Kit for detecting human membranous nephropathy related indexes, method and application thereof

Abstract

The present invention provides a kit, method and application for detecting human membranous nephropathy-related indicators. Through multiple mutation screenings of the coding nucleotide sequences of PLA2R antigen and THSD7A antigen, the most optimized sequences of PLA2R antigen and THSD7A antigen are obtained. The dialysis fluid formula in the preparation and purification processes of PLA2R antigen and THSD7A antigen, as well as the preservation fluid formula of PLA2R antigen and THSD7A antigen, are optimized. Highly pure PLA2R antigen and THSD7A antigen are prepared, and the problems of instability and batch-to-batch variation of PLA2R antigen and THSD7A antigen are solved to the greatest extent. The prepared kit can significantly improve the accuracy and sensitivity of detection, control batch-to-batch variation, and achieve rapid, accurate, simple and efficient detection of human membranous nephropathy-related indicators.

Description

[0001] This application claims the priority of a prior Chinese application with the application number 202310117302.6 and the filing date of February 15, 2023; all of its content is incorporated herein by reference and made a part of this invention. Technical Field

[0002] The present invention relates to the field of flow cytometry technology, and particularly to a kit for detecting human membranous nephropathy-related indicators, its method and application. Background Art

[0003] Among the current methods for detecting autoimmune diseases, enzyme-linked immunosorbent assay (ELISA), immunoblotting, and indirect immunofluorescence are the mainstream and commonly used methodologies. In ELISA, purified antigen is coated on a microplate to form a solid-phase antigen. A standard product with a known concentration and a test sample with an unknown concentration are successively added to the microplate coated with the antigen. After incubation, a biotin-labeled anti-IgG antibody is added, and then it binds to streptavidin labeled with horseradish peroxidase (HRP) to form an immune complex. After thorough washing, the substrate 3,3',5,5'-tetramethylbenzidine (TMB) is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and then into the final yellow under the action of an acid. The intensity of the color is positively correlated with the anti-phospholipase A2 antibody (Anti-PLA2R) in the sample. The absorbance (OD value) is measured with an enzyme-labeled instrument at a wavelength of 450 nm, and the concentration of human anti-phospholipase A2 antibody in the sample is calculated through a standard curve. The operation steps of ELISA are cumbersome, the results have poor repeatability, and the detection time is long.

[0004] Membranous nephropathy (MN), namely membranous glomerulonephritis, specifically refers to nephrotic syndrome caused by the excessive deposition of immune complexes in glomerular capillaries, and it is one of the most common pathological types in adult nephrotic syndrome. Clinically, MN is mainly manifested as massive proteinuria, hypoproteinemia, edema, and hyperlipidemia. According to different etiologies, MN can be divided into idiopathic membranous nephropathy (IMN) and secondary membranous nephropathy (SMN). Among them, IMN is the main type of MN, accounting for about 70% - 80%. Its etiology is still unclear, and it is also known as primary membranous nephropathy (PMN). Most scholars believe that PMN is an antibody-mediated autoimmune glomerular disease. The target antigen located on podocytes is recognized by autoantibodies and binds to form immune complexes, which deposit under the podocytes of the basement membrane, activate the complement system, cause podocyte damage and detachment, lead to an increase in the permeability of the basement membrane, and then massive proteinuria appears.

[0005] Phospholipase A2 receptor (PLA2R) is divided into N type and M type. Human M-type PLA2R is mainly expressed in renal tissue, belongs to the C-type exogenous lectin family, is a type I transmembrane protein, and can participate in the pathogenic processes of inflammation and acute kidney injury under pathological conditions. In 2009, Beck et al. found PLA2R deposition in the kidneys of patients with idiopathic membranous nephropathy (IMN). Immunofluorescence showed that it was mainly co-deposited with IgG4 subtype antibodies, accompanied by granular deposition of C3 along the glomerular basement membrane. The antibody against this target antigen in serum was also of IgG4 subtype, with a positive rate as high as 70%. As the main target antigen of IMN, PLA2R can be used as an index for differentiating idiopathic membranous nephropathy from other glomerular diseases.

[0006] Thrombospondin type-1 domain-containing 7A (THSD7A) has the same biochemical characteristics as PLA2R and is expressed on the surface of podocytes in humans and rodents. Anti-THSD7A antibodies can be detected in 2.5%-5% of IMN patients, and the specific antibody is mainly of IgG4 subtype, which binds to the antibody only under non-reducing conditions. After the antigen-antibody binding, immune complexes are formed and deposited under the glomerular epithelium, further activating the complement system, resulting in podocyte damage or apoptosis and forming proteinuria.

[0007] PLA2R and THSD7A have been confirmed as the two main pathogenic target antigens of IMN, and the antibodies in serum are mainly of IgG4 subtype. Research shows that the detection rate of anti-PLA2R antibodies in patients' serum is about 70%. In the detection of anti-THSD7A antibodies in patients with negative anti-PLA2R antibody screening for IMN, it was found that anti-THSD7A antibodies were present in the serum of 2.5%-5% of the patients. Although the positive rate of anti-THSD7A antibodies in IMN patients is low, its specificity is extremely strong. Therefore, the combined detection of the two can improve the diagnostic efficiency of idiopathic membranous nephropathy.

[0008] The detection of PLA2R and THSD7A requires the use of PLA2R antigen and THSD7A antigen. However, the preparation process of PLA2R antigen and THSD7A antigen is complex, and it is difficult to obtain high-purity PLA2R antigen and THSD7A antigen. Moreover, they are unstable, prone to a decrease in activity during storage, and there are serious batch-to-batch differences, resulting in relatively low accuracy in the detection of PLA2R and THSD7A. Currently, in the detection technology of membranous nephropathy, PLA2R and THSD7A are generally detected separately, with a long detection time, complex operation, and poor detection sensitivity and accuracy. The detection time of enzyme-linked immunosorbent assay, immunoblotting, and indirect immunofluorescence assay is relatively long. It generally takes 2-3 hours from the start of detection to the issuance of the report, and it is impossible to detect individual samples at any time. The sensitivity is insufficient, and it can even only be qualitative and not quantitative.

[0009] Therefore, it is necessary to provide a new detection kit for human membranous nephropathy-related indicators, as well as its preparation method and usage method, to solve the above problems existing in the prior art. Summary of the Invention

[0010] Aiming at the problems existing in the prior art, the present invention provides a detection kit for human membranous nephropathy-related indicators, as well as its method and application. Through multiple mutation screenings of the coding nucleotide sequences of PLA2R antigen and THSD7A antigen, the most optimized PLA2R antigen and THSD7A antigen sequences are obtained, and the dialysis fluid formula and preservation fluid formula in the preparation and purification processes of PLA2R antigen and THSD7A antigen are optimized. High-purity PLA2R antigen and THSD7A antigen are prepared, and the problems of instability and batch-to-batch differences of PLA2R antigen and THSD7A antigen are solved to the greatest extent. The prepared kit can significantly improve the detection accuracy and sensitivity, control the batch-to-batch difference, and achieve rapid, accurate, simple, and efficient detection of human membranous nephropathy-related indicators.

[0011] On the one hand, the present invention provides a detection kit for human membranous nephropathy-related indicators, including antigen proteins, the antigen proteins include PLA2R antigen and THSD7A antigen, the amino acid sequence of the PLA2R antigen is as shown in SEQ ID NO:1, and the amino acid sequence of the THSD7A antigen is as shown in SEQ ID NO:2; the human membranous nephropathy-related indicators include PLA2R antibody and THSD7A antibody.

[0012] Existing recombinant proteins for PLA2R and / or THSD7A detection have serious batch - to - batch differences. At the same time, due to the instability of the PLA2R antigen and the THSD7A antigen, the antigen activity decreases significantly after storage for a period of time, further affecting the medical diagnosis results. In the present invention, through multiple mutation screening of the coding nucleotide sequences of the PLA2R antigen and the THSD7A antigen, the mutated nucleic acid molecules are inserted into eukaryotic expression vectors to respectively obtain recombinant PLA2R antigen and THSD7A antigen. By screening the amino acid sequences of the signal peptides of the optimal PLA2R antigen and THSD7A antigen, the cross - influence caused by non - specific reactions can be significantly improved, the batch - to - batch difference can be controlled, and the accuracy and sensitivity of the detection results can be improved.

