A kit for measuring anti-sperm antibody igg and use thereof

Abstract

The application discloses a kit for determining anti-sperm antibody IgG and application thereof, and comprises sperm membrane antigen coated carboxyl magnetic beads, an analysis buffer and an enzyme working solution, wherein the sperm membrane antigen coated carboxyl magnetic beads are labeled with biotin, streptavidin and streptavidin antibody; in the application, the streptavidin antibody is added in the coating process of the carboxyl magnetic beads, a carboxyl magnetic bead-streptavidin antibody-streptavidin-biotinized antigen is formed, the connecting length between the antigen and the magnetic beads is increased, the steric hindrance is reduced, and the interference of some sample detection is avoided.

Description

Technical Field

[0001] This invention relates to the field of biological detection kit technology, specifically to a kit for measuring antisperm antibody IgG and its application. Background Technology

[0002] Immunological infertility is one of the main causes of infertility, accounting for approximately 10% to 30% of infertility cases. Among these, reproductive disorders caused by antisperm antibodies are the most common. Sperm is a cryptic antigen that, upon contact with the body's immune system, triggers an autoimmune or alloimmune response, leading to the production of antisperm antibodies. There are three main types of antisperm antibodies, which can be classified into three subtypes: IgG, IgM, and IgA. The IgG subtype has a higher proportion of antisperm antibodies produced. The mechanisms of action of antisperm antibodies include (1) affecting sperm motility and inhibiting sperm penetration of cervical mucus; (2) affecting sperm capacitation and acrosome reaction; (3) affecting penetration of the zona pellucida and sperm-egg fusion; and (4) affecting embryonic development and implantation. Research data shows that the positive rate of antisperm antibodies is significantly higher in patients with primary and secondary infertility. Positive antisperm antibody test results have been confirmed as one of the diagnostic indicators of immunological infertility. Clinically, it is mainly used as an auxiliary diagnostic tool for decreased fertility or infertility.

[0003] Existing assay kits use an indirect method to detect antisperm antibody IgG. The molecular weight of the sperm membrane antigen coated on the magnetic bead ends is smaller than that of the antisperm antibody IgG, which can easily cause steric hindrance during the detection process, leading to abnormal sample detection.

[0004] Therefore, this patent application is filed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that sample interference exists in the existing kits for detecting antisperm antibody IgG.

[0006] The first objective of this invention is to provide a kit for determining antisperm antibody IgG, comprising carboxyl magnetic beads coated with sperm membrane antigen, analytical buffer, and enzyme working solution, wherein the carboxyl magnetic beads coated with sperm membrane antigen are labeled with biotin, streptavidin, and streptavidin antibody.

[0007] In this embodiment of the invention, streptavidin antibody is added during the coating process of carboxylated magnetic beads. By forming carboxylated magnetic beads-streptavidin antibody-streptavidin-biotinylated antigen, the connection length between the antigen and the magnetic beads is increased, the steric hindrance is reduced, and interference with the detection of some samples is avoided.

[0008] In some embodiments, the method for preparing the biotinylated sperm membrane antigen is as follows:

[0009] Step 1: Preparation of biotin-labeled sperm membrane antigen: Dilute the sperm membrane antigen and biotin-label the diluted sperm membrane antigen to obtain biotinylated antigen;

[0010] Step 2: Preparation of carboxyl magnetic beads coated with sperm membrane antigen: Dilute carboxyl magnetic beads, activate carboxyl magnetic beads, mix streptavidin antibody with carboxyl magnetic beads and react to obtain the first solution;

[0011] Streptavidin was mixed with the first solution and reacted to obtain the second solution;

[0012] Add the biotinylated antigen from step one to the second solution to obtain carboxyl magnetic beads coated with sperm membrane antigen.

[0013] In this embodiment of the invention, the sperm membrane antigen is first biotinylated to obtain biotinylated antigen; then, streptavidin antibody is added to the activated carboxylated magnetic beads, and then reacts with streptavidin to form a carboxylated magnetic bead-streptavidin antibody-streptavidin-biotinylated antigen structural system, which increases the connection length between the antigen and the magnetic beads, reduces the steric hindrance, and avoids interference in the detection of some samples.

