CN118068012A

CN118068012A - Human membranous nephropathy related index detection kit, method and application thereof

Info

Publication number: CN118068012A
Application number: CN202410151867.0A
Authority: CN
Inventors: 陈善问; 周延庆; 沈承昊; 章旭日
Original assignee: Hangzhou Saiji Biotechnology Co ltd
Current assignee: Hangzhou Saiji Biotechnology Co ltd
Priority date: 2023-02-15
Filing date: 2024-02-02
Publication date: 2024-05-24
Also published as: CN116482375A

Abstract

The invention provides a detection kit for related indexes of human membranous nephropathy and a method and application thereof, wherein the detection kit is used for carrying out mutation screening for a plurality of times aiming at coding nucleotide sequences of PLA2R antigen and THSD7A antigen to obtain the most preferable PLA2R antigen and THSD7A antigen sequences, optimizing a dialysate formula in the preparation and purification processes of the PLA2R antigen and the THSD7A antigen and a preservation liquid formula of the PLA2R antigen and the THSD7A antigen, preparing the PLA2R antigen and the THSD7A antigen with high purity, solving the problems of instability and inter-batch difference of the PLA2R antigen and the THSD7A antigen to the greatest extent, obviously improving the detection accuracy and sensitivity, controlling the inter-batch difference, and realizing rapid, accurate, simple and efficient detection of related indexes of human membranous nephropathy.

Description

Human membranous nephropathy related index detection kit, method and application thereof

The application claims a prior application in China, application number: 202310117302.6, priority of day 2023, month 2 and day 15; all of which are included as part of the present application.

Technical Field

The invention relates to the technical field of flow cytometry, in particular to a detection kit for related indexes of human membranous nephropathy, a method and application thereof.

Background

In the existing method for detecting autoimmune diseases, an enzyme-linked immunosorbent method, an immunoblotting method and an indirect immunofluorescence method are the mainstream and commonly used methods, wherein the enzyme-linked immunosorbent method is used for preparing solid-phase antigens by coating purified antigens on a micro-pore plate, sequentially adding a standard substance with known concentration and a sample to be detected with unknown concentration into the micro-pore coated with the antigens, incubating, adding a biotin-labeled anti-IgG antibody, combining with horseradish peroxidase HRP-labeled streptavidin to form an immune complex, and adding a substrate 3,3', 5' -tetramethyl benzidine (TMB) for color development after thorough washing. TMB is converted to blue under the catalysis of HRP enzyme and to final yellow under the action of acid. The shade of color positively correlated with the Anti-phospholipase A2 antibody (Anti-PLA 2R) in the sample. The absorbance (OD value) was measured at a wavelength of 450nm using a microplate reader and the concentration of human antiphospholipid A2 antibody in the sample was calculated by a standard curve. The ELISA method has the advantages of complicated operation steps, poor result repeatability and long detection time.

Membranous nephropathy (membranous nephropathy, MN), membranous glomerulonephritis, in particular nephrotic syndrome caused by excessive deposition of immune complexes in glomerular capillaries, is one of the most common pathological types in adult nephrotic syndrome. Clinically MN is mainly manifested by massive proteinuria, hypoproteinemia, edema and hyperlipidemia. Depending on the etiology, MN can be classified into idiopathic membranous nephropathy (idiopathic membranousnephropathy, IMN) and secondary membranous nephropathy (secondary membranous nephropathy, SMN), wherein IMN is the main type of MN, accounting for about 70% -80%, whose etiology is not yet clear, also known as primary membranous nephropathy (primarymembranous nephropathy, PMN). Most scholars consider PMN as an autoimmune glomerular disease mediated by antibodies, and target antigens located on podocytes are recognized by autoantibodies and combined with the autoantibodies to form immune complexes deposited under basal membrane podocytes, and activation of the complement system causes podocyte injury and shedding, resulting in increased basal membrane permeability and thus occurrence of a large amount of proteinuria.

P Phospholipase A2 receptor (PLA 2R) is divided into N type and M type, and human M type PLA2R is mainly expressed in kidney tissue, belongs to C type lectin family, is type I transmembrane protein, and can be involved in inflammation and acute kidney injury pathogenic process under pathological state. In 2009 Beck et al found PLA2R deposition in the kidneys of patients with idiopathic membranous nephropathy (idiopathic membranous nephropathy, IMN), immunofluorescence was seen to be predominantly co-deposited with IgG4 subtype antibodies, accompanied by C3-like deposition along glomerular basement membrane particles. Antibodies to the target antigen in serum are also of the IgG4 subtype, with a positive rate of up to 70%. PLA2R is used as a main target antigen of IMN and can be used as an index for identifying idiopathic membranous nephropathy and other glomerular diseases.