[0013] Further, the coding nucleotide sequence of the PLA2R antigen is as shown in SEQ ID NO:3, and the coding nucleotide sequence of the THSD7A antigen is as shown in SEQ ID NO:4.

[0014] Further, it also includes a detection antibody. The detection antibody includes an anti - human IgG antibody complex, and the anti - human IgG antibody complex is a phycoerythrin - labeled anti - human IgG antibody.

[0015] Further, the PLA2R antigen and the THSD7A antigen are respectively conjugated with microspheres to prepare a PLA2R antigen solution conjugated with microspheres and a THSD7A antigen solution conjugated with microspheres; the fluorescent - labeled IgG antibody solution is a phycoerythrin - labeled anti - human IgG antibody solution; the kit also includes a sample diluent.

[0016] On the other hand, the present invention provides a preparation method of a kit for detecting human membranous nephropathy - related indicators. The method includes the following steps:

[0017] Step (1): Prepare the PLA2R antigen and the THSD7A antigen respectively; the coding nucleotide sequence of the PLA2R antigen is as shown in SEQ ID NO:3, and the coding nucleotide sequence of the THSD7A antigen is as shown in SEQ ID NO:4;

[0018] Step (2): Dialyze the PLA2R antigen with the first antigen dialysis solution and then store it with the first antigen preservation solution to obtain a PLA2R antigen solution containing the first antigen preservation solution; dialyze the THSD7A antigen with the second antigen dialysis solution and then store it with the second antigen preservation solution to obtain a THSD7A antigen solution containing the second antigen preservation solution;

[0019] Step (3): Prepare the PLA2R antigen solution for coupling microspheres using the PLA2R antigen solution containing the first antigen preservation solution; prepare the THSD7A antigen solution for coupling microspheres using the THSD7A antigen solution containing the second antigen preservation solution.

[0020] Further, the first antigen dialysis solution includes tris (hydroxymethyl) aminomethane buffer, bovine serum albumin, mannitol, L - histidine, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, dithiothreitol, 4 - (2 - aminoethyl) benzenesulfonyl fluoride hydrochloride; the first antigen preservation solution includes tris (hydroxymethyl) aminomethane buffer, bovine serum albumin, mannitol, L - histidine, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, dithiothreitol, 4 - (2 - aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol, ProClin300.

[0021] Further, the second antigen dialysis solution includes sodium citrate dihydrate, bovine serum albumin, mannitol, L - glycine, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, dithiothreitol, 4 - (2 - aminoethyl) benzenesulfonyl fluoride hydrochloride; the second antigen preservation solution includes sodium citrate dihydrate, bovine serum albumin, mannitol, L - glycine, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, dithiothreitol, 4 - (2 - aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol, ProClin300.

[0022] By optimizing the antigen dialysis solution and antigen preservation solution of PLA2R antigen and THSD7A antigen, the problem of poor stability of PLA2R antigen and THSD7A antigen can be better solved, enabling the long - term stable preservation of PLA2R antigen, THSD7A antigen, the PLA2R antigen solution of the coupling microspheres and the THSD7A antigen solution of the coupling microspheres in the kit, and contributing to improving the accuracy and sensitivity of detection.

[0023] Further, in step (1), the preparation of PLA2R antigen and THSD7A antigen respectively: construct a recombinant vector containing the nucleotide sequence encoding PLA2R antigen and a recombinant vector containing the nucleotide sequence encoding THSD7A antigen, and express them through a eukaryotic expression system to prepare recombinant PLA2R antigen and recombinant THSD7A antigen.

[0024] On the other hand, the present invention provides a method for detecting human membranous nephropathy - related indicators, which is used for non - disease diagnosis purposes and is detected using the kit as described above, including the following steps:

[0025] Step (a): Dilute the sample with the sample diluent, and the dilution factor is 50 times.

[0026] Step (b): Mix the diluted sample, the PLA2R antigen solution conjugated with microspheres, and the THSD7A antigen solution conjugated with microspheres, and incubate in the dark to obtain the first complex;

[0027] Step (c): Add the detection antibody solution to the first complex and incubate in the dark to obtain the complex to be detected;

[0028] Step (d): Detect the complex to be detected using a flow cytometer.

[0029] Research has shown that the sample diluted by the diluent by an appropriate multiple should be detected to achieve better detection accuracy and sensitivity. The reason is that the optimal reaction ratio of antigen and antibody can improve the detection accuracy and sensitivity.

[0030] On the other hand, the present invention provides the use of a group of antigen preservation solutions for preparing reagents for improving the detection sensitivity of human membranous nephropathy-related indicators. The human membranous nephropathy-related indicators include PLA2R antibody and THSD7A antibody. The antigen preservation solutions include a first antigen preservation solution and a second antigen preservation solution. The first antigen preservation solution is used to preserve the PLA2R antigen solution conjugated with microspheres, and the second antigen preservation solution is used to preserve the THSD7A antigen solution conjugated with microspheres.

[0031] On the other hand, the present invention provides the use of a group of antigen dialysis solutions for preparing reagents for improving the detection sensitivity of human membranous nephropathy-related indicators. The human membranous nephropathy-related indicators include PLA2R antibody and THSD7A antibody. The antigen dialysis solutions include a first antigen dialysis solution and a second antigen dialysis solution. The first antigen dialysis solution is used for dialysis to prepare PLA2R antigen, and the second antigen dialysis solution is used for dialysis to prepare THSD7A antigen.

[0032] The kit, method and application for detecting human membranous nephropathy-related indicators based on a flow cytometer provided by the present invention have the following

[0033] Beneficial effects:

[0034] 1. Based on the flow cytometer, the simultaneous and accurate detection of PLA2R and THSD7A can be achieved, and the detection lower limit of PLA2R reaches 4 RU / mL, and the blank limit is not higher than 0.4 RU / mL; the detection lower limit of THSD7A reaches 3 ng / mL, and the blank limit is not higher than 0.6 ng / mL;

[0035] 2. Screen and optimize the nucleotide coding sequences of PLA2R antigen and THSD7A antigen, and express and prepare recombinant antigens through a eukaryotic cell expression system, significantly improving the cross-influence caused by non-specific reactions, controlling the batch-to-batch difference, and improving the accuracy and sensitivity of detection results;

[0036] 3. Optimize the antigen dialysis solution formula and antigen preservation solution formula for PLA2R antigen and THSD7A antigen, solve the problem of poor stability of PLA2R antigen and THSD7A antigen, enable the long-term stable preservation of PLA2R antigen, THSD7A antigen, the PLA2R antigen solution coupled with microspheres and the THSD7A antigen solution coupled with microspheres in the kit, which can be stably preserved for more than 1 year, and contribute to improving the detection accuracy and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the detection principle of the kit for detecting human membranous nephropathy-related indicators in Example 1;

[0038] Figure 2 It is the distribution of captured fluorescent microspheres in sample detection in Example 1;

[0039] Figure 3 It is the calibration curve for detecting anti-PLA2R antibody by flow cytometry fluorescence in Example 1;

[0040] Figure 4 It is the calibration curve for detecting anti-THSD7A antibody by flow cytometry fluorescence in Example 1;

[0041] Figure 5 It is the PLA2R linear evaluation result in Example 1;

[0042] Figure 6 It is the THSD7A linear evaluation result in Example 1;

[0043] Figure 7 It is the comparison result diagram between healthy samples and PLA2R-positive samples in Example 1;

[0044] Figure 8 It is the comparison result diagram between healthy samples and THSD7A-positive samples in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0045] To describe the present invention more specifically, the technical solutions of the present invention will be described in detail below in conjunction with the drawings and specific embodiments. These descriptions only show how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0046] Example 1: Preparation, Use and Effect Evaluation of the Kit

[0047] I. Preparation of the Kit

[0048] The specific composition is shown in Table 1:

[0049] Table 1. Composition of the Kit

[0050]

[0051] The preparation method is as follows:

[0052] 1. Preparation of PLA2R antigen and THSD7A antigen

[0053] The recombinant proteins of PLA2R antigen and THSD7A antigen contain an efficient signal peptide sequence at the N-terminus and a His-tag purification tag composed of 10 histidines, and are transiently transfected and expressed using the pcDNA3.4 vector and Expi293F cell expression system.

