Detection kit for detecting anti-phospholipase A2 receptor antibody IgG as well as preparation method and application thereof

Abstract

The invention discloses a detection kit for detecting an antiphospholipase A2 receptor antibody I gG as well as a preparation method and application of the detection kit. The detection kit comprises a reagent component 1, a reagent component 2 and a reagent component 3. The reagent component 1 is a magnetic particle buffer solution which is coated on toluenesulfonyl magnetic particles and is connected with a phospholipase A2 receptor antigen; the reagent component 2 is a biotin labeled mouse anti-human I gG specific monoclonal antibody; and the reagent component 3 is a marker of streptavidin and alkaline phosphatase. According to the present invention, the method has advantages of high sensitivity, high specificity, good stability, low interference rate and appropriate cost on the PLA2R I gG detection, can reduce the PLA2R I gG detection difficulty, can increase the accuracy of the detection result, and can provide the great effect in the fields of idiopathic membranous nephropathy auxiliary diagnosis, treatment effect evaluation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of immunoassay reagents, and in particular to a detection kit for detecting anti-phospholipase A2 receptor antibody IgG, and a preparation method and application thereof. Background Art

[0002] Membranous nephropathy (MN) is an autoimmune glomerular disease characterized by the formation of in situ immune complexes, which are deposited under the epithelium of glomerular podocytes and destroy the glomerular structure. At present, scientists have identified a new term with the progress of research: neutral endopeptidase, namely M-type phospholipase A2 receptor (PLA2R). PLA2R and type I thrombospondin 7A (THSD7A) are deposited in the renal glomeruli as target antigens and form a new immune antigen-antibody complex with anti-PLA2R antibodies in the renal circulation, which leads to the occurrence of idiopathic membranous nephropathy (70% to 75%). PLA2R is a type I transmembrane glycoprotein and a member of the mannitol receptor (MR) family. The main feature of the immune response of membranous nephropathy is that Th2 cells are activated to produce certain cytokines, especially IL-4, IL-10, IL-13, and IL-14, which activate B lymphocytes to synthesize IgG4, which is the main immunoglobulin of membranous nephropathy. IgG4 will bind to the conformational epitope of PLA2R on the surface of podocytes to form intraepithelial immune complexes. The formation of immune complexes and the deposition of electron-dense materials destroy the functional integrity of the glomerular filtration barrier. However, IgG4 does not activate complement. Mannose-binding lectin (MBL) can be recognized by the glomeruli of patients with membranous nephropathy, but C1q does not recognize it. Therefore, complement activation through the MBL pathway is the main pathway for activating the immune system. The lack of galactose side chains in the IgG4 molecule of anti-PLA2R antibodies also shows that immune complexes activate the complement system mainly through the MBL pathway. MBL will activate two serine proteases (MASP1 / 2, MBL-related serine proteases), which are expressed similarly to Cr and Cs. Cs is hydrolyzed from (4A and C4B) and C2 (CA and CB). C4b and C2a combine to form the C3 convertase, which, like the classical pathway, ultimately forms the membrane attack complex (MAC). In membranous nephropathy, MAC activates podocytes, leading to a series of adverse reactions. Excessive ROS (reactive oxygen species) ultimately lead to changes in the glomerular basement membrane (GBM) and the synthesis of certain metalloproteinases through the upregulation of NADPH oxidase, thereby destroying the GBM.

[0003] A study reported that by comparing the titers of anti-PLA2R antibodies in the membranous nephropathy group, the disease control group, and the healthy control group, 17 cases of anti-PLA2R antibodies were positive in the membranous nephropathy group (positive rate 50%), and 17 cases were negative. Both the disease control group and the healthy control group were negative, and the specificity and positive predictive value were 100%. It was concluded that the difference between the membranous nephropathy group and the other two groups was statistically significant. This shows that anti-PLA2R antibodies have a high positive rate and specificity in the diagnosis of membranous nephropathy. When patients cannot undergo renal biopsy, they can be used as a necessary specific serological quantitative test, which is helpful for the diagnosis of the disease.

[0004] The sensitivity of PLA2R in different regions shows great differences, among which the population in my country is 71% to 89%. Serum anti-PLA2R antibodies in patients with non-membranous nephropathy are almost all negative. The degree of reduction in anti-PLA2R antibody titer is significantly positively correlated with the degree of proteinuria reduction, and is significantly negatively correlated with the increase in serum albumin. There is also a certain time difference between the changes in anti-PLA2R antibody titer and urine protein. The decrease in antibody titer levels often precedes the decrease in urine protein and the relief of other test indicators. It is clinically used for auxiliary diagnosis of idiopathic membranous nephropathy.