The thrombospondin type 1 7A domain (Thrombospondin type-1domain-containing 7A, THSD 7A) has the same biochemical characteristics as PLA2R and is expressed on the podocyte surface of humans and rodents. 2.5% -5% of IMN patients can detect anti-THSD 7A antibodies, and the specific antibodies are mainly of IgG4 subtype, and only bind with the antibodies under non-reducing condition, and form immune complexes after antigen-antibody binding, and deposit under glomerular epithelium, and further activate complement system to cause podocyte injury or apoptosis, thus forming proteinuria.

PLA2R and THSD7A have been demonstrated to be the major two pathogenic target antigens for IMN, and antibodies in serum are both predominantly of the IgG4 subtype. Studies have shown that the detection rate of anti-PLA 2R antibodies in patient serum is about 70%. In anti-THSD 7A antibody assays for patients negative for IMN screening for anti-PLA 2R antibodies, anti-THSD 7A antibodies were found to be present in 2.5% -5% of the patient serum. Although anti-THSD 7A antibodies have a low positive rate in IMN patients, they are extremely specific. Therefore, the combined detection of the two can improve the diagnosis efficiency of idiopathic membranous nephropathy.

The PLA2R antigen and the THSD7A antigen need to be used for detecting the PLA2R antigen and the THSD7A antigen, but the preparation process of the PLA2R antigen and the THSD7A antigen is complex, the PLA2R antigen and the THSD7A antigen with high purity are difficult to prepare, the PLA2R antigen and the THSD7A antigen are unstable, the activity is easy to be reduced in the storage process, and serious inter-batch difference problems exist, so that the detection accuracy of the PLA2R and the THSD7A is lower. The existing membranous nephropathy detection technology is generally used for independently detecting PLA2R and THSD7A, and has the defects of long detection time, complex operation, poor detection sensitivity and poor accuracy. The ELISA, immunoblotting and indirect immunofluorescence methods have long detection time, generally take 2-3 hours from the beginning of detection to the report, and cannot detect a single sample at any time, so that the sensitivity is insufficient, and even only qualitative and quantitative detection is possible.

Therefore, it is necessary to provide a novel detection kit for related indexes of human membranous nephropathy, and a preparation method and a use method thereof, so as to solve the above problems in the prior art.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides a detection kit for related indexes of human membranous nephropathy, a method and application thereof, and aims at carrying out multiple mutation screening on coding nucleotide sequences of PLA2R antigen and THSD7A antigen to obtain the most preferable PLA2R antigen and THSD7A antigen sequences, optimizing a dialysate formula in the preparation and purification processes of the PLA2R antigen and the THSD7A antigen and a preservation liquid formula of the PLA2R antigen and the THSD7A antigen, preparing the PLA2R antigen and the THSD7A antigen with high purity, solving the problems of instability and inter-batch difference of the PLA2R antigen and the THSD7A antigen to the greatest extent, and the prepared kit can obviously improve the detection accuracy and sensitivity, control inter-batch difference and realize rapid, accurate, simple and efficient detection of related indexes of human membranous nephropathy.

In one aspect, the invention provides a detection kit for related indexes of human membranous nephropathy, which comprises an antigen protein, wherein the antigen protein comprises a PLA2R antigen and a THSD7A antigen, the amino acid sequence of the PLA2R antigen is shown as SEQ ID NO.1, and the amino acid sequence of the THSD7A antigen is shown as SEQ ID NO. 2; the related indicators of human membranous nephropathy include PLA2R antibodies and THSD7A antibodies.

The existing recombinant proteins for PLA2R and/or THSD7A detection have serious inter-batch difference problems, and meanwhile, due to the instability of PLA2R antigen and THSD7A antigen, the activity of the antigen is obviously reduced after the recombinant proteins are stored for a period of time, and the medical diagnosis result is further influenced. According to the invention, through carrying out multiple mutation screening on the coding nucleotide sequences of the PLA2R antigen and the THSD7A antigen, inserting the mutated nucleic acid molecules into eukaryotic expression vectors to respectively prepare and recombine the PLA2R antigen and the THSD7A antigen, screening the optimal amino acid sequences of the signal peptides of the PLA2R antigen and the THSD7A antigen, the cross influence caused by non-specific reaction can be obviously improved, the inter-batch difference is controlled, and the accuracy and the sensitivity of the detection result are improved.

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

Further, detection antibodies are also included, including anti-human IgG antibody complexes, which are phycoerythrin-labeled anti-human IgG antibodies.

Further, the PLA2R antigen and the THSD7A antigen are respectively coupled with the microsphere to prepare a PLA2R antigen solution of the coupled microsphere and a THSD7A antigen solution of the coupled microsphere; the fluorescent-labeled IgG antibody solution is phycoerythrin-labeled anti-human IgG antibody solution; the kit further comprises a sample diluent.