[0054] Transient transfection and expression: On the day of transfection, adjust the density of Expi293F cells in the logarithmic growth phase to 2.5×10 6 / mL, and the medium used is Expi293 TM Expression Medium (Thermo Fisher Scientific (China) Co., Ltd., A1435101). For 100 mL of transfected cells, 100 μg of the aforementioned recombinant plasmid is required, and 270 μL of the transfection reagent ExpiFectamine TM 293 (Thermo Fisher Scientific (China) Co., Ltd., A14524) is added. After culturing at 37 °C and 8% carbon dioxide for 4 - 5 days, centrifuge at 3000G for 20 minutes to collect the medium, and filter it through a 0.22 μM filter membrane.

[0055] Purification:

[0056] Purify using Ni-NTA affinity chromatography medium (GenScript Corporation, product number L00250), with equilibration buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, pH 8.0) and elution buffer (300 mM NaCl, 50 mM NaH2PO4, 250 mM imidazole, pH 8.0). Subsequently, desalt using a HiTrap desalting column (Cytiva, product number 29048684) in buffer A (20 mM Tris-HCl, pH 8.0). Next, use a HiTrap Capto Q (Cytiva, product number 11001303) anion exchange chromatography column, equilibrate with buffer A, and elute with a gradient of buffer B (1 M NaCl, 20 mM Tris-HCl, pH 8.0). Finally, use a Superdex 200 Increase 10 / 300 GL (Cytiva, product number 28990944) gel filtration chromatography column and equilibrate with buffer C (150 mM NaCl, 10 mM NaH2PO4, pH 7.4).

[0057] 1.1 Preparation of PLA2R antigen

[0058] Synthesize the gene sequence SEQ ID NO:3 encoding the recombinant protein PLA2R and clone it into the vector pcDNA3.4. Express and purify the recombinant protein PLA2R using the aforementioned method, dialyze it in the first dialysis solution for 12 - 24 h, and then it can be stored long-term (-80 °C) in the first storage solution or immediately conjugated to microspheres.

[0059] Preparation of the first dialysis solution: Dissolve 6.057 g of tris(hydroxymethyl)aminomethane, 1 g of bovine serum albumin, 10 mL of mannitol, 0.776 g of L-histidine, 5.844 g of sodium chloride, 7.4448 g of disodium ethylenediaminetetraacetate dihydrate, 0.309 g of dithiothreitol, and 0.24 g of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in 900 mL of water in sequence, make up the volume to 1000 mL, and adjust the pH to 7.5 using NaOH (1 M) and concentrated hydrochloric acid. Filter it with a 0.22 μM filter membrane and store it at 2 - 8 °C for later use.

[0060] Preparation of the first storage solution: Dissolve 6.057 g of tris(hydroxymethyl)aminomethane, 1 g of bovine serum albumin, 10 mL of mannitol, 0.776 g of L-histidine, 5.844 g of sodium chloride, 7.4448 g of disodium ethylenediaminetetraacetate dihydrate, 0.309 g of dithiothreitol, 0.24 g of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 100 mL of glycerol, and 1 mL of ProClin300 in 800 mL of water in sequence, make up the volume to 1000 mL, and adjust the pH to 7.5 using NaOH (1 M) and concentrated hydrochloric acid. Filter it with a 0.22 μM filter membrane and store it at 2 - 8 °C for later use.

[0061] 1.2. Preparation of THSD7A antigen

[0062] Synthesize the gene sequence SEQ ID NO:4 encoding the recombinant protein THSD7A and clone it into the vector pcDNA3.4. Express and purify the recombinant protein THSD7A using the aforementioned method, dialyze it in the second dialysis solution for 12 - 24 h, and then it can be stored long-term (-80 °C) in the second storage solution or immediately conjugated to microspheres.

[0063] Preparation of the second dialysis solution: Dissolve 5.882 g of sodium citrate dihydrate, 1 g of bovine serum albumin, 10 mL of mannitol, 0.751 g of L-glycine, 17.533 g of sodium chloride, 3.722 g of disodium ethylenediaminetetraacetate dihydrate, 0.309 g of dithiothreitol, and 0.24 g of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride in 900 mL of water in sequence, make up the volume to 1000 mL, and adjust the pH to 8.0 using NaOH (1 M) and concentrated hydrochloric acid. Filter it with a 0.22 μM filter membrane and store it at 2 - 8 °C for later use.

[0064] Preparation of the second preservation solution: Dissolve 5.882 g of sodium citrate dihydrate, 1 g of bovine serum albumin, 10 mL of mannitol, 0.751 g of L-glycine, 17.533 g of sodium chloride, 3.722 g of disodium ethylenediaminetetraacetate dihydrate, 0.309 g of dithiothreitol, 0.24 g of 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 100 mL of glycerol, and 1 mL of ProClin300 in 800 mL of water in sequence, make up the volume to 1000 mL, and adjust the pH to 8.0 using NaOH (1 M) and concentrated hydrochloric acid. Filter with a 0.22 μM filter membrane and store at 2 - 8 °C for later use.

[0065] 2. Preparation of the antigen solution for coupling microspheres

[0066] Take 0.1 mL of fluorescent microspheres with a concentration of 5×10 6 per mL (manufacturer Biolegend, model 740168), add PBST buffer for washing twice. Add 100 μg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 50 μg of N-hydroxysulfosuccinimide (NHS) to the washed first fluorescent microspheres and let stand for 30 minutes to activate the microspheres. Add 10 μL of PLA2R antigen solution and react by rotating at room temperature for 5 hours. After washing the first fluorescent microspheres to remove the excess antigen, add 5% skim milk powder by mass for blocking for 30 minutes. After removing the skim milk powder, add 500 μL of Tris buffer with a pH of 7.2 for storage, and prepare 0.5 mL of the PLA2R antigen solution for coupling microspheres;

[0067] According to the same method, prepare 0.5 mL of the THSD7A antigen solution for coupling microspheres using fluorescent microspheres (manufacturer Biolegend, model 740170);

[0068] Take 1 μL each of the PLA2R antigen solution for coupling microspheres and the THSD7A antigen solution for coupling microspheres, and prepare the antigen solution for coupling microspheres.

[0069] 3. Preparation of the detection antibody solution

[0070] Couple the anti-human IgG antibody with phycoerythrin to obtain the detection antibody solution. The mass ratio of the anti-human IgG antibody to phycoerythrin in the detection antibody solution is 1:1. The anti-human IgG antibody is purchased from Maidian Biotechnology Co., Ltd., product number Z01236M.

[0071] 4. Preparation of the sample diluent

[0072] Dissolve 3.0275 g of tris(hydroxymethyl)aminomethane (Tris) and 9.0 g of sodium chloride (NaCl) in 800 mL of pure water, add 1 mL of ProClin300, adjust the pH to 6.8. After complete dissolution, add 20 g of bovine serum albumin (BSA). After complete dissolution, make up the volume to 1000 mL and adjust the pH to 6.8 for standby.

[0073] 5. Prepare washing buffer (1×)

[0074] Dissolve 2.4 g of potassium dihydrogen phosphate (KH2PO4), 36.32 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), 8 g of NaCl, and 2 g of potassium chloride (KCl) in 1000 mL of pure water. Add 25 g of BSA, 1% (by mass) of ProClin300 preservative, and 0.5% (by mass) of Tween-20 to obtain washing buffer (10×) for standby. Optionally, equilibrate the washing buffer (10×) to room temperature. After all salts are dissolved, take 10 mL of the washing buffer (10×) and add it to 90 mL of pure water to obtain washing buffer (1×).

