Anti-THSD7A antibody detection kit and application thereof

Abstract

The invention discloses an anti-THSD7A antibody detection kit and application thereof, the anti-THSD7A antibody detection kit comprises a magnetic bead working solution, an analysis buffer solution and an enzyme working solution; the magnetic bead working solution is formed by mixing a Tris-HCl buffer solution added with 1-3% of sorbitol and toluenesulfonyl magnetic beads coated with THSD7A antigen, and the enzyme working solution is added with a blocking agent ALPIW2 with the concentration of 12.5-50 [mu] g / mL. On one hand, the sorbitol is added into the magnetic bead working solution, on the other hand, the blocking agent ALPIW2 is added into the enzyme buffer solution, through the combined action of the sorbitol and the blocking agent ALPIW2, the overall stability and precision of the reagent are improved, non-specific interference is reduced, and the clinical coincidence rate is greatly improved.

Description

Technical Field

[0001] This invention relates to the field of immunoassay reagent technology, specifically to an anti-THSD7A antibody detection kit and its application. Background Technology

[0002] Membranous nephropathy (MN) is a common glomerular disease characterized by thickening of the glomerular basement membrane and deposition of immune complexes. It is one of the leading causes of nephrotic syndrome in adults. Its main feature is the deposition of immune complexes in the subepithelial region of the glomerular basement membrane, leading to basement membrane thickening and damage to the filtration barrier, resulting in proteinuria. MN can be divided into two main categories based on its etiology: idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN).

[0003] The main autoantigens in idiopathic membranous nephropathy (IMN) include PLA2R and THSD7A. THSD7A is an important autoantigen in IMN, and anti-THSD7A antibodies can be detected in the serum of approximately 5%-10% of IMN patients, especially in patients who are negative for anti-PLA2R antibodies. Therefore, detecting anti-THSD7A antibodies is very important in IMN.

[0004] Currently, existing anti-THSD7A antibody assay kits (chemiluminescence method) are mainly affected by two factors when testing clinical samples: first, the reagents are unstable and have poor precision; second, the binding of the sample and the reagent is subject to non-specific interference, leading to false positive results and affecting clinical use.

[0005] In view of the above, this application is hereby submitted. Summary of the Invention

[0006] To address the aforementioned issues, this application provides an anti-THSD7A antibody detection kit and its application. On one hand, it adds stabilizers and solubilizers to the magnetic bead buffer; on the other hand, it adds a dedicated blocking agent to the enzyme buffer. Through the combined action of the stabilizers, solubilizers, and blocking agents, the overall stability and precision of the reagent are improved, non-specific interference is reduced, and the clinical concordance rate is greatly improved.

[0007] The objective of this invention is achieved through the following technical solution: an anti-THSD7A antibody detection kit, comprising a magnetic bead working solution, an analytical buffer, and an enzyme working solution; the magnetic bead working solution is prepared by mixing and adjusting the volume of Tris-HCl buffer containing 1-3% sorbitol with toluenesulfonyl magnetic beads coated with THSD7A antigen, and the enzyme working solution contains 12.5-50 μg / mL of the blocking agent ALPIW2.

[0008] The amount of sorbitol added to the magnetic bead buffer is 2%, and the amount of ALPIW2 inhibitor added to the enzyme working solution is 25 μg / mL.

[0009] The Tris-HCl buffer solution contains Triton, Tween, trehalose, SDS, and bovine serum albumin.

[0010] The analytical buffer solution is a Tris system buffer solution with a pH of 7.4.

[0011] The enzyme working solution contains alkaline phosphatase-labeled mouse anti-human IgG antibody.

[0012] The anti-THSD7A antibody detection kit also includes calibrators and quality control products, both of which contain anti-THSD7A antibodies.

[0013] The preparation of the magnetic bead working solution includes the following steps:

[0014] (1) Prepare Tris-HCl buffer and add 1-3% sorbitol to Tris-HCl buffer to obtain magnetic bead buffer;

[0015] (2) Take the toluenesulfonyl magnetic bead solution and perform water bath sonication in a borate buffer solution at pH 9.5;

[0016] (3) Add THSD7A antigen to the solution in step (2) and mix well. Then add potassium phosphate aqueous solution with pH 9.5 and mix well. Incubate overnight at 37°C.