In another aspect, the invention provides a method for preparing a detection kit for related indexes of human membranous nephropathy, which comprises the following steps:

Step (1), preparing PLA2R antigen and THSD7A antigen respectively; the coding nucleotide sequence of the PLA2R antigen is shown as SEQ ID NO.3, and the coding nucleotide sequence of the THSD7A antigen is shown as SEQ ID NO. 4;

step (2), dialyzing the PLA2R antigen by using a first antigen dialysate, and preserving the PLA2R antigen by using a first antigen preserving fluid to prepare a PLA2R antigen solution containing the first antigen preserving fluid; dialyzing the THSD7A antigen with a second antigen dialysate, and preserving the THSD7A antigen with a second antigen preserving solution to obtain a THSD7A antigen solution containing the second antigen preserving solution;

Step (3), preparing PLA2R antigen solution of the coupled microsphere by adopting PLA2R antigen solution containing the first antigen preservation solution; the THSD7A antigen solution of the coupled microspheres was prepared using a THSD7A antigen solution containing a second antigen preservation solution.

Further, the first antigen dialysate comprises tris buffer, bovine serum albumin, mannitol, L-histidine, sodium chloride, disodium edetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride; the first antigen preservation solution comprises a tris buffer solution, bovine serum albumin, mannitol, L-histidine, sodium chloride, ethylenediamine tetraacetic acid disodium salt dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol and ProClin300,300.

Further, the second antigen dialysate comprises sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediamine tetraacetic acid disodium salt dihydrate, dithiothreitol and 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride; the second antigen preservation solution comprises sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediamine tetraacetic acid disodium salt dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol and ProClin300,300.

Through optimizing the antigen dialysate and the antigen preservation solution of the PLA2R antigen and the THSD7A antigen, the problem of poor stability of the PLA2R antigen and the THSD7A antigen can be better solved, so that the PLA2R antigen and the THSD7A antigen, and the PLA2R antigen solution of the coupling microsphere and the THSD7A antigen solution of the coupling microsphere in the kit can be stably preserved for a long time, and the detection accuracy and the detection sensitivity can be improved.

Further, step (1) separately prepares PLA2R antigen and THSD7A antigen: recombinant vectors containing nucleotide sequences for encoding PLA2R antigen and THSD7A antigen are respectively constructed, and the recombinant vectors containing nucleotide sequences for encoding THSD7A antigen are expressed by a eukaryotic expression system to prepare recombinant PLA2R antigen and recombinant THSD7A antigen.

In yet another aspect, the present invention provides a method for detecting an index associated with human membranous nephropathy, for non-disease diagnostic use, using a kit as described above, comprising the steps of:

diluting the sample with a sample diluent, wherein the dilution factor is 50 times;

step (b), mixing the diluted sample, the PLA2R antigen solution of the coupled microsphere and the THSD7A antigen solution of the coupled microsphere, and incubating in a dark place to obtain a first compound;

step (c), adding a detection antibody solution into the first complex, and incubating in a dark place to obtain a complex to be detected;

And (d) detecting the complex to be detected by using a flow cytometer.

Research proves that better detection accuracy and sensitivity can be realized only by detecting a sample diluted by a proper multiple by the diluent, and the detection accuracy and sensitivity can be improved under the optimal reaction proportion of the antigen and the antibody.

In yet another aspect, the invention provides the use of a set of antigen-preserving fluids comprising a first antigen-preserving fluid for preserving a PLA2R antigen solution of coupled microspheres and a second antigen-preserving fluid for preserving a THSD7A antigen solution of coupled microspheres for the preparation of a reagent for increasing the sensitivity of detection of a human membranous kidney disease-related indicator comprising a PLA2R antibody and a THSD7A antibody.

In yet another aspect, the invention provides the use of a set of antigen dialysate for the preparation of a reagent for increasing the sensitivity of detection of indicators related to human membranous nephropathy, including PLA2R antibodies and THSD7A antibodies, comprising a first antigen dialysate for dialysis preparation of PLA2R antigen and a second antigen dialysate for dialysis preparation of THSD7A antigen.

The invention provides a kit for detecting related indexes of human membranous nephropathy based on a flow cytometer, a method and application thereof, and has the following advantages of

The beneficial effects are that:

1. The simultaneous and accurate detection of PLA2R and THSD7A can be realized based on a flow cytometer, and the detection lower limit of the PLA2R reaches 4RU/mL, and the blank limit is not higher than 0.4RU/mL; the detection lower limit of THSD7A reaches 3ng/mL, and the blank limit is not higher than 0.6ng/mL;

2. Screening and optimizing nucleotide coding sequences of PLA2R antigen and THSD7A antigen, preparing recombinant antigen by eukaryotic cell expression system expression, remarkably improving cross influence caused by non-specific reaction, controlling inter-batch difference, and improving accuracy and sensitivity of detection result;

3. The PLA2R antigen and THSD7A antigen dialysate formulation and antigen preservation solution formulation are optimized, the problem of poor stability of the PLA2R antigen and THSD7A antigen is solved, and the PLA2R antigen and THSD7A antigen, and the PLA2R antigen solution of the coupled microsphere and the THSD7A antigen solution of the coupled microsphere in the kit can be stably preserved for a long period of time, can be stably preserved for more than 1 year, and are favorable for improving detection accuracy and sensitivity.