[0014] In some embodiments, the method for preparing the sperm membrane antigen-coated carboxyl magnetic beads further includes step three, which is:

[0015] The carboxyl magnetic beads coated with sperm membrane antigen obtained in step two were blocked with blocking solution, and after washing and dilution with analytical buffer, the free biotinylated antigen obtained in step one was added.

[0016] In this embodiment of the invention, free biotinylated antigen is added to carboxyl magnetic beads coated with sperm membrane antigen, which improves the problem of signal loss of magnetic beads under multiple ultrasound in the prior art and effectively improves the precision of the reagent.

[0017] In some embodiments, the activation of the carboxyl magnetic beads in step (2) is performed using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide sodium salt.

[0018] In some embodiments, the mass ratio of streptavidin antibody to carboxylated magnetic beads is 10 μg / mg.

[0019] In some embodiments, the mass ratio of streptavidin antibody to streptavidin is 1 mg / mg.

[0020] In some embodiments, the amount of free biotinylated antigen added in step three is based on the ratio of free biotinylated antigen to carboxyl magnetic beads = 0.5 μg / mg.

[0021] In some embodiments, the method for preparing carboxyl magnetic beads coated with sperm membrane antigen is as follows:

[0022] (1) Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen.

[0023] (2) Dilute the carboxyl magnetic beads to 10 mg / mL, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide sodium salt to activate the carboxyl magnetic beads for 15 minutes. After activation, mix streptavidin antibody with the carboxyl magnetic beads and react for 2 hours to obtain the first solution; the mass ratio of streptavidin antibody to carboxyl magnetic beads is 10 μg / mg.

[0024] (3) Mix streptavidin with the first solution and react for 2 hours to obtain the second solution; the mass ratio of streptavidin antibody to streptavidin is 1 mg / mg;

[0025] (4) Add the biotinylated antigen from step (1) to the second solution;

[0026] (5) The carboxyl magnetic beads coated with sperm membrane antigen were blocked with blocking solution, washed three times with analytical buffer, diluted, and then the free biotinylated antigen obtained in step (1) was added; free biotinylated antigen: carboxyl magnetic beads = 0.5 μg / mg.

[0027] In this embodiment of the invention, by designing a reasonable mass ratio of streptavidin antibody to carboxylated magnetic beads and a reasonable mass ratio of streptavidin antibody to streptavidin, the kit signal is higher, while the improvement of abnormal samples is significant, the detection rate of abnormal samples is significantly reduced, and the detection precision and magnetic bead retention rate are effectively improved.

[0028] In some embodiments, the analytical buffer is a Tris-HCl buffer and the blocking solution is a vitamin H solution.

[0029] In some embodiments, the enzyme working solution is an alkaline phosphatase-labeled mouse anti-human IgG antibody. A second object of the present invention is to provide an application of a kit for determining antisperm antibody IgG, for the detection of antisperm antibody IgG.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] (1) In the embodiments of the present invention, by connecting SA to SA during the coating process of carboxy magnetic beads, the connection length between the antigen and the magnetic beads is increased by forming carboxy magnetic beads-streptavidin antibody-streptavidin-biotinylated antigen, thereby reducing spatial steric hindrance, avoiding interference in the detection of some samples, and reducing the detection rate of abnormal samples.

[0032] (2) In the embodiments of the present invention, when free biotin antigen is further added to the coated magnetic beads, the detection rate of abnormal reagent samples is significantly reduced, and the detection precision and magnetic bead retention rate are effectively improved. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0034] Figure 1 This is a graph showing the detection signal values ​​of the kits coated with different ratios of SA and SA antibodies in Experiment 2 of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. The illustrative embodiments and descriptions of this invention are only used to explain this invention and are not intended to limit this invention.

[0036] Example 1:

[0037] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads containing SA antibody (streptavidin antibody) and SA (streptavidin), analytical buffer, and enzyme working solution, wherein the carboxyl magnetic beads are labeled with biotin.

[0038] The preparation method of adding SA antibody and biotin-labeled carboxyl magnetic beads (i.e., carboxyl magnetic beads coated with sperm membrane antigen) is as follows:

[0039] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0040] B. Add the SA antibody according to the mass ratio of SA antibody to carboxyl magnetic beads = 10 μg / mg, mix thoroughly and react for 2 hours;

[0041] C. Take SA and add it to the solution after the reaction in step B. The mass ratio of SA antibody to SA = 1 mg / mg. Mix thoroughly and react for 2 hours.