[0075] 6. Calibrator

[0076] The calibrator consists of PLA2R antibodies (manufacturer: CREATIVE DIAGNOSTICS, model: CABT-B2145) and THSD7A antibodies (manufacturer: CREATIVE DIAGNOSTICS, model: DPABH-15118) with 7 different concentrations (specific concentrations are PLA2R: 0 RU / mL, 5 RU / mL, 20 RU / mL, 55 RU / mL, 170 RU / mL, 500 RU / mL, 1450 RU / mL; THSD7A: 0 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, 100 ng / mL, 190 ng / mL), and blank serum (added with 20% mouse serum without endogenous detection targets) to simulate the matrix environment in human serum for establishing a calibration curve.

[0077] 7. Quality control sample

[0078] The quality control sample consists of PLA2R antibodies and THSD7A antibody solution matrix solution.

[0079] II. Usage method of the kit

[0080] Using the kit prepared in this example, the usage method is as follows:

[0081] (a) Dilute 5 μL of the sample to be tested with the sample diluent, and control the volume ratio of the sample to be tested to the sample diluent to be 1:51;

[0082] (b) Add 20 μL of the antigen solution conjugated with microspheres and 20 μL of the diluted sample to be tested into the sample tube respectively, and incubate the obtained first complex in the dark at room temperature for 0.5 h; Resuspend the first complex after dark incubation with 1000 μL of washing buffer (1×), then centrifuge at 400 g for 5 min and retain the precipitate;

[0083] (c) Add 20 μL of the detection antibody solution to the obtained precipitate, mix the complex to be detected evenly, and incubate in the dark at room temperature for 0.5 h; Resuspend the complex to be detected after dark incubation with 1000 μL of washing buffer (1×), then centrifuge at 400 g for 5 min and retain the precipitate;

[0084] (d) Resuspend the obtained precipitate with 100 μL of washing buffer (1×), and detect the fluorescence type and fluorescence signal intensity of the resuspended mixture on a DxFLEX flow cytometer produced by Beckman Coulter. The specific detection process is a conventional technical means for those skilled in the art and will not be elaborated here.

[0085] This kit requires a small amount of sample, only 5 - 20 μL of serum is needed.

[0086] Figure 1 It is a schematic diagram of the detection principle of the kit for detecting human membranous nephropathy - related indicators. Among them, 1 is the microsphere, 2 is the antigen, 3 is the antibody to be detected, 4 is the detection antibody, 5 is the phycoerythrin. The antigen 2 binds to the carboxyl - group - containing polystyrene microsphere 1 to form the first complex. The detection antibody 4 and the phycoerythrin 5 bind to form the second complex. The first complex, the antibody to be detected 3 and the second complex bind to form the immune complex. Two beams of excitation light with different wavelengths emitted by the flow cytometer irradiate the immune complex. The type of detection index is determined by the fluorescence intensity of different polystyrene fluorescent - coded microspheres, and the content of each detection index is determined by the fluorescence intensity of the phycoerythrin.

[0087] Figure 2 It is the distribution of captured fluorescent microspheres for sample detection. Refer to Figure 2 , with the fluorescence signal value in the forward scatter (FSC) channel as the ordinate and the fluorescence signal value in the allophycocyanin (APC) channel as the abscissa. The position of the microsphere size is determined by the ordinate, and the APC fluorescence intensity carried by different microspheres is distinguished by the abscissa. Two types of polystyrene microspheres coated with PLA2R antigen and THSD7A antigen can be distinguished. Among them, P1 represents the distribution of the fluorescence signal values in the forward scatter channel and the allophycocyanin channel of the polystyrene microsphere coated with PLA2R antigen, and P2 represents the distribution of the fluorescence signal values in the forward scatter channel and the allophycocyanin channel of the polystyrene microsphere coated with THSD7A antigen.

[0088] III. Analysis of Detection Results

[0089] 1. Calibration Curve

[0090] Seven calibration curve points, namely Calibrators A, B, C, D, E, F, and G, which have been prepared, are provided. Each calibration curve point has a corresponding concentration value. According to the fluorescence measurement values and the corresponding concentration values of each point, the calibration curve of the corresponding index can be plotted. Figure 3 This is the calibration curve for the flow cytometry fluorescence detection of anti-PLA2R antibody in this example. Figure 4 This is the calibration curve for the flow cytometry fluorescence detection of anti-THSD7A antibody in this example.

[0091] 2. Linearity Evaluation

[0092] For linearity, high-concentration samples at the upper limit of the linear range are diluted into at least seven samples with different concentrations. Each concentration is tested three times, and the mean value of the detected result concentration is calculated respectively. Taking the dilution concentration x as the independent variable and the mean value of the detected concentration y as the dependent variable, the linear regression equation is obtained. The correlation coefficient R2 of the linear regression is calculated. Figure 5 This is the linearity evaluation result of PLA2R in this example. Figure 6 This is the linearity evaluation result of THSD7A in this example. Refer to Figure 5 、 Figure 6 For PLA2R, the linear regression equation is y = 1.0838x + 10.107, and R2 = 0.9964; for THSD7A, the linear regression equation is y = 1.0405x + 5.1614, and R2 = 0.9933.

[0093] Using the kit for detecting human membranous nephropathy-related indicators of the present invention, the detectable linear range of PLA2R is not narrower than [4, 1500] RU / mL, and the detectable linear range of THSD7A is not narrower than [3, 200] ng / mL. Within the linear range, the linear correlation coefficient |R2| is not less than 0.990.

[0094] 3. Blank Limit Evaluation

[0095] The blank samples of PLA2R and THSD7A are selected and detected 20 times repeatedly. The average value M and the standard deviation SD value of the measurement results are calculated. The blank limit is M + 2SD. Table 4 shows the blank limit results of this example. Referring to Table 4, the blank limits of PLA2R and THSD7A are 0.21 RU / mL and 0.57 ng / mL respectively.

[0096] 4. Repeatability Evaluation

[0097] Detect two calibration products of PLA2R and THSD7A at different concentration levels, conduct 10 parallel detections, calculate the coefficient of variation CV, which can characterize the repeatability of the detection kit for membranous nephropathy-related indicators. Table 2 shows the results of the repeatability evaluation. Referring to Table 2, in the within-batch experiment, the coefficients of variation CV of the high-value and low-value calibration products of PLA2R and THSD7A are both within 7%.

[0098] Table 2. Repeatability test results

[0099]

[0100] 5. Evaluation of inter-batch difference

[0101] Take three batches of the kit, 10 for each batch, and repeat the detection of the same calibration product respectively. Calculate the average value M and standard deviation SD of the 30 measurement results, and calculate the coefficient of variation CV. The results of the inter-batch difference evaluation are shown in Table 3. It can be seen that the CVs of the three batches of inter-batch experiments of PLA2R and THSD7A are both within 10%.

[0102] Table 3. Results of inter-batch difference evaluation

[0103]

[0104]

[0105] From the evaluation of repeatability and inter-batch difference, it can be known that the detection kit for membranous nephropathy-related indicators of the present inventor has good repeatability and inter-batch difference. The coefficient of variation CV of the within-batch experiment is not greater than 7%, and the CV of the inter-batch experiment is not greater than 10%.

[0106] 6. Detection of clinical samples

[0107] Use the kit in this example to detect 50 healthy human samples, 39 PLA2R-positive samples, and 30 THSD7A-positive samples, and conduct comparative analysis. Figure 7 is the comparison result graph of healthy samples and PLA2R-positive samples. Figure 8 is the comparison result graph of healthy samples and THSD7A-positive samples. Referring to Figure 7 and Figure 8 , the fluorescence intensities of anti-PLA2R antibody and anti-THSD7A antibody measured using healthy samples and positive samples are significantly distinguishable. It can be seen that the contents of anti-PLA2R antibody and anti-THSD7A antibody measured from healthy samples and positive samples are significantly distinguishable. The detection kit for human membranous nephropathy-related indicators provided in this example can simultaneously detect the contents of anti-PLA2R antibody and anti-THSD7A antibody, and has high accuracy, high precision, good specificity, stable measurement results, and is more convenient for clinical application.