Drawings

FIG. 1 is a schematic diagram of the detection principle of the detection kit for human membranous nephropathy related indicators in example 1;

FIG. 2 shows the distribution of captured fluorescent microspheres for sample detection in example 1;

FIG. 3 is a calibration curve for flow-through fluorescence detection of anti-PLA 2R antibodies in example 1;

FIG. 4 is a calibration curve for flow fluorometric detection of anti-THSD 7A antibodies in example 1;

FIG. 5 is a graph showing the PLA2R linear evaluation result in example 1;

Fig. 6 is a THSD7A linear evaluation result in example 1;

FIG. 7 is a graph of the comparison of healthy samples and PLA2R positive samples in example 1;

Fig. 8 is a graph of the comparison of healthy samples and THSD7A positive samples in example 1.

Detailed Description

In order to more particularly describe the present invention, the following detailed description of the technical scheme of the present invention is provided with reference to the accompanying drawings and the specific embodiments. These descriptions are merely illustrative of how the present invention may be implemented and are not intended to limit the specific scope of the invention. The scope of the invention is defined in the claims.

Example 1: preparation, use and effect evaluation of kit

1. Preparation of the kit

The specific composition is shown in table 1:

TABLE 1 composition of kit

The preparation method comprises the following steps:

1. preparation of PLA2R antigen and THSD7A antigen

The recombinant protein N-terminal of PLA2R antigen and THSD7A antigen contains high-efficiency signal peptide sequence and His-tag purification tag composed of 10 histidines, and the pcDNA3.4 vector and an Expi293F cell expression system are used for transient transfection expression.

Transient transfection expression: the day of transfection was adjusted to 2.5X10 ⁶/mL of the Expi293F cell density in the logarithmic growth phase using the medium Expi293 ^TM expression medium (Sesameifer technologies (China), A1435101). 100mL of the cells were transfected with 100. Mu.g of the recombinant plasmid described above, and 270. Mu.L of transfection reagent ExpiFectamine ^TM 293 (Semer Feishi technologies (China), A14524) was added. After 4-5 days of incubation at 37℃in 8% carbon dioxide, the medium was collected by centrifugation at 3000G for 20 min and filtered through a 0.22. Mu.M filter.

Purifying:

Purification using Ni-NTA affinity chromatography medium (Kirschner, cat# L00250), use of equilibration solution (300mM NaCl,50mM NaH2PO4,10mM imidazole, pH 8.0), and eluent (300mM NaCl,50mM NaH2PO4,250mM imidazole, pH 8.0). Desalting was then performed in buffer A (20 mM Tris-HCl, pH 8.0) using HITRAP DESALTING column (Silo Tuo Co., ltd., cat. No. 29048684). Next, hiTrap Capto Q (Silo Tuo Co., ltd., cat. No. 11001303) anion exchange chromatography column was used, equilibrated with buffer A and eluted with a gradient of buffer B (1M NaCl,20mM Tris-HCl, pH 8.0). Finally, the column was equilibrated with buffer C (150mM NaCl,10mM NaH2PO4,pH 7.4) using a Superdex 200Incree10/300 GL (Silo Tuo Co., ltd., cat# 28990944) gel filtration chromatography.

1.1 Preparation of PLA2R antigen

The gene sequence SEQ ID NO. 3 of the coding recombinant protein PLA2R is synthesized and cloned to a vector pcDNA3.4. Recombinant protein PLA2R was expressed and purified using the methods described above, dialyzed in the first dialysate for 12-24 hours, and then either stored in the first preservative solution for a long period (-80 ℃) or the microspheres were immediately coupled.

Preparing a first dialysis solution: 6.057g of tris, 1g of bovine serum albumin, 10mL of mannitol, 0.776g of L-histidine, 5.844g of sodium chloride, 7.4448g of disodium ethylenediamine tetraacetate dihydrate, 0.309g of dithiothreitol, 0.24g of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride were dissolved in 900mL of water in this order, the volume was set to 1000mL, and the pH was adjusted to 7.5 using NaOH (1M) and concentrated hydrochloric acid. Filtering with 0.22 μm filter membrane, and storing at 2-8deg.C.