[0042] D. Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen.

[0043] Take the labeled biotinylated antigen and add it to the solution after the reaction in step C according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0044] E. Add the magnetic bead blocking agent vitamin H solution to the solution after the reaction in D, mix thoroughly, and allow to react at room temperature for 28-33 minutes by rolling.

[0045] F. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0046] Example 2:

[0047] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads containing SA antibody and SA, an analytical buffer, and an enzyme working solution. The carboxyl magnetic beads are labeled with biotin.

[0048] The preparation method of adding SA antibody and biotin-labeled carboxyl magnetic beads (i.e., carboxyl magnetic beads coated with sperm membrane antigen) is as follows:

[0049] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0050] B. Add the SA antibody according to the mass ratio of SA antibody to carboxyl magnetic beads = 10 μg / mg, mix thoroughly and react for 2 hours;

[0051] C. Take SA and add it to the solution after the reaction in step B. The mass ratio of SA antibody to SA is 0.5 mg / mg. Mix thoroughly and react for 2 hours.

[0052] D. Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen.

[0053] Take the labeled biotinylated antigen and add it to the solution after the reaction in step C according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0054] E. Add the magnetic bead blocking agent vitamin H solution to the solution after the reaction in D, mix thoroughly, and allow to react at room temperature for 28-33 minutes by rolling.

[0055] F. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0056] Example 3:

[0057] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads containing SA antibody and SA, an analytical buffer, and an enzyme working solution. The carboxyl magnetic beads are labeled with biotin.

[0058] The preparation method of adding SA antibody and biotin-labeled carboxyl magnetic beads (i.e., carboxyl magnetic beads coated with sperm membrane antigen) is as follows:

[0059] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0060] B. Add the SA antibody according to the mass ratio of SA antibody to carboxyl magnetic beads = 10 μg / mg, mix thoroughly and react for 2 hours;

[0061] C. Take SA and add it to the solution after the reaction in step B. The mass ratio of SA antibody to SA = 2 mg / mg. Mix thoroughly and react for 2 hours.

[0062] D. Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen.

[0063] Take the labeled biotinylated antigen and add it to the solution after the reaction in step C according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0064] E. Add the magnetic bead blocking agent vitamin H solution to D, mix thoroughly, and allow to react at room temperature for 28-33 minutes.

[0065] F. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0066] Example 4:

[0067] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads containing SA antibody and SA, analytical buffer, and enzyme working solution. The carboxyl magnetic beads are labeled with biotin and free biotinylated antigen is added after coating.

[0068] The preparation method of carboxyl magnetic beads with added SA antibody and biotinylated SA, and with added free biotinylated antigen after coating, is as follows:

[0069] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0070] B. Add the SA antibody according to the mass ratio of SA antibody to carboxyl magnetic beads = 10 μg / mg, mix thoroughly and react for 2 hours;

[0071] C. Take SA and add it to the solution after the reaction in step B. The mass ratio of SA antibody to SA = 1 mg / mg. Mix thoroughly and react for 2 hours.

[0072] D. Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen.

[0073] Take the labeled biotinylated antigen and add it to the solution after step C reaction according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and react at room temperature for 28-33 minutes.

[0074] E. Add the magnetic bead blocking agent vitamin H solution to the solution after the reaction in D, mix thoroughly, and allow to react at room temperature for 28-33 minutes by rolling.

[0075] F. Use Tris-HCl analysis buffer to wash away excess biotinylated antigen and magnetic bead blocking agent. Add free biotinylated antigen to the magnetic beads at a ratio of biotinylated antigen: carboxyl magnetic beads = 0.5 μg / mg, and then dilute the magnetic beads to the set concentration.

[0076] Example 5:

[0077] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads containing SA antibody and SA, an analytical buffer, and an enzyme working solution. The carboxyl magnetic beads are labeled with biotin and contain free biotinylated antigen.

[0078] The preparation method of carboxyl magnetic beads with added SA antibody and biotinylated SA, and with the addition of free biotinylated antibody after coating is as follows:

[0079] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0080] B. Add the SA antibody according to the mass ratio of SA antibody to magnetic beads = 10 μg / mg, mix thoroughly, and react for 2 hours.

[0081] C. Take SA and add it to the solution after the reaction in step B. The mass ratio of SA antibody to SA = 1 mg / mg. Mix thoroughly and react for 2 hours.