[0005] At present, the existing methods and technologies for detecting PLA2R IgG on the market include enzyme-linked immunosorbent assay, chemiluminescence assay, time-resolved fluorescence immunoassay, etc. However, these still face some challenges in practical applications, such as poor sensitivity, low specificity, insufficient stability, and high price. Therefore, the development of a PLA2R IgG assay kit with high sensitivity, high specificity, good stability, low interference rate and appropriate cost has important practical significance and application prospects.

[0006] In view of this, this application is hereby filed. Summary of the invention

[0007] In order to solve the problems of poor sensitivity, low specificity, insufficient stability, high price, inconvenient operation, etc. of the existing PLA2R IgG assay kit. The present invention provides a detection kit for detecting anti-phospholipase A2 receptor antibody IgG, and its preparation method and application, which utilizes the high affinity of the "biotin-avidin" reaction system and the low non-specific binding characteristics with the tissue cell matrix to improve the sensitivity and specificity of the detection system, and utilizes long-arm biotin to link mouse anti-human IgG antibody to reduce steric hindrance, and streptavidin to link alkaline phosphatase to amplify the signal, which can significantly improve the sensitivity, specificity and stability of PLA2R IgG detection, and is simple to operate, can reduce the detection cost of patients, and can play a greater role in the fields of auxiliary diagnosis and treatment effect monitoring of membranous nephropathy.

[0008] The purpose of the present invention is achieved through the following technical scheme: a detection kit for detecting anti-phospholipase A2 receptor antibody IgG, comprising reagent component 1, reagent component 2, and reagent component 3.

[0009] Wherein, the reagent component 1 is a magnetic particle buffer coated on toluenesulfonyl magnetic particles and connected with phospholipase A2 receptor antigen. Specifically, the working concentration of phospholipase A2 receptor antigen in the reagent component 1 is 1-4 μg / mL, and the working concentration of toluenesulfonyl magnetic particles is 0.1-0.4 mg / mL; preferably, the working concentration of phospholipase A2 receptor antigen in the reagent component 1 is 2 μg / mL, and the working concentration of magnetic particles is 0.4 mg / mL.

[0010] In addition, in a specific embodiment, the buffer of reagent component 1 includes: Tris with a working concentration of 12.11 g / L, NaCl with a working concentration of 27.0 g / L, D-mannitol with a working concentration of 30.0 g / L, BSA with a working concentration of 10.0 g / L, sodium dodecyl sulfate with a working concentration of 2.0 g / L, Triton X-100 with a working concentration of 5.0 mL / L, Proclin300 with a working concentration of 1.0 mL / L, glycerol with a working concentration of 10.0 mL / L and 4-aminoantipyrine with a working concentration of 0.04 g / L, added into purified water; the pH value of the buffer of reagent component 1 is 7.5±0.1.

[0011] Furthermore, the reagent component 2 is a biotin-labeled mouse anti-human IgG specific monoclonal antibody. The working concentration of the mouse anti-human IgG specific monoclonal antibody in the reagent component 2 is 0.05-0.2 μg / mL; preferably, the working concentration of the mouse anti-human IgG specific monoclonal antibody in the reagent component 2 is set to 0.1 μg / mL.

[0012] During implementation, the mouse anti-human IgG specific monoclonal antibody in the reagent component 2 is linked to the biotin ester at a molar ratio of 1:10 to 1:20 to obtain a biotinylated IgG secondary antibody. Preferably, the mouse anti-human IgG specific monoclonal antibody is labeled with the biotin ester at a molar ratio of 1:15, and the biotin ester is a long-chain biotin ester.

[0013] In addition, in a specific embodiment, the buffer solution of reagent component 2 includes: Tris with a working concentration of 12.11 g / L, NaCl with a working concentration of 27.0 g / L, newborn calf serum with a working concentration of 50.0 mL / L, and Proclin300 with a working concentration of 5.0 mL / L, added to purified water; the pH value of the buffer solution of reagent component 2 is 7.5±0.1.

[0014] Furthermore, the reagent component 3 is a marker of streptavidin and alkaline phosphatase. The working concentration of the marker of streptavidin and alkaline phosphatase in the reagent component 3 is 0.1-0.4 μg / mL. Preferably, the working concentration of the marker of streptavidin and alkaline phosphatase in the reagent component 3 is 0.2 μg / mL.