Preparing a first preservation solution: 6.057g of tris, 1g of bovine serum albumin, 10mL of mannitol, 0.776g of L-histidine, 5.844g of sodium chloride, 7.4448g of disodium edetate dihydrate, 0.309g of dithiothreitol, 0.24g of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 100mL of glycerol, 1mL of ProClin300 were dissolved in 800mL of water in this order, the volume was set to 1000mL, and the pH was adjusted to 7.5 using NaOH (1M) and concentrated hydrochloric acid. Filtering with 0.22 μm filter membrane, and storing at 2-8deg.C.

1.2 Preparation of THSD7A antigen

The gene sequence SEQ ID NO.4 of the recombinant protein THSD7A is synthesized and cloned to a vector pcDNA3.4. Recombinant protein THSD7A was expressed and purified using the methods described previously, dialyzed in a second dialysate for 12-24h, and then either stored for a long period (-80 ℃) in the second preservation, or the microspheres were immediately coupled.

Preparing a second dialysate: 5.882g of sodium citrate dihydrate, 1g of bovine serum albumin, 10mL of mannitol, 0.751g L-glycine, 17.533g of sodium chloride, 3.722g of disodium ethylenediamine tetraacetate dihydrate, 0.309g of dithiothreitol, 0.24g of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride were dissolved in 900mL of water in sequence, to a volume of 1000mL, and pH was adjusted to 8.0 using NaOH (1M) and concentrated hydrochloric acid. Filtering with 0.22 μm filter membrane, and storing at 2-8deg.C.

Preparing a second preservation solution: 5.882g of sodium citrate dihydrate, 1g of bovine serum albumin, 10mL of mannitol, 0.751g L-glycine, 17.533g of sodium chloride, 3.722g of disodium edetate dihydrate, 0.309g of dithiothreitol, 0.24g of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 100mL of glycerol, 1mL of ProClin300 were dissolved in 800mL of water in this order, the volume was set to 1000mL, and the pH was adjusted to 8.0 using NaOH (1M) and concentrated hydrochloric acid. Filtering with 0.22 μm filter membrane, and storing at 2-8deg.C.

2. Preparing antigen solution of coupled microsphere

Taking 0.1mL of fluorescent microspheres (manufacturer Biolegend, model 740168) with the concentration of 5 multiplied by 10 ⁶/mL, adding PBST buffer solution for cleaning for 2 times, adding 100 mug of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and 50 mug of N-hydroxy-thiosuccinimide (NHS) into the cleaned first fluorescent microspheres, standing for 30 minutes to activate the microspheres, adding 10 mug of PLA2R antigen solution, performing rotary reaction at room temperature for 5 hours, cleaning the first fluorescent microspheres to remove redundant antigen, adding 5% of skim milk powder by mass fraction, sealing for 30 minutes, adding 500 mug of Tris buffer solution with the pH of 7.2 after removing the skim milk powder, and storing to prepare 0.5mL of PLA2R antigen solution of the coupled microspheres;

according to the same method, 0.5mL of THSD7A antigen solution of the coupled microsphere is prepared by adopting fluorescent microsphere (manufacturer Biolegend, model 740170);

Taking 1 mu L of PLA2R antigen solution of each coupling microsphere and 1 mu L of THSD7A antigen solution of each coupling microsphere to prepare the antigen solution of the coupling microsphere.

3. Preparation of detection antibody solution

The anti-human IgG antibody is coupled with phycoerythrin to obtain a detection antibody solution, wherein the mass ratio of the anti-human IgG antibody to the phycoerythrin is 1:1, and the anti-human IgG antibody is purchased from Madean Biotechnology Co., ltd., product number Z01236M.

4. Preparation of sample dilutions

3.0275G of Tris (hydroxymethyl) aminomethane (Tris) and 9.0g of sodium chloride (NaCl) are dissolved in 800mL of pure water, 1mL of ProClin is added, the pH is regulated to 6.8, 20g of Bovine Serum Albumin (BSA) is added after complete dissolution, the volume is fixed to 1000mL after complete dissolution, and the pH is regulated to 6.8 for standby.

5. Preparation of washing buffer (1×)

2.4G of potassium dihydrogen phosphate (KH ₂ PO 4), 36.32g of disodium hydrogen phosphate dodecahydrate (Na ₂HPO₄.12H2O), 8g of NaCl and 2g of potassium chloride (KCl) were dissolved in 1000mL of pure water, and 25g of BSA, a ProClin preservative with a mass fraction of 1% and Tween 20 (Tween-20) with a mass fraction of 0.5% were added to obtain a washing buffer (10X) for use. Alternatively, the wash buffer (10×) was stabilized to room temperature and 10mL of wash buffer (10×) was added to 90mL of purified water to obtain the wash buffer (1×) after all salts were dissolved.

6. Calibration material

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

7. Quality control product

The quality control product consists of PLA2R antibody and THSD7A antibody solution matrix solution.