[0082] D. Take the labeled biotinylated antigen and add it to the solution after the reaction in step C according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0083] E. Add the magnetic bead blocking agent vitamin H solution to the solution after reaction D, mix thoroughly, and allow to react at room temperature for 28-33 minutes.

[0084] F. Use Tris-HCl analysis buffer to wash away excess biotinylated antigen and magnetic bead blocking agent. Add free biotinylated antigen to the magnetic beads at a ratio of biotinylated antigen: carboxyl magnetic beads = 2 μg / mg, and dilute the magnetic beads to the set concentration.

[0085] Comparative Example 1:

[0086] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads, an analytical buffer, and an enzyme working solution, wherein the carboxyl magnetic beads are labeled with biotin.

[0087] The method for preparing the carboxyl magnetic beads is as follows:

[0088] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0089] B. Take the labeled biotinylated antigen and add it to the activated carboxyl magnetic beads in step A according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0090] C. Add the vitamin H solution of magnetic bead sealing agent to the magnetic beads that have reacted in step B, mix thoroughly, and allow to react at room temperature for 28-33 minutes.

[0091] D. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0092] Comparative Example 2:

[0093] A kit for determining antisperm antibody IgG includes pre-made SA magnetic beads, analytical buffer, and enzyme working solution, wherein the SA magnetic beads are labeled with biotin.

[0094] The SA magnetic beads are prepared by:

[0095] A. The concentration of SA magnetic beads is 10 mg / mL. Biotinylated antigen is added according to the ratio of biotinylated antigen: SA magnetic beads = 20 μg / mg. After thorough mixing, the reaction time is 28-33 minutes.

[0096] B. Add the magnetic bead blocking agent vitamin H solution to the solution after reaction A, mix thoroughly, and allow to react at room temperature for 28-33 minutes.

[0097] C. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0098] Comparative Example 3:

[0099] A kit for determining antisperm antibody IgG includes carboxyl magnetic beads with added SA, an analytical buffer, and an enzyme working solution. The carboxyl magnetic beads are labeled with biotin.

[0100] The preparation method of carboxyl magnetic beads with added SA is as follows:

[0101] A. Carboxyl magnetic beads were pre-diluted to 10 mg / mL and activated with sodium N-hydroxythiosuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0102] B. Add SA according to the mass ratio of SA: carboxyl magnetic beads = 10 μg / mg, mix thoroughly and react for 2 hours;

[0103] C. Take the labeled biotinylated antigen and add it to the carboxyl magnetic beads in step B according to the ratio of biotinylated antigen: carboxyl magnetic beads = 20 μg / mg. Mix thoroughly and allow to react at room temperature for 28-33 minutes.

[0104] D. Add the magnetic bead blocking agent vitamin H solution to the solution after reaction C, mix thoroughly, and allow to react at room temperature for 28-33 minutes.

[0105] E. Use Tris-HCl analysis buffer to wash away excess biotin antigen and magnetic bead blocking agent, and dilute the magnetic beads to the set concentration.

[0106] The following experimental results further illustrate the beneficial effects of the present invention. The commercially available reagents used for sample comparison are antisperm antibody detection reagents (enzyme-linked immunosorbent assay) produced by Shandong Kanghua Biomedical Technology Co., Ltd., and the commercially available reagents will be used uniformly in subsequent experimental contents.

[0107] Experiment 1: A comparative experiment was conducted on samples tested by the kit under the same conditions, using carboxyl magnetic beads without SA antibody and SA (Comparative Example 1), finished SA magnetic beads (Comparative Example 2), carboxyl magnetic beads with SA added (Comparative Example 3), and carboxyl magnetic beads with SA antibody and SA added (Example 1).

[0108] Experimental materials: Carboxylated magnetic beads (with and without SA antibody, commercially available SA beads, SA beads, and SA beads with and without SA antibody), ALP-labeled mouse anti-human IgG antibody, analytical buffer, anti-sperm antibody IgG calibrators cal-1 to cal-6 (Cal-1 to Cal-6 concentrations of 0, 10, 20, 50, 100, and 200 RU / mL), sample settings S1-S50, substrate solution, and washing solution. The experiment was performed on a Vovent LA2000 fully automated chemiluminescence immunoassay analyzer.