[0108] 7. Comparison with Mass Spectrometry Detection Results

[0109] For the same sample, the kit provided in this example and a mass spectrometer were used for detection respectively. The mass spectrometer (purchased from Shimadzu, model LC-MS / 8040) used 0.1% formic acid water and 0.1% formic acid acetonitrile as the mobile phase, with an ESI ion source. The nebulizing gas pressure (GS1): 45 Psi, auxiliary gas pressure: 45 Psi, curtain gas pressure: 35 Psi, temperature: 650 °C, spray voltage: 5000 V (positive ion mode), gradient elution. The sample for detection was a calibrator, and the detection results are shown in Table 4.

[0110] Table 4. Comparison of the kit provided in this example and mass spectrometry detection results

[0111] Detection index Mass spectrometry detection value Detection value of this kit PLA2R (unit: RU / mL) 20.56 21.11 THSD7A (unit: ng / mL) 8.13 8.05

[0112] Example 2: Optimization of PLA2R Antigen and THSD7A Antigen Sequences

[0113] 1. Optimization of PLA2R Antigen Sequence

[0114] The full extracellular region sequence of the PLA2R antigen (Uniprot number Q13018) consists of 10 domains, as shown in Table 5.

[0115] Table 5. PLA2R Antigen Domains

[0116] Domain name Amino acid sequence range Ricin B-type lectin 38-161 Fibronectin type-II 173-221 C-type lectin 1 238-355 C-type lectin 2 385-502 C-type lectin 3 522-643 C-type lectin 4 673-797 C-type lectin 5 819-938 C-type lectin 6 965-1096 C-type lectin 7 1121-1232 C-type lectin 8 1257-1378

[0117] Select some domains according to known literature: Ricin B-type lectin, Fibronectin type-II, C-type lectin 1, C-type lectin 2, C-type lectin 4, C-type lectin 5, C-type lectin 7, C-type lectin 8. Antibodies in most serum samples can recognize the domains Ricin B-type lectin and C-type lectin 1, while antibodies in a small part of serum samples can recognize the domains Fibronectin type-II, C-type lectin 2, C-type lectin 4, C-type lectin 5, C-type lectin 7, C-type lectin 8. Selecting these domains enables the recombinant protein to be recognized by antibodies in almost all serum samples. In this patent, for the first time, two GS linkers and an α-helix structure polypeptide (GGGSAEAAAKEAAAKASGGG) are used to connect those domains that are not adjacent in the natural sequence but are made adjacent in the recombinant sequence, and by increasing the spatial distance, the antigenic epitopes on each domain are fully exposed. An optimized signal peptide sequence (SEQ ID NO:5) and a His-tag purification tag consisting of 10 histidines (purified by affinity chromatography) are added at the N-terminus. For example, the amino acid sequence of the constructed recombinant protein (SEQ ID NO:1) can be expressed as: signal peptide sequence + His-tag + Ricin B-type lectin + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 1 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 7 + C-type lectin 8. Use the web tool provided by https: / / novoprolabs.com / tools / codon-optimization to obtain the nucleotide sequence of the recombinant protein (SEQ ID NO:3) through human codon optimization. Subsequently, express and purify the recombinant protein according to the steps in Example 1.

[0118] According to the steps described in the specification of the present invention, the following several recombinant protein preparation kits are respectively adopted:

[0119] 1. Four domains: signal peptide sequence + His-tag + Ricin B-type lectin + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 1 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 7 + C-type lectin 8 (amino acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:3);

[0120] 2. Six domains: signal peptide sequence + His-tag + Ricin B-type lectin + Fibronectin type-II + C-type lectin1 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 4 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 7 + C-type lectin 8 (amino acid sequence SEQ ID NO:6, nucleotide sequence SEQ ID NO:7);

[0121] 3. Seven domains: signal peptide sequence + His-tag + Ricin B-type lectin + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 1 + C-type lectin 2 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 4 + C-type lectin5 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 7 + C-type lectin 8 (amino acid sequence SEQ ID NO:8, nucleotide sequence SEQ ID NO:9);

[0122] 4. Four domains: signal peptide sequence + His-tag + Ricin B-type lectin + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 1 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 7 + (GGGSAEAAAKEAAAKASGGG) + C-type lectin 8 (amino acid sequence SEQ ID NO:10, nucleotide sequence SEQ ID NO:11);

[0123] 5. Recombinant protein containing the full-length sequence of PLA2R antigen (amino acid sequence SEQ ID NO:12, nucleotide sequence SEQ ID NO:13);

[0124] Fifty samples from healthy individuals and 39 samples positive for PLA2R were detected separately, and the detection results of kits prepared with 5 different recombinant PLA2R antigens were compared, as shown in Table 6. Among them, sensitivity: the proportion of samples actually positive that were detected as positive; specificity: the proportion of samples actually negative that were detected as negative; accuracy: the proportion of the sum of true positive and true negative samples in the total number of samples; each was detected 5 times repetitively.

[0125] Table 6. Detection results of recombinant protein PLA2R before and after sequence optimization

[0126]

[0127]

[0128] PLA2R antibody detection SEQ ID NO:3 SEQ ID NO:7 SEQ ID NO:9 SEQ ID NO:11 SEQ ID NO:13 Sensitivity 84.6％ 76.9％ 82.05％ 71.79％ 51.3％ Specificity 100％ 98％ 100％ 98％ 96％ Accuracy 93.3％ 88.76％ 92.13％ 86.52％％ 76.4％

[0129] It can be seen from Table 6 that there are significant differences in the experimental results among recombinant proteins with different sequences.

[0130] By comparing the 1st to 3rd ones, it can be seen that the four domains of Ricin B-type lectin, C-type lectin 1, C-type lectin 7, and C-type lectin 8 are sufficient to enable the recombinant protein to be recognized by antibodies in almost all serum samples, and adding more domains instead decreases the sensitivity. The reason may be that too many domains affect the spatial epitopes of the four domains of Ricin B-type lectin, C-type lectin 1, C-type lectin 7, and C-type lectin 8. Therefore, it is preferably to use the recombinant PLA2R antigen constructed with the four domains of Ricin B-type lectin, C-type lectin 1, C-type lectin 7, and C-type lectin 8.

[0131] By comparing the 1st and 4th ones, it can be seen that although the same four domains are used, without connecting with GS linker and α-helix structure polypeptide between the domains of C-type lectin 7 and C-type lectin 8, otherwise it will affect the detection sensitivity, specificity and accuracy. The reason may be that the natural linker peptide between C-type lectin 7 and C-type lectin 8 has a better effect than the artificial linker peptide.

[0132] 2. Optimization of THSD7A antigen sequence

[0133] The full-length extracellular region sequence of the THSD7A antigen (Uniprot number Q9UPZ6) consists of 22 domains, as shown in Table 7.

[0134] Table 7. Domains of the THSD7A antigen

[0135] Domain name Amino acid sequence range TSP type-1 1 48-116 TSP type-1 2 117-192 TSP type-1 3 193-247 Coiled coil 248-358 TSP type-1 4 359-423 TSP type-1 5 424-510 TSP type-1 6 511-575 TSP type-1 7 575-632 TSP type-1 8 633-695 TSP type-1 9 696-769 TSP type-1 10 771-831 TSP type-1 11 832-904 TSP type-1 12 905-959 TSP type-1 13 960-1033 TSP type-1 14 1034-1095 TSP type-1 15 1096-1163 TSP type-1 16 1164-1220 TSP type-1 17 1221-1284 TSP type-1 18 1285-1341 TSP type-1 19 1342-1412 TSP type-1 20 1413-1475 TSP type-1 21 1476-1535

[0136] Using the above 21 individual domains except Coiled coil (Coiled coil is common in the human body and cannot form THSD7A-specific antigenic epitopes), 30 THSD7A-positive samples were detected separately, and the test results are shown in Table 8. The results are qualitatively expressed as three situations: positive P, weakly positive Pw, and negative N.