2. Method for using kit

The kit prepared by the embodiment is used as follows:

(a) Diluting 5 mu L of a sample to be tested with a sample diluent, and controlling the volume ratio of the sample to be tested to the sample diluent to be 1:51;

(b) Respectively adding 20 mu L of the antigen solution of the coupling microsphere and 20 mu L of the diluted sample to be tested into a sample tube, and incubating the obtained first compound for 0.5h at room temperature in a dark place; the first complex after incubation in the dark was resuspended in 1000 μl of wash buffer (1×), and then 400g centrifuged for 5min to leave a pellet;

(c) Adding 20 mu L of the detection antibody solution into the obtained precipitate, uniformly mixing the obtained complex to be detected, and incubating for 0.5h at room temperature in a dark place; the complex to be detected obtained after incubation in the absence of light is resuspended in 1000. Mu.L of wash buffer (1X), and then 400g is centrifuged for 5min, leaving the precipitate;

(d) The resulting pellet was resuspended in 100. Mu.L of wash buffer (1X) and the fluorescence type and fluorescence signal intensity of the resuspended mixture was measured on a DxFLEX flow cytometer from Beckmann, inc., the specific detection procedure being a routine technical means for those skilled in the art and not described in detail herein.

The kit has small required sample size and needs 5-20 mu L of serum.

Fig. 1 is a schematic diagram of a detection principle of a detection kit for related indexes of human membranous nephropathy, wherein 1 is a microsphere, 2 is an antigen, 3 is an antibody to be detected, 4 is a detection antibody, 5 is phycoerythrin, antigen 2 is combined with polystyrene microsphere 1 with carboxyl to form a first complex, detection antibody 4 and phycoerythrin 5 are combined to form a second complex, the first complex, antibody 3 to be detected and the second complex are combined to form an immune complex, two beams of excitation light with different wavelengths emitted by a flow cytometer irradiate the immune complex, the types of detection indexes are determined by the fluorescence intensities of different polystyrene fluorescent coding microspheres, and the content of each detection index is determined by the fluorescence intensity of phycoerythrin.

FIG. 2 shows the distribution of captured fluorescent microspheres for sample detection. Referring to fig. 2, the forward angle scattered light (forwardscatter, FSC) channel fluorescence signal values are on the ordinate and the Allophycocyanin (APC) channel fluorescence signal values are on the abscissa. The ordinate is used for determining the size and the position of the microsphere, the abscissa is used for distinguishing the APC fluorescence intensity carried by different microspheres, and 2 kinds of polystyrene microspheres combined with PLA2R antigen and THSD7A antigen can be distinguished, wherein P1 represents the forward angle scattering light channel fluorescence signal value and allophycocyanin channel fluorescence signal value distribution of the polystyrene microsphere coated with PLA2R antigen, and P2 represents the forward angle scattering light channel fluorescence signal value and allophycocyanin channel fluorescence signal value distribution of the polystyrene microsphere coated with THSD7A antigen.

3. Analysis of detection results

1. Calibration curve

And providing a total of 7 calibration curve points of the prepared calibrator A, B, C, D, E, F, G, wherein each calibration curve point has a corresponding concentration value, and drawing a calibration curve of a corresponding index according to the fluorescence measured value and the corresponding concentration value of each point. Fig. 3 is a calibration curve of the flow type fluorescent detection anti-PLA 2R antibody of the present embodiment, and fig. 4 is a calibration curve of the flow type fluorescent detection anti-THSD 7A antibody of the present embodiment.

2. Linear evaluation

And linearly selecting a high-concentration sample at the upper limit of the linear range, diluting the high-concentration sample into at least 7 samples with different concentrations, testing each concentration 3 times, and respectively solving the average value of the concentration of the detection result. And (3) taking the dilution concentration x as an independent variable and taking the detection concentration mean value y as a dependent variable to calculate a linear regression equation. And calculating a correlation coefficient R2 of the linear regression. Fig. 5 shows the PLA2R linear evaluation result of this example, and fig. 6 shows the THSD7A linear evaluation result of this example. Referring to fig. 5 and 6, the play linear regression equation is y=1.0838x+10.107, r2= 0.9964; THSD7A linear regression equation is y=1.0405x+5.1614, r2= 0.9933.

By adopting the human membranous nephropathy related index detection kit, the linear interval of PLA2R can be detected to be not less than [4,1500] RU/mL, the linear interval of THSD7A is not less than [3,200] ng/mL, and the linear correlation coefficient |R2| in the linear interval is not less than 0.990.

3. Blank Limit evaluation

And (3) selecting PLA2R, THSD A blank samples, repeatedly detecting for 20 times, and calculating the average value M and the standard deviation SD value of the measurement result, wherein the blank limit is M+2SD. Table 4 shows the blank results of this example. Referring to table 4, the PLA2R, THSD7A blank limits are respectively: 0.21RU/mL, 0.57ng/mL.