[0109] Using the methods of Comparative Examples 1, 2, 3, and 1, the obtained magnetic beads were combined with analytical buffer and enzyme working solution to form kits, which were used to measure samples S1-S50 after calibration with ASA-IgG calibrators cal-1 to cal-6, and the sample detection results of different kits were observed.

[0110] The reaction procedure was as follows: First, 50 μL of calibrator, 50 μL of magnetic beads, and 100 μL of analytical buffer were added and incubated together at 37°C for 10 min. Then, 100 μL of enzyme working solution was added and incubated at 37°C for 10 min. After washing with washing buffer three times in a magnetic field, substrate solution was added and incubated at 37°C for 5 min. Finally, the reaction was detected using a fully automated chemiluminescence analyzer. The experimental results are shown in Table 1.

[0111] Table 1 shows the detection results of various samples using different magnetic beads.

[0112]

[0113]

[0114]

[0115] In Table 1, "+" indicates that antisperm antibody IgG is positive, and "-" indicates that antisperm antibody IgG is negative.

[0116] Table 2 (Comparison of positive and negative test results between finished SA magnetic beads and commercially available reagent samples)

[0117]

[0118] Table 3 (Comparison of positive and negative concordance rates between samples without SA antibodies, SA magnetic beads, and commercially available reagents)

[0119]

[0120] Table 4 (Comparison of positive and negative result concordance rates between samples with added SA magnetic beads and commercially available reagents)

[0121]

[0122] Table 5 (Comparison of positive and negative concordance rates between samples with added SA antibodies and SA magnetic beads and commercially available reagents)

[0123]

[0124] Table 1

[0125] As can be seen from Tables 1 to 5 above, under the same conditions, the magnetic beads with added SA antibody in Example 1 showed the best positive and negative concordance rate with the samples measured by commercially available reagents, while the samples without added SA antibody and with SA magnetic beads showed the worst concordance rate.

[0126] Experiment 2: A comparative experiment on the test kit's performance in detecting samples when different proportions of SA antibody and SA were added.

[0127] During the magnetic bead coating process, the SA antibody: carboxyl magnetic beads = 10 μg / mg was fixed, and the ratio of added SA to SA antibody was adjusted to SA antibody: SA = 0.5 mg / mg, 1 mg / mg, and 2 mg / mg. The antisperm antibody IgG samples were measured according to the method in Experiment 1, and the batch-to-batch difference was observed.

[0128] Experimental materials: Carboxylated magnetic beads (SA antibody: SA = 0.5 mg / mg, 1 mg / mg, 2 mg / mg), ALP-labeled mouse anti-human IgG antibody, analysis buffer, anti-sperm antibody IgG calibrators cal-1 to cal-6 (Cal-1 to Cal-6 concentrations of 0, 10, 20, 50, 100, 200 RU / mL), samples S1-S50, substrate solution, washing solution, and the analysis was performed using a Vovent LA2000 fully automated chemiluminescence immunoassay analyzer.

[0129] The experimental method was the same as in Experiment 1, and the experimental results are shown in Table 6:

[0130] Table 6 shows the detection results when different ratios of SA antibody to SA were used during the coating process.

[0131]

[0132]

[0133]

[0134] Table 7 (Comparison of positive and negative concordance rates between SA antibody and SA ratio of 0.5 mg / mg and commercially available reagent samples)

[0135]

[0136] Table 8 (Comparison of positive and negative concordance rates between SA antibody and SA ratio of 1 mg / mg and commercially available reagent samples)

[0137]

[0138] Table 9 (Comparison of positive and negative concordance rates between SA antibody and SA ratio of 2 mg / mg and commercially available reagent samples)

[0139]

[0140] Table 10 shows the detection signal values ​​of the kits under different SA antibody to SA ratios during the coating process.

[0141] Observation of detection signals of samples with different numbers 0.5mg / mg 1mg / mg 2mg / mg S1 15657 23410 24042 S2 21036 25064 27064 S3 30826 37323 38381 S4 139622 163289 159109 S5 141167 213793 209005 S6 223454 251664 272708 S7 311055 371769 356224 S8 281182 414807 404584 S9 310304 423516 401938 S10 388630 526575 545636

[0142] From Tables 6-10 and Figure 1 ( Figure 1 Y1 to Y10 in the table correspond to samples S1 to S10 with different numbers in Table 10. As shown, when both SA and anti-SA are added simultaneously, although the addition ratios differ, the concordance rates with commercially available reagents are generally good. Furthermore, the kit prepared using the same labeling method exhibits the best overall performance when the SA to anti-SA ratio is 1 mg / mg, showing good concordance with third-party reagents, and the detection signal is higher than that at ratios of 0.5 mg / mg and 2.0 mg / mg.