[0137] Table 8. Results of separately detecting positive samples with different domains of THSD7A

[0138]

[0139] According to the results of separately detecting positive samples with different domains, one or more of the 14 domains of TSP type-1 1 / TSP type-1 2 / TSPtype-1 5 / TSP type-1 7 / TSP type-1 9 / TSP type-1 10 / TSP type-1 11 / TSP type-1 12 / TSP type-1 14 / TSP type-1 15 / TSP type-1 18 / TSP type-1 19 / TSP type-1 20 / TSPtype-1 21 can be recognized by the existing positive serum samples (the proportion of positive samples measured is the highest). Therefore, these 14 domains were selected to construct the recombinant protein of the THSD7A antigen. An optimized signal peptide sequence (SEQ ID NO:14) and a His-tag purification tag consisting of 10 histidines were added to the N-terminus. For example, the amino acid sequence of the constructed recombinant protein (SEQ ID NO:2) can be expressed as: signal peptide sequence + His-tag + TSP type-1 1 + TSP type-1 2 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 9 + TSP type-1 10. Using the web tool provided by https: / / novoprolabs.com / tools / codon-optimization, the nucleotide sequence of the recombinant protein of the THSD7A antigen (SEQ ID NO:4) was obtained through human codon optimization. Subsequently, the recombinant protein was expressed and purified according to the steps in Example 1.

[0140] According to the steps described in the specification of the present invention, the following several kinds of kits for preparing THSD7A antigen recombinant proteins are respectively adopted:

[0141] 1. Four domains: signal peptide sequence + His-tag + TSP type-1 1 + TSP type-1 2 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 9 + TSP type-1 10 (amino acid sequence SEQ ID NO:2, nucleotide sequence SEQ ID NO:4);

[0142] 2. Ten domains: signal peptide sequence + His-tag + TSP type-1 1 + TSP type-1 2 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 5 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 7 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 9 + TSP type-1 10 + (GGGSAEAAAKEAAAKASGGG) + TSPtype-1 14 + TSP type-1 15 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 18 + TSP type-1 19 (amino acid sequence SEQ ID NO:15, nucleotide sequence SEQ ID NO:16);

[0143] 3. Fourteen domains: signal peptide sequence + His-tag + TSP type-1 1 + TSP type-1 2 + TSP type-13 + Coiled coil + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 6 + TSP type-1 7 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 9 + TSP type-1 10 + TSP type-1 11 + TSP type-1 12 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 18 + TSP type-1 19 + TSP type-1 20 + TSP type-121 (amino acid sequence SEQ ID NO:17, nucleotide sequence SEQ ID NO:18);

[0144] 4. Four domains: signal peptide sequence + His-tag + TSP type-1 1 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 2 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 9 + (GGGSAEAAAKEAAAKASGGG) + TSP type-1 10 (amino acid sequence SEQ ID NO:19, nucleotide sequence SEQ ID NO:20);

[0145] 5. Recombinant protein containing the full-length sequence of THSD7A antigen (amino acid sequence SEQ ID NO:21, nucleotide sequence SEQ ID NO:22)

[0146] Fifty healthy human samples and thirty THSD7A-positive samples were detected respectively, and the detection results of kits prepared with five different recombinant proteins of THSD7A antigen were compared, as shown in Table 9. Among them, sensitivity: the proportion of samples detected as positive among those actually positive; specificity: the proportion of samples detected as negative among those actually negative; accuracy: the proportion of the sum of true positive and true negative samples in the total number of samples; each was detected 5 times repetitively.

[0147] Table 9. Detection results of recombinant protein THSD7A before and after sequence optimization

[0148]

[0149] THSD7A antibody detection SEQ ID NO:4 SEQ ID NO:16 SEQ ID NO:18 SEQ ID NO:20 SEQ ID NO:22 Sensitivity 93.3％ 83.3％ 83.3％ 80％ 73.3％ Specificity 98％ 94％ 94％ 92％ 90％ Accuracy 96.3％ 90％ 90％ 87.5％ 83.6％

[0150] As can be seen from Table 9, there are significant differences in the experimental results among THSD7A recombinant proteins with different sequences. By comparing the first to the third types, it can be seen that the four domains of TSP type-1 1 / TSP type-1 2 / TSP type-1 9 / TSP type-1 10 are sufficient to enable the THSD7A recombinant protein to be recognized by antibodies in almost all serum samples. Moreover, adding more domains decreases the sensitivity and specificity. The reason may be that excessive domains affect the spatial epitopes of these four domains of THSD7A. Therefore, it is preferred to use the THSD7A antigen recombinant protein constructed with the four domains of TSP type-1 1 / TSP type-1 2 / TSP type-1 9 / TSP type-1 10. By comparing the first and the fourth types, it can be seen that although the same four domains are used, between the domains of TSP type-1 1 / TSP type-1 2 and between TSP type-1 9 / TSP type-1 10, they are not connected with GS linkers and α-helix structure polypeptides, otherwise it will affect the detection sensitivity, specificity and accuracy. The reason may be that the connecting peptides in the natural sequence are more conducive to the protein forming the correct folding structure.

[0151] Example 3: Screening of the first antigen dialysis solution and the second antigen dialysis solution

[0152] 1. Screening of the first antigen dialysis solution

[0153] In this example, a kit for detecting human membranous nephropathy-related indicators was prepared according to the method provided in Example 1. During the preparation of the PLA2R antigen, six different antigen dialysis solutions shown in Table 10-1 and the first antigen dialysis solution used in Example 1 were respectively adopted, and NaOH (1M) and concentrated hydrochloric acid were used to adjust the pH to 7.5, which were respectively used to dialyze and prepare the PLA2R antigen. Then, according to the method provided in Example 1, a microsphere-conjugated antigen solution was prepared, and a corresponding kit for detecting human membranous nephropathy-related indicators was prepared for the detection of PLA2R. The test sample was positive serum (the concentration of PLA2R detected by mass spectrometry was 26.15 RU / mL). After the prepared kit was stored at 2-8°C for 0 days, 1 month, 3 months, 6 months, and 12 months, it was detected according to the method provided in Example 1. The influence of different first antigen dialysis solutions on the detection results of PLA2R was investigated, and 10 repeated detections were carried out, and the average value was taken and the CV value was calculated.

[0154] Table 10-1. Composition of different first antigen dialysis solutions (the proportional relationship is according to Example 1)

[0155]

[0156] Table 10-2. Influence of Different First Antigen Dialysates on the Stability of PLA2R

[0157]

[0158]

[0159] As can be seen from Table 10-2, after optimizing the PLA2R antigen sequence, the formulation of the PLA2R antigen dialysate also needs to be further screened to achieve better detection sensitivity. Moreover, the PLA2R antigen prepared with different first antigen dialysates will not only affect the stability of the PLA2R antigen but also affect the detection results of PLA2R. The reason may be that the dialysis effects of different antigen dialysates are different, resulting in different purities of the prepared PLA2R antigen, which in turn affects the stability during storage. Therefore, it is necessary to screen the optimal first antigen dialysate formulation.

[0160] When comparing the PLA2R antigens prepared with different antigen dialysates, their sensitivities for PLA2R detection are different, and the detection values after being placed for a period of time are also different. The most preferred first antigen dialysate formulation is the one prepared according to Example 1: Tris(hydroxymethyl)aminomethane + Bovine Serum Albumin + Mannitol + L-Histidine + Sodium Chloride + Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate + Dithiothreitol + 4-(2-Aminoethyl)benzenesulfonyl Fluoride Hydrochloride. At this time, both the detection sensitivity and the antigen stability are significantly better.