[0017] (4) Take out the toluenesulfonyl magnetic beads after overnight reaction in step (3), add buffer containing Tris-HCl to wash them, and place them on a magnetic rack until the magnetic bead solution becomes clear. Remove the supernatant, add buffer containing Tris-HCl again, and place them in a 37°C environment to roll overnight.

[0018] (5) Place the magnetic bead solution from step (4) on a magnetic rack until the magnetic bead solution becomes clear, remove the supernatant, and add the magnetic bead buffer solution prepared in step (1) to make up the volume to obtain the magnetic bead working solution.

[0019] The preparation of the enzyme working solution includes the following steps:

[0020] A: The activated mouse anti-human IgG antibody and the activated alkaline phosphatase were linked at a molar ratio of 1:1 to 1:3 to prepare an enzyme conjugate mother solution.

[0021] B: Dilute the enzyme conjugate stock solution with phosphate-containing buffer;

[0022] C: Add 12.5–50 μg / mL of the inhibitor ALPIW2 to the solution in step B to prepare the enzyme working solution.

[0023] The preparation of the analytical buffer includes the following steps:

[0024] It is prepared by purifying with a phosphate-containing buffer solution and then filtering with a filter membrane.

[0025] The anti-THSD7A antibody detection kit of the present invention can be used for the detection of anti-THSD7A antibodies.

[0026] Compared with the prior art, this application has the following beneficial effects: On the one hand, the present invention adds sorbitol to the magnetic bead working solution, and on the other hand, it adds the blocking agent ALPIW2 to the enzyme buffer. Through the combined action of sorbitol and the blocking agent ALPIW2, the overall stability and precision of the reagent are improved, non-specific interference is reduced, and the clinical compliance rate is greatly improved. Detailed Implementation

[0027] Example

[0028] This embodiment discloses an anti-THSD7A antibody detection kit, which includes magnetic bead working solution, analytical buffer, enzyme working solution, calibrator and quality control.

[0029] The preparation method of the anti-THSD7A antibody detection kit in this embodiment is as follows:

[0030] The specific steps for preparing the magnetic bead working solution are as follows:

[0031] (1) Prepare four Tris-HCl buffer solutions, each containing 0.1% Triton, 0.1% Tween, 1% trehalose, 1% SDS, and 1% bovine serum albumin. Then, add 1% sorbitol, 2% sorbitol, and 3% sorbitol to three of the Tris-HCl buffer solutions respectively. The last Tris-HCl buffer solution should not contain sorbitol. Label these solutions as a1, a2, a3, and a, respectively. Specifically, adding 1% sorbitol to the Tris-HCl buffer solution means adding 1g of sorbitol per 100ml of Tris-HCl buffer solution.

[0032] (2) Take the toluenesulfonyl magnetic bead solution and perform water bath sonication in a borate buffer solution at pH 9.5, repeating twice.

[0033] (3) Add THSD7A antigen to the solution in step (2) at a ratio of 1 mg magnetic beads to 5 μg antigen. After mixing at room temperature for 5 minutes, add potassium phosphate aqueous solution with pH 9.5 and mix well. Then place it on a rolling mixer and incubate overnight at 37°C.

[0034] (4) Take out the toluenesulfonyl magnetic beads after overnight reaction in step (3), add buffer containing Tris-HCl and wash on a rolling mixer for 10 minutes, and place on a magnetic rack until the magnetic bead solution becomes clear. Remove the supernatant and repeat twice; then add buffer containing Tris-HCl and place on a rolling mixer at 37°C overnight.