[0015] In addition, the buffer of reagent component 3 includes: Tris with a working concentration of 12.11 g / L, NaCl with a working concentration of 27.0 g / L, D-trehalose with a working concentration of 10.0 g / L, casein with a working concentration of 5.0 g / L, BSA with a working concentration of 20.0 g / L, Proclin300 with a working concentration of 3.0 mL / L, Triton X-100 with a working concentration of 1.0 mL / L, 4-aminoantipyrine with a working concentration of 0.04 g / L, glycerol with a working concentration of 10.0 mL / L and fruit green pigment with a working concentration of 0.05 g / L, which are added to purified water; the pH value of the buffer of reagent component 3 is 7.5±0.1.

[0016] As another implementation, the detection kit for detecting anti-phospholipase A2 receptor antibody IgG also includes a reagent calibrator and a reagent quality control. Specifically, the reagent calibrator includes anti-phospholipase A2 receptor antibody IgG solutions at concentrations of 0RU / mL, 20RU / mL, 50RU / mL, 200RU / mL, 500RU / mL and 1500RU / mL. The reagent quality control includes anti-phospholipase A2 receptor antibody IgG solutions at concentrations of 50RU / mL and 500RU / mL.

[0017] In addition, the buffer solutions of the reagent calibrator and reagent quality control products include: Tris with a working concentration of 12.11 g / L, NaCl with a working concentration of 9.0 g / L, D-mannitol with a working concentration of 10.0 g / L, BSA with a working concentration of 30 g / L, Triton X-100 with a working concentration of 0.5 mL / L, Proclin 300 with a working concentration of 2.0 mL / L, and propylene glycol with a working concentration of 10 mL / L, which are added to purified water; the pH values ​​of the buffer solutions of the reagent calibrator and reagent quality control products are both 7.2±0.1.

[0018] The present invention also provides a method for preparing the above-mentioned detection kit for detecting anti-phospholipase A2 receptor antibody IgG, wherein the preparation process of the reagent component 1 comprises the following steps:

[0019] (1) Dilute the phospholipase A2 receptor antigen with PBS, dialyze it against PBS, and recover the antigen after dialysis.

[0020] (2) Ultrasonic cleaning and activation of the tosyl magnetic particles.

[0021] Specifically, the tosyl magnetic particles are taken and cleaned by water bath ultrasonic cleaning with a borate buffer having a pH value of 9.5, and then the borate buffer having a pH value of 9.5 is added to the tosyl magnetic particles again.

[0022] (3) Magnetic particle coating: Take the dialyzed phospholipase A2 receptor antigen and the activated tosyl magnetic particles for linking reaction. The specific method is as follows:

[0023] (3.1) Add phospholipase A2 receptor antigen to the tosyl magnetic particle solution and mix by rolling at room temperature for 5 minutes.

[0024] (3.2) Add potassium phosphate solution of pH 9.5 to the solution treated in step (3.1), vortex quickly to mix, and incubate on a roller mixer at 37°C for 4 hours.

[0025] (4) The coated magnetic particles are washed and sealed, and then redissolved to obtain reagent component 1. The specific method is as follows:

[0026] (4.1) After the magnetic particles that have been incubated in step (3) are taken out, Tris-HCl buffer at pH 7.4 is added, and the mixture is rolled on a roller mixer for 10 minutes. The mixture is placed on a magnetic rack until the magnetic particle solution becomes clear, and the supernatant is removed. This is repeated twice. Tris-HCl buffer at pH 7.4 is added again, and the mixture is placed on a roller mixer and rolled and incubated at 37° C. for 4 hours.

[0027] (4.2) Take out the magnetic particle solution treated in step (4.1) and place it on a magnetic rack until the magnetic particle solution becomes clear, remove the supernatant, make up the volume with the buffer of reagent component 1, and store it in an environment of 2-8°C.

[0028] The preparation process of the reagent component 2 comprises the following steps:

[0029] A: Mouse anti-human IgG specific monoclonal antibody was first diluted with 0.02 MOL / L PBS, dialyzed in PBS for 15 hours, and after changing the solution once, the dialyzation was continued for 24 hours. After the dialysis was completed, the antibody was recovered into a centrifuge tube for later use; the pH of PBS was 7.4.