[0161] 2. Screening of the Second Antigen Dialysate

[0162] In this example, a kit for detecting human membranous nephropathy-related indicators was prepared according to the method provided in Example 1. During the preparation of the THSD7A antigen, 6 different antigen dialysates shown in Table 11-1 and the second antigen dialysate used in Example 1 were respectively adopted, and NaOH (1M) and concentrated hydrochloric acid were used to adjust the pH to purify and prepare the THSD7A antigen. Then, according to the method provided in Example 1, a microsphere-conjugated antigen solution was prepared, and a corresponding kit for detecting human membranous nephropathy-related indicators was prepared for the detection of THSD7A. The test sample was positive serum (with a concentration of 8.52 ng / mL detected by mass spectrometry). Detection was carried out according to the method provided in Example 1 to investigate the influence of different second antigen dialysates on the detection results of THSD7A, and 10 repeated detections were performed, and the average value was taken.

[0163] Table 11-1. Composition of Different Second Antigen Dialysates (Proportion Relationship According to Example 1)

[0164]

[0165] Table 11-2. Influence of Different Second Antigen Dialysates on the Stability of THSD7A

[0166]

[0167]

[0168]

[0169] As can be seen from Table 11-2, the THSD7A antigen prepared with different second antigen dialysis solutions will not only affect the stability of the THSD7A antigen, but also affect the detection results of THSD7A. Therefore, it is necessary to optimize the formula of the second antigen dialysis solution to improve the detection sensitivity of THSD7A and at the same time improve the stability of the THSD7A antigen. As can be seen from Table 11-2, only by adopting a formula different from that of the first antigen dialysis solution and different pH values can it be applicable to the stable preservation of the THSD7A antigen and at the same time improve the detection sensitivity. The most preferred formula of the second antigen dialysis solution is prepared according to Example 1: sodium citrate dihydrate + bovine serum albumin + mannitol + L-glycine + sodium chloride + disodium ethylenediaminetetraacetate dihydrate + dithiothreitol + 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and the pH is adjusted to 8.0. At this time, the detection sensitivity and antigen stability are both significantly better.

[0170] Example 4: Influence of antigen preservation solution

[0171] 1. Influence of antigen preservation solution on the stability of PLA2R antigen and THSD7A antigen

[0172] This example proves through research that there are problems with the poor stability of the PLA2R antigen and the THSD7A antigen. Therefore, after the PLA2R antigen and the THSD7A antigen are prepared, they need to be stored in an antigen preservation solution. At the initial stage of the research, the same preservation solution (directly using the first antigen dialysis solution as the preservation solution for both antigens) was used for the PLA2R antigen and the THSD7A antigen, and the following three groups of situations were investigated respectively:

[0173] The first group: Without using an antigen preservation solution, directly replaced with water;

[0174] The second group: Using Tris buffer solution as the antigen preservation solution (pH 8.0);

[0175] The third group: Using: tris(hydroxymethyl)aminomethane buffer solution, bovine serum albumin, mannitol, L-histidine, sodium chloride, disodium ethylenediaminetetraacetate dihydrate, dithiothreitol, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, PH = 7.5;

[0176] The antigen was prepared and placed at 2-8°C for 0 days, 1 day, 10 days, 30 days, 60 days, and 90 days respectively. The kit was prepared according to the method provided in Example 1, and the activity of PLA2R antigen was detected using PLA2R calibrator (20 RU / mL); the activity of THSD7A antigen was detected using THSD7A calibrator (10 ng / mL). The detection results are shown in Table 12.

[0177] Table 12. Stability study of PLA2R antigen and THSD7A antigen

[0178]

[0179] It can be seen from Table 12 that the stabilities of PLA2R antigen and THSD7A antigen in the first group / second group are both poor. After being placed for 1 day, the activity decreased significantly. The main reason is the oxidation of the side chains of protein molecules in the solution. After adding the antigen preservation solution, the reducing agent in it can significantly improve its stability.

[0180] 2. Influence of antigen preservation solution on the stability of PLA2R antigen solution coupled with microspheres and THSD7A antigen solution coupled with microspheres

[0181] Due to the stability problems of PLA2R antigen and THSD7A antigen, in this example, an attempt was also made to prepare the PLA2R antigen solution coupled with microspheres and the THSD7A antigen solution coupled with microspheres immediately on the day of obtaining the PLA2R antigen and THSD7A antigen, to investigate whether there are still stability problems after coupling with microspheres, and the influence of adding the antigen preservation solution on the prepared antigen solution coupled with microspheres.

[0182] The following two groups of situations were investigated respectively:

[0183] The first group: Without using the antigen preservation solution, the antigen was immediately used to prepare the antigen solution coupled with microspheres on the day of antigen preparation;

[0184] The second group: After adding the antigen preservation solution (the third group in this example) to the prepared antigen, it was immediately used to prepare the antigen solution coupled with microspheres on the day;

[0185] The antigen solutions coupled with microspheres prepared were respectively placed at 2-8°C for 0 days, 1 day, 5 days, 10 days, 60 days, and 90 days, and the fluorescence signal values (MFI) of PLA2R and THSD7A were detected. The detection results are shown in Table 13.

[0186] Table 13. Stability study of PLA2R antigen coupled with microspheres and THSD7A antigen coupled with microspheres

[0187]

[0188] As can be seen from Table 13, after the PLA2R antigen and the THSD7A antigen are conjugated to microspheres, there will still be stability problems and it is difficult to store them stably, which will seriously affect the shelf life of the kit. Therefore, an antigen preservation solution must be added immediately after the preparation of the PLA2R antigen and the THSD7A antigen to improve the stability of the PLA2R antigen and the THSD7A antigen, thereby also improving the stability of the antigen solution of the conjugated microspheres in the kit and significantly extending the shelf life of the kit.

[0189] Example 5: Screening of the first antigen preservation solution and the second antigen preservation solution

[0190] This example proves through research that the PLA2R antigen and the THSD7A antigen need to be stored using different antigen preservation solutions respectively, which can more effectively improve the stability of the PLA2R antigen and the THSD7A antigen, thereby extending the shelf life of the kit.

[0191] 1. Screening of the first antigen preservation solution

[0192] In this example, a kit for detecting human membranous nephropathy-related indicators was prepared according to the method provided in Example 1. For the PLA2R antigen, the prepared PLA2R antigen was stored using 6 different first antigen preservation solutions shown in Table 14-1 and the first antigen preservation solution used in Example 1 respectively, and the pH was adjusted to 7.5 using NaOH (1M) and concentrated hydrochloric acid. The THSD7A antigen was stored according to the second antigen preservation solution provided in Example 1. The others were prepared into an antigen solution conjugated to microspheres according to the method provided in Example 1, and corresponding kits for detecting human membranous nephropathy-related indicators were prepared. They were placed at 2-8°C for 0 days, 30 days, 90 days, 180 days, and 360 days respectively to examine the activity of the PLA2R antigen conjugated to the microspheres, and it was also used for the detection of PLA2R and THSD7A. The test samples were PLA2R-positive serum (detected by mass spectrometry, with a content of 23.35 RU / mL) and THSD7A-positive serum (detected by mass spectrometry, with a content of 11.76 ng / mL). Detection was carried out according to the method provided in Example 1 to examine the influence of different first antigen preservation solutions on the detection results of PLA2R and THSD7A, and 10 repeated detections were carried out and the average value was taken. The results are shown in Table 14.