[0035] (5) Place the magnetic bead solution from step (4) on a magnetic rack until the magnetic bead solution becomes clear, remove the supernatant, and obtain toluenesulfonyl magnetic beads coated with THSD7A antigen; finally, divide the toluenesulfonyl magnetic beads coated with THSD7A antigen into 4 equal parts, and add the a1 magnetic bead buffer, a2 magnetic bead buffer, a3 magnetic bead buffer and a magnetic bead buffer prepared in step (1) to the 4 parts of toluenesulfonyl magnetic beads coated with THSD7A antigen to adjust the volume to the working concentration (magnetic bead concentration is 0.4 mg / mL), and obtain 4 magnetic bead working solutions, which are labeled as A1 magnetic bead working solution, A2 magnetic bead working solution, A3 magnetic bead working solution and A magnetic bead working solution, respectively, and store them in a 2℃ refrigerator for later use.

[0036] The specific steps for preparing the enzyme working solution are as follows:

[0037] A: The activated mouse anti-human IgG antibody and the activated alkaline phosphatase were linked at a molar ratio of 1:3 to prepare an enzyme conjugate mother solution.

[0038] B: Dilute the enzyme conjugate stock solution to 1:5000 with a phosphate-containing buffer solution; the phosphate buffer solution contains 5% mannitol and 2% casein.

[0039] C: Divide the solution from step B into 4 equal parts, and add 12.5 μg / mL ALPIW2, 25 μg / mL ALPIW2, and 50 μg / mL ALPIW2 to 3 of the solutions respectively. Do not add ALPIW2 to the last solution to obtain 4 enzyme working solutions, which are labeled as b1 enzyme working solution, b2 enzyme working solution, b3 enzyme working solution and b enzyme working solution respectively.

[0040] The specific method for preparing the analytical buffer is as follows:

[0041] The solution was prepared by adding 2% mannitol and 1% BSA to a phosphate-containing buffer solution and filtering it through a 0.22 μm filter membrane.

[0042] The specific methods for preparing calibrators and quality control samples are as follows:

[0043] Anti-THSD7A antibody was dissolved in phosphate buffer containing 1% BSA to prepare calibrators with concentrations of 0 ng / mL, 20 ng / mL, 50 ng / mL, 200 ng / mL, 500 ng / mL, and 1500 ng / mL, labeled as S0, S1, S2, S3, S4, and S5, respectively; and quality control samples with concentrations of 50 ng / mL and 500 ng / mL, labeled as QC1 and QC2, respectively.

[0044] Performance verification

[0045] Under the same conditions, the above magnetic bead working solution was combined with enzyme working solution and analytical buffer to form a kit. After calibration with its calibrators, quality control samples QC1 and QC2 were tested. At the same time, the reagents were placed in a 37℃ oven to observe their stability. The experimental results are as follows:

[0046] Table 1 Precision Verification

[0047]

[0048]

[0049] Table 2 Stability Verification

[0050]

[0051]

[0052] The experimental data above show that the precision of QC1 and QC2 tests of the magnetic bead working solutions in groups A1-A3 is better after adding sorbitol compared to the precision of the working solutions in group A without sorbitol. Specifically, the precision of QC1 and QC2 tests is best when 2% sorbitol is added to the working solutions in group A2. However, in group A3, the precision does not improve further with increasing sorbitol addition, and even slightly decreases. Furthermore, observations of reagent tests at 4℃ and 37℃ show that the stability retention rate of the working solutions in groups A1-A3 is higher after adding sorbitol compared to the working solutions in group A without sorbitol. The stability retention rate of groups A2 and A3 shows little change after adding 2% and 3% sorbitol, respectively. Therefore, adding 2% sorbitol to the working solutions in group A2 is the optimal choice.

[0053] The optimal A2 group magnetic bead working solution was combined with enzyme working solutions b1, b2, b3, and b to create four kits, labeled B1, B2, B3, and B, respectively. Forty samples were tested after calibration with calibrators, and the results were compared with the Euromon THSD7A antibody ELISA kit. The experimental data are as follows:

[0054] Table 3 Comparison of Clinical Samples

[0055]

[0056]

[0057] The results from the above samples show that adding 25 μg / mL and 50 μg / mL of ALPIW2 inhibitor to the enzyme working solution yields the same effect, meaning the B2 kit and the B3 kit are equally effective. This eliminates interference from samples 1, 2, 23, and 28, consistent with the results obtained from Euromon assays, significantly improving reagent specificity. Considering economics and cost, adding 25 μg / mL ALPIW2 to the b2 enzyme working solution meets the reagent performance requirements. Therefore, the B2 kit, consisting of A2 magnetic bead working solution, b2 enzyme working solution, and analysis buffer, is the optimal choice.