[0030] B: Biotin-labeled antibody: Take 6.7 mg of long-chain biotin ester in a centrifuge tube, add 1 mL of dimethyl sulfoxide to dissolve, and prepare a long-chain biotin ester solution with a concentration of 6.7 mg / mL; add the long-chain biotin ester to the antibody recovered in step A at a ratio of 10 μL of long-chain biotin ester to 1 mg of antibody, react for 30 minutes, and then add 1 MOL / L of tris(hydroxymethyl)aminomethane buffer to terminate the reaction.

[0031] C: Dialyze the cell for 9 hours in 0.02 MOL / L PBS. After changing the medium once, dialysis was continued for 15 hours. The pH of PBS was 7.4.

[0032] D: After dialysis, the biotin-labeled antibody is recovered into a centrifuge tube for later use.

[0033] E: Prepare the biotinylated antibody to 0.1 μg / mL using the buffer of reagent component 2 and store at 2-8°C.

[0034] The preparation process of the reagent component 3 comprises the following steps:

[0035] The markers of streptavidin and alkaline phosphatase were diluted with the buffer of reagent component 3 to a concentration of 0.2 μg / mL to obtain reagent component 3.

[0036] The detection kit of the present invention can be used to detect anti-phospholipase A2 receptor antibody IgG.

[0037] The operation process and reaction principle of the detection kit for detecting anti-phospholipase A2 receptor antibody IgG of the present invention are as follows:

[0038] (1) Add 20 μL of sample, 50 μL of reagent component 1 and 50 μL of reagent component 2, mix and incubate at 37°C for 10 minutes, the PLA2R antigen in reagent component 1 reacts with the PLA2R IgG molecules in the sample to be tested and the mouse anti-human IgG in reagent component 2 to form an immune complex; after washing, add 50 μL of reagent component 3, mix and incubate at 37°C for 10 minutes, the streptavidin in reagent component 3 reacts with the biotin labeled on the mouse anti-human IgG, and finally forms a solid phase-PLA2R antigen-mouse anti-human PLA2R IgG antibody-biotin-streptavidin-alkaline phosphatase immune complex, and the immune complex is placed in a magnetic field for washing. The washing solution contains tris(hydroxymethylaminomethane), Tween-20, Proclin300, and sodium azide.

[0039] (2) Add a substrate solution containing AMPPD to the immune complex, and alkaline phosphatase catalyzes the luminescence of the substrate AMPPD, and the luminescence intensity is detected. The concentration of PLA2R IgG is positively correlated with the luminescence intensity.

[0040] (3) A concentration-luminescence intensity four-parameter fitting curve is drawn based on the calibrator of known concentration, and the luminescence value obtained in step (2) is substituted into the drawn fitting curve to obtain the concentration value of PLA2R IgG in the sample to be tested.

[0041] The detection kit for detecting anti-phospholipase A2 receptor antibody IgG of the present invention has a correlation of more than 95% between sample detection and domestically marketed imported products, and meets the following performance requirements:

[0042] A. Blank limit: The blank limit is not higher than 0.50RU / mL.

[0043] B. Linearity: In the range of 2.00RU / mL to 1500.00RU / mL, the linear correlation coefficient r is not less than 0.9900.

[0044] C. Accuracy: Relative deviation is within ±10.0%.

[0045] D. Precision: The intra-analytical coefficient of variation (CV) is no more than 8.0%.

[0046] E. Stability: Accelerated stability at 37°C for 9 days is ≤10%, and long-term stability for 1-16 months is ≤10%.

[0047] Compared with the prior art, this application has the following beneficial effects:

[0048] (1) The detection kit of the present invention utilizes the high affinity of the "biotin-avidin" reaction system and the low non-specific binding characteristics with tissue cell matrix to improve the sensitivity and specificity of the detection system.

[0049] (2) The detection kit of the present invention achieves a high-sensitivity and high-specificity detection effect by optimizing the reagent formula, while requiring a small amount of sample and having a short reaction time.

[0050] (3) The detection kit of the present invention has the advantages of high sensitivity, high specificity, good stability, low interference rate and reasonable cost for the detection of PLA2R IgG, which can reduce the difficulty of detecting PLA2R IgG and increase the accuracy of the test results, and play a great role in the auxiliary diagnosis of idiopathic membranous nephropathy, treatment effect evaluation and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The exemplary embodiments of the present application and their descriptions are used to explain the present application and do not constitute a limitation of the present application. In each figure, the same reference numeral represents the same component.

[0052] Figure 1 This is a comparison chart of the measured values ​​of the PLA2R IgG kit of the present invention and the samples of imported products that have been marketed in China. DETAILED DESCRIPTION

[0053] Example 1

[0054] This embodiment discloses a detection kit for detecting anti-phospholipase A2 receptor antibody IgG, which includes reagent component 1, reagent component 2, reagent component 3, reagent calibrator and reagent quality control product.