[0193] Table 14-1. Composition of different first antigen preservation solutions (the proportional relationship is according to Example 1)

[0194]

[0195] Table 14-2. Influence of different first antigen preservation solutions on the detection results

[0196]

[0197] As can be seen from Table 14-2, the formulation of the antigen preservation solution also needs to be further optimized to achieve better detection sensitivity. Moreover, using different first antigen preservation solutions will not only affect the stability of the PLA2R antigen and the detection results of PLA2R, but also affect the detection results of THSD7A. The reason may be that during the detection process, the antigen solutions of the two coupled microspheres need to be mixed together, so there is an interaction. The most preferred formulation of the first antigen preservation solution is: Tris(hydroxymethyl)aminomethane + Bovine Serum Albumin + Mannitol + L-Histidine + Sodium Chloride + Ethylenediaminetetraacetic Acid Disodium Salt Dihydrate + Dithiothreitol + 4-(2-Aminoethyl)benzenesulfonyl Fluoride Hydrochloride + Glycerol + ProClin300, with the pH adjusted to 7.5.

[0198] 2. Screening of the second antigen preservation solution

[0199] In this example, a kit for detecting human membranous nephropathy-related indicators was prepared according to the method provided in Example 1. For the THSD7A antigen, 6 different second antigen preservation solutions shown in Table 15-1 and the second antigen preservation solution used in Example 1 were respectively used to preserve the prepared THSD7A antigen, and NaOH (1M) and concentrated hydrochloric acid were used to adjust the pH to 8.0. The PLA2R antigen was preserved according to the first antigen preservation solution provided in Example 1, and then prepared into a microsphere-coupled antigen solution according to the method provided in Example 1, and a corresponding kit for detecting human membranous nephropathy-related indicators was prepared. It was placed at 2-8°C for 0 days, 30 days, 90 days, 180 days, and 360 days respectively to investigate the activity of the THSD7A antigen coupled to the microspheres, and it was also used for the detection of PLA2R and THSD7A. The test samples were PLA2R-positive serum (detected by mass spectrometry, with a content of 23.35 RU / mL) and THSD7A-positive serum (detected by mass spectrometry, with a content of 11.76 ng / mL). Detection was carried out according to the method provided in Example 1 to investigate the influence of different second antigen preservation solutions on the detection results of PLA2R and THSD7A, and 10 repeated detections were carried out, and the average value was taken. The results are shown in Table 15.

[0200] Table 15-1. Composition of different second antigen preservation solutions (the proportional relationship is according to Example 1)

[0201]

[0202] Table 15-2. Influence of different second antigen preservation solutions on the detection results

[0203]

[0204] As can be seen from Table 15, the formulation of the antigen preservation solution also needs to be further optimized to achieve better detection sensitivity. Moreover, using different second antigen preservation solutions will not only affect the stability of the THSD7A antigen and the detection results of THSD7A, but also affect the detection results of PLA2R. Therefore, the most preferred formulation of the second antigen preservation solution is: sodium citrate dihydrate + bovine serum albumin + mannitol + L-glycine + sodium chloride + disodium ethylenediaminetetraacetate dihydrate + dithiothreitol + 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride + glycerol + ProClin300, adjusting the pH to 8.0.

[0205] Example 6: Comparison with existing kits

[0206] The kit provided in Example 1 was used respectively, and compared with the existing commercially available detection kits related to human membranous nephropathy. The test sample was positive serum (the concentration of PLA2R was 41.05 RU / mL and the concentration of THSD7A was 27.01 ng / mL detected by mass spectrometry), and the detection was carried out simultaneously. The detection results are shown in Table 16.

[0207] Table 16. Comparison with existing kits

[0208]

[0209]

[0210] As can be seen from Table 16, the detection kit related to human membranous nephropathy provided by the present invention can significantly improve the detection accuracy and sensitivity of PLA2R and THSD7A, and at the same time can eliminate the batch-to-batch difference, and the detection results are more stable and reliable.

[0211] Although the present invention is disclosed as above, the present invention is not limited thereto. Any person skilled in the art can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be subject to the scope defined by the claims.

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

Claims

1. A human membranous nephropathy-related index detection kit, characterized in that: It comprises antigen proteins, wherein the antigen proteins comprise PLA2R antigen and THSD7A antigen, the amino acid sequence of the PLA2R antigen is shown in SEQ ID NO:1, and the amino acid sequence of the THSD7A antigen is shown in SEQ ID NO:2; and the human membranous nephropathy-related indicators comprise PLA2R antibodies and THSD7A antibodies.

2. The human membranous nephropathy-related index detection kit according to claim 1, characterized in that: The encoding nucleotide sequence of the PLA2R antigen is shown in SEQ ID NO:3, and the encoding nucleotide sequence of the THSD7A antigen is shown in SEQ ID NO:

4.

3. The human membranous nephropathy-related index detection kit according to claim 2, characterized in that: It also includes a detection antibody, which includes an anti-human IgG antibody complex, and the anti-human IgG antibody complex is an anti-human IgG antibody labeled with phycoerythrin.

4. The human membranous nephropathy-related index detection kit according to claim 3, characterized in that: The PLA2R antigen and THSD7A antigen are coupled to microspheres respectively to prepare PLA2R antigen solution coupled to microspheres and THSD7A antigen solution coupled to microspheres; the detection antibody solution is a phycoerythrin-labeled anti-human IgG antibody solution; and the kit also includes a sample diluent.

5. A method for preparing a kit for detecting human membranous nephropathy-related indicators, characterized in that: The following steps are involved: Step (1), preparing PLA2R antigen and THSD7A antigen respectively; the encoding nucleotide sequence of the PLA2R antigen is shown in SEQ ID NO:3, and the encoding nucleotide sequence of the THSD7A antigen is shown in SEQ ID NO:4; the amino acid sequence of the PLA2R antigen is shown in SEQ ID NO:1; the amino acid sequence of the THSD7A antigen is shown in SEQ ID NO:2; Step (2), dialyzing the PLA2R antigen with a first antigen dialysate to obtain a PLA2R antigen solution; dialyzing the THSD7A antigen with a second antigen dialysate to obtain a THSD7A antigen solution; Step (3), using the PLA2R antigen solution containing the first antigen preservation solution to prepare the PLA2R antigen solution coupled to the microspheres; The THSD7A antigen solution coupled to the microspheres was prepared using the THSD7A antigen solution containing the second antigen preservative solution.

6. The preparation method according to claim 5, characterized in that: The components of the first antigen dialysis solution include tris(hydroxymethyl)aminomethane buffer, bovine serum albumin, mannitol, L-histidine, sodium chloride, ethylenediaminetetraacetic acid disodium salt dihydrate, dithiothreitol, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, and the pH value is 7.5; the components of the first antigen preservation solution include tris(hydroxymethyl)aminomethane buffer, bovine serum albumin, mannitol, L-histidine, sodium chloride, ethylenediaminetetraacetic acid disodium salt dihydrate, dithiothreitol, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, glycerol, and ProClin300, and the pH value is 7.

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7. The preparation method according to claim 6, characterized in that: The components of the second antigen dialysis solution include sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediaminetetraacetic acid disodium salt dihydrate, dithiothreitol, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, pH=8.0; the components of the second antigen preservation solution include sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediaminetetraacetic acid disodium salt dihydrate, dithiothreitol, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, glycerol, ProClin300, pH=8.

0.

8. The preparation method according to claim 7, characterized in that: The step (1) of preparing the PLA2R antigen and the THSD7A antigen separately comprises constructing a recombinant vector containing a nucleotide sequence encoding the PLA2R antigen and a recombinant vector containing a nucleotide sequence encoding the THSD7A antigen, respectively, and expressing them through a eukaryotic expression system to prepare recombinant PLA2R antigen and recombinant THSD7A antigen.

9. A method for detecting human membranous nephropathy-related indicators, characterized in that: The method is used for non-disease diagnosis purposes, and the detection is performed using the kit as claimed in claim 4, comprising the following steps: Step (a), diluting the sample with a sample diluent, the dilution factor being 50 times; Step (b), mixing the diluted sample, the PLA2R antigen solution coupled to the microspheres, and the THSD7A antigen solution coupled to the microspheres, and incubating in the dark to obtain a first complex; Step (c), adding a detection antibody solution to the first complex, incubating in the dark, to obtain a complex to be detected; Step (d), detecting the complex to be detected using a flow cytometer.

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