4. Repeatability evaluation

2 Parts of PLA2R, THSD A calibrator with different concentration levels are detected, 10 times of parallel detection are carried out, the coefficient of variation CV is calculated, and the repeatability of the detection kit for the related indexes of the membranous nephropathy can be characterized. Table 2 shows the results of the repeatability evaluation, and referring to table 2, in the in-batch experiment, the variation coefficient CV of the PLA2R, THSD a high-value and low-value calibrators was 7% or less.

TABLE 2 repeatability test results

5. Evaluation of the differences between batches

Three batches of the kit are taken, 10 batches of the kit are repeatedly detected, 1 calibrator is used for detecting the same, the average value M and the standard deviation SD of 30 measurement results are calculated, and the coefficient of variation CV is calculated. Referring to Table 3, it is clear that the CV of the batch-to-batch experiments for the 3 batches of PLA2R, THSD A were within 10%.

TABLE 3 results of batch-to-batch differential assessment

As shown by the repeatability and the inter-batch difference evaluation, the detection kit for the related indexes of the membranous nephropathy has good repeatability and inter-batch difference, the variation coefficient CV of the intra-batch experiment is not more than 7%, and the CV of the inter-batch experiment is not more than 10%.

6. Clinical sample detection

50 Healthy human samples were tested with 39 PLA2R positive samples and 30 THSD7A positive samples using the kit of this example, and compared and analyzed. Fig. 7 is a graph of comparison results of a healthy sample and a PLA2R positive sample, fig. 8 is a graph of comparison results of a healthy sample and a THSD7A positive sample, and with reference to fig. 7 and 8, the fluorescence intensities of the anti-PLA 2R antibody and the anti-THSD 7A antibody measured by using the healthy sample and the positive sample are clearly differentiated, and the contents of the anti-PLA 2R antibody and the anti-THSD 7A antibody measured by the healthy sample and the positive sample are clearly differentiated. The detection kit for the related indexes of the human membranous nephropathy provided by the embodiment can detect the contents of the anti-PLA 2R antibody and the anti-THSD 7A antibody at the same time, and has the advantages of high accuracy, high precision, good specificity, stable detection result and more convenient clinical application.

7. Comparison with Mass Spectrometry detection results

For the same samples, detection was performed using the kit and mass spectrometer provided in this example, respectively, and the mass spectrometer (purchased from shimadzu, model LC-MS/8040) using 0.1% formic acid water and 0.1% acetonitrile formate as mobile phases, using ESI ion source, atomizing gas pressure (GS 1): 45Psi, auxiliary air pressure: 45Psi, air curtain pressure: 35Psi, temperature: 650 ℃, spray voltage: 5000V (positive ion mode), gradient elution, and the sample detected was a calibrator, and the detection results are shown in Table 4.

Table 4, comparison of the results of the kit and Mass Spectrometry provided in this example

Detection index	Mass spectrum detection value	The detection value of the kit
			PLA2R (unit is RU/mL)	20.56	21.11
THSD7A (in ng/mL)	8.13	8.05

Example 2: optimization of PLA2R antigen and THSD7A antigen sequences

1. Optimization of PLA2R antigen sequences

The full length extracellular region sequence of PLA2R antigen (Uniprot number Q13018) consists of 10 domains, as shown in table 5.

Table 5, PLA2R antigen domain.

Structural 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

The selection of partial domains ：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. according to known literature, wherein a large portion of the antibodies in the serum sample recognize domains Ricin B-TYPE LECTIN, C-TYPE LECTIN 1, and a small portion of the antibodies in the serum sample recognize domains Fibronectin type-II、C-type lectin 2、C-type lectin 4、C-type lectin 5、C-type lectin 7、C-type lectin 8., allows the recombinant protein to be recognized by antibodies in nearly all serum samples. The two GS linkers and one alpha helical structure polypeptide (GGGSAEAAAKEAAAKASGGG) are used for connecting domains which are not adjacent in a natural sequence but are adjacent in a recombination sequence for the first time, and the epitope on each domain is fully exposed by a method of increasing the space distance. The N-terminal was added with an optimized signal peptide sequence (SEQ ID NO: 5) and a His-tag purification tag consisting of 10 histidines (affinity chromatography purification). For example, the constructed recombinant protein amino acid sequence (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. the nucleotide sequence of the recombinant protein (SEQ ID NO: 3) was obtained by human codon optimization using the web tool provided by https:// novoprolabs. The recombinant protein was then expressed and purified according to the procedure in example 1.