[0055] The preparation method of the detection kit for detecting anti-phospholipase A2 receptor antibody IgG is as follows:

[0056] (1) Prepare the buffer solution of reagent component 1, the specific steps are as follows:

[0057] (1.1) Prepare instruments for weighing and measuring reagents, and adjust the weighing balance to zero.

[0058] (1.2) Add 800 mL of purified water to a beaker, weigh 12.11 g of Tris and 27.0 g of NaCl, and stir thoroughly to dissolve.

[0059] (1.3) Adjust the pH value of the solution to within the range of 7.5±0.1.

[0060] (1.4) Weigh and add 30 g D-mannitol, 10 g BSA, 2.0 g sodium dodecanedionyl sulfate, and 0.04 g 4-aminoantipyrine in sequence, and stir until fully dissolved.

[0061] (1.5) Add 5.0 mL of Triton X-100, 1.0 mL of Proclin 300, and 10.0 mL of glycerol in sequence and stir until fully dissolved.

[0062] (1.6) The dissolved solution is diluted to 1 L with purified water to prepare a buffer solution of reagent component 1 with a pH of 7.5.

[0063] (2) Prepare reagent component 1, the specific steps are as follows:

[0064] (2.1) Tosyl magnetic particles are taken and ultrasonically cleaned in a water bath with a borate buffer having a pH of 9.5 for 5 minutes. After the water bath treatment, the borate buffer is again added to the tosyl magnetic particles to replace the buffer component of the tosyl magnetic particles with the borate buffer having a pH of 9.5.

[0065] (2.2) Add PLA2R antigen to the treated tosyl magnetic microparticle solution at a ratio of 1 mg magnetic beads to 5 μg antigen, and mix by rolling at room temperature for 5 minutes.

[0066] (2.3) Add potassium phosphate solution of pH 9.5 to the magnetic particle solution prepared in step (2.2), mix quickly and thoroughly, and roll and mix at 37°C for 4 hours.

[0067] (2.4) After the reaction is completed, the magnetic particle solution is taken out and added with pH 7.4 Tris-HCl buffer, and the mixture is rolled and mixed at room temperature for 10 minutes. The solution is placed on a magnetic rack until the magnetic particle solution becomes clear, and the supernatant is removed. Repeat this process twice. The solution is then added with pH 7.4 Tris-HCl buffer and the mixture is rolled and mixed at 37°C for 4 hours.

[0068] (2.5) After the magnetic particle solution in step (2.4) has been reacted, it is repeatedly washed three times with Tris-HCl buffer at pH 7.4, and then the buffer of reagent component 1 is added to reconstitute it to the working concentration (magnetic particles 0.4 mg / mL), and stored at 5°C.

[0069] (3) Prepare the buffer solution of reagent component 2, the specific steps are as follows:

[0070] (3.1) Prepare instruments for weighing and measuring reagents, and adjust the weighing balance to zero.

[0071] (3.2) Add 800 mL of purified water to a beaker, weigh 12.11 g of Tris and 27.0 g of NaCl, and stir thoroughly to dissolve.

[0072] (3.3) Adjust the pH value to within the range of 7.5±0.1.

[0073] (3.4) Add 50.0 mL of newborn calf serum and 5.0 mL of Proclin 300 in sequence and stir until fully dissolved.

[0074] (3.5) The dissolved solution is diluted to 1 L with purified water to prepare a buffer solution of reagent component 2 with a pH of 7.5.

[0075] (4) Prepare reagent component 2, the specific steps are as follows:

[0076] (4.1) Take 0.1 mg of mouse anti-human PLA2R IgG specific monoclonal antibody and dialyze it in 5 L of 0.02 M PBS (pH 7.4) for 15 hours. After changing the liquid once, continue dialysis for 24 hours. After the dialysis is completed, recover the antibody into a centrifuge tube for later use.

[0077] (4.2) Biotin-labeled antibody: 6.7 mg of long-chain biotin ester was placed in a centrifuge tube and dissolved in 1 mL of dimethyl sulfoxide to prepare a long-chain biotin ester solution with a concentration of 6.7 mg / mL; the long-chain biotin ester was added to the dialyzed mouse anti-human IgG antibody at a ratio of 10 μL of long-chain biotin ester per 1 mg of antibody, and the reaction was allowed to proceed for 30 minutes, and then 1 MOL / L of tris(hydroxymethyl)aminomethane buffer was added to terminate the reaction.