[0058] The anti-THSD7A antibody detection kit provided by this invention incorporates sorbitol, which significantly improves the dispersibility and compatibility of reagent components, enhancing the overall precision and stability of the kit. The use of the blocking agent ALPIW2 significantly reduces false positives, preventing an increase in false results. This anti-THSD7A antibody detection kit achieves high precision and specificity while greatly improving overall reagent stability. Furthermore, this anti-THSD7A antibody detection kit has a wide range of applications, meeting the current market demand for efficient and convenient detection kits, covering existing clinical testing needs, and providing strong support for medical diagnostics, scientific research, and other fields.

[0059] It should be noted that all features disclosed in this specification, or all steps in all methods or processes disclosed, may be combined in any way, except for mutually exclusive features and / or steps.

[0060] Furthermore, the specific embodiments described above are exemplary. Those skilled in the art can devise various solutions inspired by the disclosure of this invention, and these solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification is illustrative and does not constitute a limitation on the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A detection kit for anti-THSD7A antibodies, characterized in that, It includes magnetic bead working solution, analytical buffer, and enzyme working solution; the magnetic bead working solution is prepared by mixing and adjusting the volume of Tris-HCl buffer containing 1-3% sorbitol with toluenesulfonyl magnetic beads coated with THSD7A antigen, and the enzyme working solution contains 12.5-50 μg / mL of the blocking agent ALPIW2.

2. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The amount of sorbitol added to the magnetic bead buffer is 2%, and the amount of ALPIW2 inhibitor added to the enzyme working solution is 25 μg / mL.

3. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The Tris-HCl buffer solution contains Triton, Tween, trehalose, SDS, and bovine serum albumin.

4. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The analytical buffer solution is a Tris system buffer solution with a pH of 7.

4.

5. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The enzyme working solution contains alkaline phosphatase-labeled mouse anti-human IgG antibody.

6. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, It also includes calibrators and quality control products, both of which contain anti-THSD7A antibodies.

7. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The preparation of the magnetic bead working solution includes the following steps: (1) Prepare Tris-HCl buffer and add 1-3% sorbitol to Tris-HCl buffer to obtain magnetic bead buffer; (2) Take the toluenesulfonyl magnetic bead solution and perform water bath sonication in a borate buffer solution at pH 9.5; (3) Add THSD7A antigen to the solution in step (2) and mix well. Then add potassium phosphate aqueous solution with pH 9.5 and mix well. Incubate overnight at 37°C. (4) Take out the toluenesulfonyl magnetic beads after overnight reaction in step (3), add buffer containing Tris-HCl to wash them, and place them on a magnetic rack until the magnetic bead solution becomes clear. Remove the supernatant, add buffer containing Tris-HCl again, and place them in a 37°C environment to roll overnight. (5) Place the magnetic bead solution from step (4) on a magnetic rack until the magnetic bead solution becomes clear, remove the supernatant, and add the magnetic bead buffer solution prepared in step (1) to make up the volume to obtain the magnetic bead working solution.

8. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The preparation of the enzyme working solution includes the following steps: A: The activated mouse anti-human IgG antibody and the activated alkaline phosphatase were linked at a molar ratio of 1:1 to 1:3 to prepare an enzyme conjugate mother solution. B: Dilute the enzyme conjugate stock solution with phosphate-containing buffer; C: Add 12.5–50 μg / mL of the inhibitor ALPIW2 to the solution in step B to prepare the enzyme working solution.

9. The anti-THSD7A antibody detection kit according to claim 1, characterized in that, The preparation of the analytical buffer includes the following steps: It is prepared by purifying with a phosphate-containing buffer solution and then filtering with a filter membrane.

10. The application of an anti-THSD7A antibody detection kit as described in any one of claims 1-9, characterized in that, Used for the detection of anti-THSD7A antibodies.

Images