[0143] Experiment 3: Comparison of precision and magnetic bead drop rate between kits with and without added free biotin antigen.

[0144] Experimental materials: Carboxyl magnetic beads containing anti-SA and SA (with added free biological antigen, without added free biotin antigen), ALP-labeled mouse anti-human IgG antibody, anti-sperm antibody IgG calibrators cal-1 to cal-6 (Cal-1 to Cal-6 concentrations of 0, 20, 50, 100, 200, 400 RU / mL), substrate solution, washing solution, and the assay was performed using a Vovent LA2000 fully automated chemiluminescence immunoassay analyzer.

[0145] The methods of Examples 1, 4, and 5 were used. In Example 4, anti-SA and SA carboxyl magnetic beads were added, and the free biotinylated antigen: magnetic beads = 0.5 μg / mg; in Example 5, anti-SA and SA carboxyl magnetic beads were added, and the free biotinylated antigen: magnetic beads = 2 μg / mg (Example 1 did not add free biotinylated antigen). Kits were prepared using the three conditions described in Examples 1, 4, and 5, along with analytical buffer and enzyme working solution. ASA-IgG positive samples were continuously tested 50 times, and the precision and magnetic bead drop rate of different kits were observed. In this example, the concept of magnetic bead drop rate was introduced to verify the performance of the kits, representing the difference in signal detected from the first to the last test of the same sample in a complete 50-test kit. Magnetic bead drop rate = 1 - (mean of the last 10 test signals) / (mean of the first 10 test signals), and the obtained value should range from 0% to 10%.

[0146] Simultaneously, the antisperm antibody IgG samples were tested using the method described in Experiment 1 to observe whether the addition of free biotin antigen affected the sample detection. The test results are shown in Table 11.

[0147] Table 11. Signal values ​​detected by each kit at different numbers of repeated tests.

[0148]

[0149]

[0150]

[0151] Table 12 shows the test results of multiple samples using each kit.

[0152]

[0153]

[0154]

[0155] Table 13 (Comparison of positive and negative concordance rates between samples without added free biotin antigen and commercially available reagents)

[0156]

[0157] Table 14 (Comparison of positive and negative concordance rates between free biotinylated antigen (magnetic beads = 0.5 μg / mg) and commercially available reagent samples)

[0158]

[0159] Table 15 (Comparison of positive and negative concordance rates between free biotinylated antigen: magnetic beads = 2 μg / mg and commercially available reagent samples)

[0160]

[0161]

[0162] Tables 11-15 show that whether or not free biotinylated antigen is added after the magnetic beads are coated has a significant impact on the precision and drop rate of the magnetic beads. The overall magnetic bead drop rate and precision of the kit with added free biotinylated antigen are within ±10%, which is far superior to the group without added antigen. Furthermore, at the addition ratio of free biotinylated antigen: carboxyl magnetic beads = 0.5 μg / mg, the impact on the reagent signal is minimal, and there is no significant difference in the detected samples compared to the group without added antigen.

[0163] Based on the above four experiments, we can conclude that:

[0164] (1) In the embodiments of the present invention, by connecting SA to SA during the coating process of carboxy magnetic beads, the connection length between the antigen and the magnetic beads is increased by forming carboxy magnetic beads-streptavidin antibody-streptavidin-biotinylated antigen, thereby reducing spatial steric hindrance, avoiding interference in the detection of some samples, and reducing the detection rate of abnormal samples.

[0165] (2) In the embodiments of the present invention, when free biotin antigen is further added to the coated magnetic beads, the detection rate of abnormal reagent samples is significantly reduced, and the detection precision and magnetic bead retention rate are effectively improved.

[0166] Anything not mentioned or described in detail in the embodiments of this invention is something that can be obtained using existing technology.