[0078] (4.3) The cell was dialyzed against 0.02 M PBS (pH 7.4) for 9 hours. After changing the medium once, the dialyzation was continued for 15 hours.

[0079] (4.4) Recover the antibodies. After dialysis, recover the antibodies into a centrifuge tube for later use.

[0080] (4.5) The antibody recovered in step (4.4) was prepared into 0.1 μg / mL using the buffer of reagent component 2 and stored at 5°C.

[0081] (5) Prepare the buffer solution of reagent component 3, the specific steps are as follows:

[0082] (5.1) Prepare instruments for weighing and measuring reagents, and adjust the weighing balance to zero.

[0083] (5.2) Add 800 mL of purified water to a beaker, weigh 12.11 g of Tris and 27.0 g of NaCl, and stir thoroughly to dissolve.

[0084] (5.3) Adjust the pH value to within the range of 7.5±0.1.

[0085] (5.4) Weigh and add 10.0 g D-trehalose, 5.0 g casein, 20.0 g BSA, 0.04 g 4-aminoantipyrine, and 0.05 g 4-fruit green pigment in sequence and stir until fully dissolved.

[0086] (5.5) Add 3.0 mL Proclin 300, 1.0 mL Triton X-100, and 10.0 mL glycerol in sequence and stir until fully dissolved.

[0087] (5.6) The dissolved solution is diluted to 1 L with purified water to prepare a buffer solution of reagent component 3 with a pH of 7.5.

[0088] (6) Prepare the buffer solution of reagent component 3, the specific method is as follows:

[0089] Dilute the streptavidin-labeled alkaline phosphatase to a working concentration of 0.2 μg / mL using the buffer of reagent component 3 and store it at 2-8°C.

[0090] (7) Prepare the buffer solution for reagent calibration and the buffer solution for reagent quality control. The specific steps are as follows:

[0091] (7.1) Prepare instruments for weighing and measuring reagents, and adjust the weighing balance to zero.

[0092] (7.2) Add 800 mL of purified water to a beaker, weigh 12.11 g of Tris and 9.0 g of NaCl, and stir thoroughly to dissolve.

[0093] (7.3) Adjust the pH value to within the range of 7.2±0.1.

[0094] (7.4) Weigh and add 10.0 g D-mannitol and 30.0 g BSA in sequence and stir until fully dissolved.

[0095] (7.5) Add 2.0 mL Proclin 300, 0.5 mL Triton X-100, and 10.0 mL glycerol in sequence and stir until fully dissolved.

[0096] (7.6) Add purified water to make up to 1 L of the dissolved solution to prepare a pH 7.2 buffer for calibration and quality control.

[0097] (8) Prepare reagent calibrators and reagent quality controls as follows:

[0098] (8.1) Take one PLA2R IgG tube and use the reagent calibrator buffer to prepare reagent calibrators with concentrations of 0RU / mL, 20RU / mL, 50RU / mL, 200RU / mL, 500RU / mL and 1500RU / mL.

[0099] (8.2) Take one PLA2R IgG and prepare reagent control products with concentrations of 50 RU / mL and 500 RU / mL respectively using the reagent control product buffer.

[0100] Example 2

[0101] This example provides a method for drawing a calibration curve using the detection kit for detecting anti-phospholipase A2 receptor antibody IgG in Example 1. The following matters should be noted when drawing the calibration curve:

[0102] A. Invert the test kit several times to mix before loading it into the instrument.

[0103] B. The calibrators should be equilibrated to room temperature before use.

[0104] C. Check that the instrument status meets the experimental requirements.

[0105] D. Follow the instrument operating steps, add the calibration material for on-machine testing, and obtain the luminescence value of each point of the calibration material.

[0106] Calibrator curve drawing: The reagent calibrator concentration and its detected luminescence value obtained by the test are fitted with four parameters to obtain the calibration curve of calibrator concentration-luminescence value. The detection luminescence value of the reagent quality control product or the analyte is brought into the calibration curve of calibrator concentration-luminescence value for calculation to obtain the corresponding concentration value of the reagent quality control product or the analyte. The concentrations and detection luminescence values ​​of the calibrators at each point of the kit are shown in Table 1.

