C for anti-sperm antibody IgM.

[0085] Example 6

[0086] Performance evaluation of chemiluminescence kit for anti-sperm antibody IgM

[0087] Repeatability test:

[0088] Two quality control products of different concentrations were tested using the same batch of test kits, each test was repeated 10 times, and the coefficient of variation (CV) was no greater than 10.0%.

[0089] Table 1 Repeatability test results of kit A (unit: RU / mL)

[0090] Number of tests Sample 1 Sample 2 1 9.78 84.12 2 9.65 80.26 3 9.98 80.80 4 10.05 80.33 5 10.21 81.60 6 10.30 84.85 7 10.21 83.83 8 9.97 83.66 9 10.16 79.33 10 9.70 78.36 CV(%) 2.28％ 2.76％

[0091] Table 2 Repeatability test results of kit B (unit: RU / mL)

[0092] Number of tests Sample 1 Sample 2 1 9.84 78.09 2 10.35 81.62 3 10.56 82.95 4 10.34 81.54 5 10.20 80.77 6 10.51 80.42 7 10.38 78.63 8 10.42 81.95 9 10.30 81.16 10 9.83 83.89 CV(%) 2.45％ 2.18％

[0093] Table 3 Repeatability test results of kit C (unit: RU / mL)

[0094] Number of tests Sample 1 Sample 2 1 10.22 81.08 2 10.31 80.97 3 10.05 79.99 4 10.42 83.45 5 10.12 82.65 6 10.43 82.42 7 10.09 81.58 8 9.85 80.54 9 10.26 79.50 10 9.93 82.78 CV(%) 1.92％ 1.60％

[0095] The results showed that the coefficients of variation of the test results of high and low value samples of kits A, B, and C were all within 10% and lower than 3%. Among them, the repeatability test results of kit C were slightly better than those of kits A and B.

[0096] Intrinsic interference detection:

[0097] Table 4 Endogenous interference test results of kit A (unit: RU / mL)

[0098]

[0099] Table 5 Endogenous interference test results of kit B (unit: RU / mL)

[0100]

[0101]

[0102] Table 6 Test results of endogenous interference of kit C (unit: RU / mL)

[0103]

[0104] The results showed that kits A, B, and C had good anti-interference ability to bilirubin, hemoglobin, and triglycerides, and kit C also had good anti-interference ability to biotin.

[0105] The test kits A, B, and C in this example were used together with the Guilin Uni-Tech chemiluminescent immunoassay analyzer IA260 to form a detection system and compared with the Kanghua anti-sperm antibody detection reagent (enzyme-linked immunosorbent assay). The specific experiments are as follows:

[0106] Table 7 Clinical test results of kit A

[0107]

[0108] Table 8 Clinical test results of kit B

[0109]

[0110]

[0111] Table 9 Kit C clinical test results

[0112]

[0113] The results showed that kit C had the best clinical compliance rate with the comparison manufacturer's reagents.

[0114] Based on the above results, Kit C has the best performance and clinical expression, and can better solve the problem of biotin interference.

[0115] Beneficial effects:

[0116] 1. The preparation method of the anti-sperm antibody IgM chemiluminescent kit provided by the present invention greatly shortens the production cycle, reduces the process difficulty, and is beneficial to the production work of enterprises.

[0117] 2. The present invention provides an anti-sperm antibody IgM chemiluminescence kit, with a test repeatability CV of <3%; a test sample with a biotin interference concentration of 50 ng / mL was tested, and the results showed no obvious interference; 50 clinical samples were tested and compared with the results of the comparison manufacturer, and the total compliance rate was 98%.

[0118] 3. The present invention provides an anti-sperm antibody IgM chemiluminescent kit, which is combined with Guilin Ulite chemiluminescent immunoassay analyzer IA260 to form a detection system, with a single hour test volume of 130 tests. Compared with traditional colloidal gold immunochromatography and enzyme-linked immunosorbent assay, it greatly improves the test speed and optimizes the result accuracy.

[0119] 4. The present invention improves the existing detection method of anti-sperm antibody IgM, adopts chemiluminescence immunoassay, and develops a new detection kit with high detection sensitivity and good repeatability, and can better solve the problem of biotin interference, thereby ensuring accurate test results.

[0120] The above disclosure is only a preferred embodiment of an anti-sperm antibody IgM chemiluminescent kit and a preparation method thereof of the present invention. Of course, this cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiments and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. An anti-sperm antibody IgM chemiluminescent kit, characterized in that: The method comprises reagent M, reagent R2 and reagent R1, wherein the reagent M is a magnetic particle working solution containing sperm antigen coupling, the reagent R2 is an enzyme-labeled working solution containing anti-human IgM antibody labeled with alkaline phosphatase, and the reagent R1 is a phosphate buffer solution containing a blocking agent and streptavidin.

2. The anti-sperm antibody IgM chemiluminescent kit according to claim 1, characterized in that: The reagent M includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, sugar molecule 5-25 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and biotin-labeled sperm antigen 0.5 μg / mL-2 μg / mL.

3. The anti-sperm antibody IgM chemiluminescent kit according to claim 2, characterized in that: The reagent R2 includes MES buffer 0.05-0.1 mol / L, pH value in the range of 6.0-8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, and alkaline phosphatase-labeled anti-human IgM antibody 0.5 μg / mL-2 μg / mL.

4. The anti-sperm antibody IgM chemiluminescent kit according to claim 3, characterized in that: The reagent R1 includes phosphate buffer 0.05-0.1 mol / L, pH value in the range of 6.0±8.0, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 5-10 g / L, non-ionic surfactant 0.5 g / L-5 g / L, preservative 0.5 g / L-1 g / L, blocking agent 50 μg / mL-100 μg / mL, and streptavidin 0.5 μg / mL-1 μg / mL.

5. A method for preparing an anti-sperm antibody IgM chemiluminescent kit, used for preparing the anti-sperm antibody IgM chemiluminescent kit according to any one of claims 1 to 4, characterized in that: The following steps are involved: The biotin-labeled sperm antigen is coated with magnetic beads to prepare reagent M; The alkaline phosphatase-labeled anti-human IgM antibody is added at a concentration of 0.5 μg / mL-2 μg / mL to 0.05-0.1 mol / L MES buffer, zinc chloride, magnesium chloride, 0.1 g / L-10 g / L protein stabilizer, 1-5 g / L carbohydrate molecule, 0.5 g / L-5 g / L nonionic surfactant and 0.5 g / L-1 g / L preservative to prepare reagent R2; Adding blocking agent BR0110-100 μg / mL, HBR-PLUS10-100 μg / mL, and streptavidin 0.5-1 μg / mL to phosphate buffer 0.05-0.1 mol / L, protein stabilizer 0.1 g / L-10 g / L, carbohydrate molecule 1-5 g / L, nonionic surfactant 0.5 g / L-5 g / L, and preservative 0.5 g / L-1 g / L to prepare reagent R1; The ASA-IgM chemiluminescence kit is composed based on the reagent M, the reagent R2 and the reagent R1.

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