[0167] The above specific embodiments further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are merely specific embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for determining antisperm antibody IgG, characterized in that, The product includes carboxyl magnetic beads coated with sperm membrane antigen, analytical buffer, and enzyme working solution, wherein the carboxyl magnetic beads coated with sperm membrane antigen are labeled with biotin, streptavidin, and streptavidin antibody; In preparing the carboxyl magnetic beads coated with the sperm membrane antigen, the sperm membrane antigen is first biotinylated to obtain biotinylated antigen; then streptavidin antibody is added to the activated carboxyl magnetic beads, and then reacted with streptavidin to form a carboxyl magnetic bead-streptavidin antibody-streptavidin-biotinylated antigen structural system. After obtaining carboxyl magnetic beads coated with sperm membrane antigen, the magnetic beads are further blocked with blocking solution, and then free biotinylated antigen is added.

2. The kit for determining antisperm antibody IgG according to claim 1, characterized in that, The preparation method of carboxyl magnetic beads coated with sperm membrane antigen is as follows: Step 1: Preparation of biotin-labeled sperm membrane antigen: Dilute the sperm membrane antigen and biotin-label the diluted sperm membrane antigen to obtain biotinylated antigen; Step 2: Preparation of carboxyl magnetic beads coated with sperm membrane antigen: Dilute carboxyl magnetic beads, activate carboxyl magnetic beads, mix streptavidin antibody with carboxyl magnetic beads and react to obtain the first solution; Streptavidin was mixed with the first solution and reacted to obtain the second solution; Add the biotinylated antigen from step one to the second solution to obtain carboxyl magnetic beads coated with sperm membrane antigen.

3. The kit for determining antisperm antibody IgG according to claim 2, characterized in that, The method for preparing the carboxyl magnetic beads coated with sperm membrane antigens further includes step three, which is: The carboxyl magnetic beads coated with sperm membrane antigen obtained in step two were blocked with blocking solution, and after washing and dilution with analytical buffer, the free biotinylated antigen obtained in step one was added.

4. A kit for determining antisperm antibody IgG according to claim 2, characterized in that, In step two, the activation of carboxyl magnetic beads is carried out using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide sodium salt.

5. A kit for determining antisperm antibody IgG according to claim 2, characterized in that, The mass ratio of streptavidin antibody to carboxylated magnetic beads was 10 μg / mg.

6. A kit for determining antisperm antibody IgG according to claim 2, characterized in that, The mass ratio of streptavidin antibody to streptavidin is 0.5 mg / mg to 2 mg / mg.

7. A kit for determining antisperm antibody IgG according to claim 3, characterized in that, In step three, the amount of free biotinylated antigen added is based on the ratio of free biotinylated antigen to carboxyl magnetic beads = 0.5 μg / mg.

8. A kit for determining antisperm antibody IgG according to claim 1, characterized in that, The preparation method of carboxyl magnetic beads coated with sperm membrane antigen is as follows: (1) Dilute the sperm membrane antigen to 1 mg / mL, and biotinylate the diluted sperm membrane antigen to obtain biotinylated antigen; (2) Dilute the carboxyl magnetic beads to 10 mg / mL, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide sodium salt to activate the carboxyl magnetic beads for 15 minutes. After activation, mix streptavidin antibody with the carboxyl magnetic beads and react for 2 hours to obtain the first solution. The mass ratio of streptavidin antibody to carboxyl magnetic beads is 10 μg / mg. (3) Mix streptavidin with the first solution and react for 2 hours to obtain the second solution; the mass ratio of streptavidin antibody to streptavidin is 1 mg / mg; (4) Add the biotinylated antigen from step (1) to the second solution; (5) The carboxyl magnetic beads coated with sperm membrane antigen were blocked with blocking solution, washed three times with analytical buffer, diluted, and then the free biotinylated antigen obtained in step (1) was added; free biotinylated antigen: carboxyl magnetic beads = 0.5 μg / mg.

9. A kit for determining antisperm antibody IgG according to claim 7, characterized in that, The analytical buffer was Tris-HCl buffer, and the blocking solution was vitamin H solution.

10. A kit for determining antisperm antibody IgG according to claim 1, characterized in that, The enzyme working solution is an alkaline phosphatase-labeled mouse anti-human IgG antibody.

11. The application of the kit for determining antisperm antibody IgG according to any one of claims 1 to 9, characterized in that, The kit is used for the detection of antisperm antibody IgG.

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