[0107] Table 1: Results of luminescence values ​​(RLU) of calibration samples

[0108] Calibrators Concentration (RU / mL) RLU 1 RLU 2 AVE CV S0 0.00 6002 5942 5972 0.71％ S1 20.00 254722 251302 253012 0.96％ S2 50.00 628245 614222 621233.5 1.60％ S3 200.00 2484702 2491301 2488001.5 0.19％ S4 500.00 6265648 6299496 6282572 0.38％ S5 1500.00 18552963 18498823 18525893 0.21％

[0109] Example 3

[0110] This example evaluates the performance of the detection kit for detecting anti-phospholipase A2 receptor antibody IgG in Example 1:

[0111] A. Blank limit

[0112] The zero-concentration calibrator was tested 20 times, and the adjacent-concentration calibrator was tested 3 times. The linear equation was fitted with the concentrations of the two calibrators, the average of the luminescence values ​​of the zero-concentration calibrator, and the average of the luminescence values ​​of the adjacent-concentration calibrators. The average of the luminescence values ​​of the zero-concentration calibrator + 2 times the standard deviation was then substituted into the linear equation to obtain the fitted concentration, which is the blank limit, which is 0.01RU / mL, as shown in Table 2.

[0113] Table 2: Blank limit luminescence value (RLU) results

[0114]

[0115]

[0116] B. Linear

[0117] The high-value samples close to the upper limit of the measurement range were diluted to 8 concentrations in a certain ratio using the buffer of the calibration product, among which the low-value concentration samples must be close to the lower limit of the linear range. The samples of each concentration were measured 3 times, and the average value was calculated. The average value of the result and the dilution ratio or theoretical concentration were linearly fitted using the least squares method, and the linear correlation coefficient r was calculated to be 0.9997, as shown in Table 3.

[0118] Table 3: Linearity sample determination results

[0119]

[0120] C. Accuracy

[0121] The calibration sample was tested three times, and the relative deviation of the concentration at each point of the calibration sample was calculated. The results are shown in Table 4.

[0122] Table 4: Accuracy sample test results

[0123]

[0124]

[0125] D. Precision

[0126] The quality control samples were taken for determination, each point was repeated 10 times, and the coefficient of variation (CV) was calculated. The determination results are shown in Table 5.

[0127] Table 5: Intra-batch precision determination results

[0128]

[0129]

[0130] E. Stability

[0131] a. Acceleration stability

[0132] The reagent components were placed at 37°C for heat treatment and taken out on the 3rd, 6th and 9th days respectively. The overall luminescence value of the reagent placed at 37°C and the overall luminescence value of the test kit at 2-8°C were tested, and the relative deviation was calculated. The measurement results are shown in Tables 6 and 7.

[0133] Table 6: Results of accelerated stability test of the kit

[0134]

[0135] Table 7: Results of accelerated stability test of calibrators

[0136]

[0137] b. Long-term stability

[0138] The reagent components were placed at 2-8°C for long-term stability observation for 13 months. The calibrators were tested at 1, 3, 6, 9, 12, and 13 months, and the relative deviation from the detection signal at the time of placement was calculated. The measurement results are shown in Table 8.

[0139] Table 8: Long-term stability test results

[0140]

[0141] Example 4

[0142] The detection kit in Example 1 and the anti-phospholipase A2 receptor antibody IgG detection kit (enzyme-linked immunosorbent assay) of the imported company Ou Meng were used to test 80 clinical sera collected from the hospital laboratory. The test results are shown in Table 9. The measured values ​​of the Ou Meng company's kit are the horizontal axis and the measured values ​​of the detection kit in Example 1 are the vertical axis. The comparison analysis of the two test results is shown in the attached figure. Figure 1 As shown; the relevant equation is: y = 1.0109x-1.6195, the correlation coefficient R 2 =0.9995. This shows that the kit of the present invention has a good correlation with the imported kit.

[0143] Table 9: Sample comparison test results

[0144]

[0145]

[0146] In summary, the PLA2R IgG kit provided by the present invention has significant innovation and practicality. The solid phase is coated with a specific PLA2R antigen, and the high affinity of the "biotin-avidin" reaction system is utilized, and the non-specific binding to the tissue cell matrix is ​​low, so as to improve the sensitivity and specificity of the detection system. The long-chain biotin is used to link the mouse anti-human IgG antibody to reduce the steric hindrance, and the streptavidin is linked to the alkaline phosphatase to amplify the signal. By optimizing the reagent formula, a high-sensitivity and high-specificity detection effect is achieved, and at the same time, the sample requirement is small and the reaction time is short. In addition, the detection of PLA2R IgG has the advantages of high sensitivity, high specificity, good stability, low interference rate and appropriate cost, which can reduce the difficulty of PLA2R IgG detection, increase the accuracy of the test results, and play a greater role in the fields of auxiliary diagnosis and treatment effect evaluation of idiopathic membranous nephropathy.

[0147] It should be noted that all features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any manner.

[0148] In addition, the above specific embodiments are exemplary, and those skilled in the art can come up with various solutions inspired by the disclosure of the present invention, and these solutions also belong to the disclosure scope of the present invention and fall within the protection scope of the present invention. Those skilled in the art should understand that the present invention description and its drawings are illustrative and do not constitute a limitation of the claims. The protection scope of the present invention is defined by the claims and their equivalents.

Claims

1. A detection kit for detecting anti-phospholipase A2 receptor antibody IgG, characterized in that: Including reagent component 1, reagent component 2, reagent component 3: The reagent component 1 is a magnetic particle buffer coated on toluenesulfonyl magnetic particles and connected to a phospholipase A2 receptor antigen; The reagent component 2 is a biotin-labeled mouse anti-human IgG specific monoclonal antibody; The reagent component 3 is a marker of streptavidin and alkaline phosphatase.

2. The detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 1, characterized in that: The working concentration of the phospholipase A2 receptor antigen in the reagent component 1 is 1-4 μg / mL, and the working concentration of the tosyl magnetic particles is 0.1-0.4 mg / mL.

3. The detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 1, characterized in that: The working concentration of the mouse anti-human IgG specific monoclonal antibody in the reagent component 2 is 0.05-0.2 μg / mL.

4. The detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 1, characterized in that: The working concentrations of the markers of streptavidin and alkaline phosphatase in the reagent component 3 are 0.1-0.4 μg / mL.

5. The detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 1, characterized in that: Also includes reagent calibrators and reagent quality controls; The reagent calibrator comprises anti-phospholipase A2 receptor antibody IgG solution at concentrations of 0RU / mL, 20RU / mL, 50RU / mL, 200RU / mL, 500RU / mL and 1500RU / mL; The reagent quality control product comprises anti-phospholipase A2 receptor antibody IgG solution with a concentration of 50RU / mL and 500RU / mL.

6. The detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 1, characterized in that: The buffer solution of the reagent component 1 comprises the following components: tris(hydroxymethyl)aminomethane, NaCl, D-mannitol, BSA, sodium dodecyl sulfate, Triton X-100, glycerol and 4-aminoantipyrine; The buffer solution of the reagent component 2 comprises the following components: tris(hydroxymethyl)aminomethane, NaCl, newborn calf serum and Proclin300; The buffer solution of the reagent component 3 includes the following components: tris(hydroxymethyl)aminomethane, NaCl, D-trehalose, casein, BSA, Proclin 300, Triton X-100, 4-aminoantipyrine, glycerol and chlorophyll.

7. A method for preparing a detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to any one of claims 1 to 6, characterized in that: The preparation process of the reagent component 1 comprises the following steps: (1) diluting the phospholipase A2 receptor antigen with PBS, dialyzing it against PBS, and recovering the antigen after dialysis; (2) ultrasonically cleaning and activating the tosyl magnetic particles; (3) Magnetic particle coating: the dialyzed phospholipase A2 receptor antigen is linked to the activated tosyl magnetic particles; (4) The coated magnetic particles are washed and sealed, and then redissolved to obtain reagent component 1.

8. The method for preparing a detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 7, characterized in that: Preparation process of the reagent component 2 The following steps are involved: A: Dilute mouse anti-human IgG specific monoclonal antibody with PBS, dialyze against PBS, and recover the antibody; B: Biotin-labeled antibody: Take biotin ester in a centrifuge tube, add dimethyl sulfoxide to dissolve, and prepare a biotin ester solution; add the antibody recovered in step A to the biotin ester solution for reaction, and then add tris(hydroxymethylaminomethane) buffer to terminate the reaction; C: dialyzed in PBS; D: Recover biotin-labeled antibodies; E: Prepare the antibody using the buffer of reagent component 2 to obtain reagent component 2.

9. The method for preparing a detection kit for detecting anti-phospholipase A2 receptor antibody IgG according to claim 7, characterized in that: The preparation process of the reagent component 3 comprises the following steps: The markers of streptavidin and alkaline phosphatase are diluted with the buffer of reagent component 3 to obtain reagent component 3.

10. A use of the detection kit according to any one of claims 1 to 6, characterized in that: Used to detect anti-phospholipase A2 receptor antibody IgG.

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