According to the steps described in the present specification, several recombinant protein preparation kits were used, respectively:

1. 4 domains: the amino acid sequence of the signal peptide sequence +His-tag+Ricin B-type lectin+(GGGSAEAAAKEAAAKASGGG)+C-type lectin 1+(GGGSAEAAAKEAAAKASGGG)+C-type lectin 7+C-type lectin 8( is SEQ ID NO. 1, and the nucleotide sequence is SEQ ID NO. 3);

2. 6 domains: the amino acid sequence of the 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( is SEQ ID NO. 6, and the nucleotide sequence is SEQ ID NO. 7);

3. 7 domains: the amino acid sequence of the 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( is SEQ ID NO. 8, and the nucleotide sequence is SEQ ID NO. 9);

4.4 domains: the amino acid sequence of the signal peptide sequence +His-tag+Ricin B-type lectin+(GGGSAEAAAKEAAAKASGGG)+C-type lectin 1+(GGGSAEAAAKEAAAKASGGG)+C-type lectin 7+(GGGSAEAAAKEAAAKASGGG)+C-type lectin 8( is SEQ ID NO. 10, and the nucleotide sequence is SEQ ID NO. 11);

5. Recombinant proteins comprising the full length sequence of PLA2R antigen (amino acid sequence SEQ ID NO:12, nucleotide sequence SEQ ID NO: 13);

50 healthy human samples and 39 PLA2R positive samples were detected respectively, and the detection results of the kit prepared by 5 different PLA2R antigen recombinant proteins were compared, as shown in Table 6. Wherein, sensitivity: the proportion of positive samples is detected; specificity: detecting the proportion of negative in the sample which is actually negative; accuracy: the ratio of the sum of the number of true positive and true negative samples to the total number of samples; each replicate was tested 5 times.

TABLE 6 recombinant protein PLA2R detection results before and after sequence optimization

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

As can be seen from table 6, there is a significant difference in experimental results between recombinant proteins of different sequences.

As can be seen from a comparison of the four domains Ricin B-TYPE LECTIN, C-TYPE LECTIN 1, C-TYPE LECTIN, C-TYPE LECTIN 8 are sufficient to allow the recombinant protein to be recognized by antibodies in almost all serum samples, and the addition of more domains instead reduces the sensitivity, probably because the excess domains affect the spatial epitopes of the four domains Ricin B-TYPE LECTIN, C-TYPE LECTIN 1, C-TYPE LECTIN7, C-TYPE LECTIN 8, and therefore the PLA2R antigen recombinant proteins constructed using the four domains Ricin B-TYPE LECTIN, C-TYPE LECTIN 1, C-TYPE LECTIN7, C-TYPE LECTIN 8 are preferred.

As can be seen by comparing the 1 st and 4 th, although the same four domains are used, the GS linker and the alpha helix polypeptide are not used for connection between the domains C-TYPE LECTIN and C-TYPE LECTIN 8, otherwise the sensitivity, specificity and accuracy of detection are affected, probably because the effect of the natural connecting peptide between C-TYPE LECTIN 7 and C-TYPE LECTIN is better than that of the artificial connecting peptide.

2. Optimization of THSD7A antigen sequence

The full-length extracellular region sequence of THSD7A antigen (Uniprot number Q9UPZ 6) consists of 22 domains, as shown in table 7.

TABLE 7 THSD7A antigen Domain

Structural 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

Using 21 separate domains (common in Coiled coil humans, incapable of forming THSD 7A-specific epitopes) except Coiled coil, 30 cases of THSD7A positive samples were detected separately, and the detection results are shown in table 8. The results are qualitatively expressed as positive P, weak positive Pw, negative N.

TABLE 8 detection of positive sample results by THSD7A different Domains alone

The single detection of positive samples based on different domains results ,TSP type-1 1/TSP type-1 2/TSP type-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/TSP type-1 21 one or more of these 14 domains can be recognized by existing positive serum samples (the highest proportion of positive samples is measured), and thus these 14 domains were selected to construct THSD7A antigen recombinant proteins. The N-terminus was augmented with an optimized signal peptide sequence (SEQ ID NO: 14) and a His-tag purification tag consisting of 10 histidines. For example, the constructed recombinant protein amino acid sequence (SEQ ID NO: 2) can be expressed as: signal peptide sequence +His-tag +TSP type-1 +TSP type-1 2+ (GGGSAEAAAKEAAAKASGGG) +TSP type-1-9 +TSP type-1-10. The nucleotide sequence of the recombinant protein of THSD7A antigen (SEQ ID NO: 4) was obtained by human codon optimization using the web tool provided by https:// novoprolabs. The recombinant protein was then expressed and purified according to the procedure in example 1.

According to the steps described in the specification of the present invention, several THSD7A antigen recombinant protein preparation kits were used, respectively:

1. 4 domains: signal peptide sequence +His-tag +TSP type-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);

2. 10 domains: the amino acid sequence of the 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)+TSP type-1 14+TSP type-1 15+(GGGSAEAAAKEAAAKASGGG)+TSP type-1 18+TSP type-1 19( is SEQ ID NO. 15, and the nucleotide sequence is SEQ ID NO. 16);

3. 14 domains: the amino acid sequence of the signal peptide sequence +His-tag+TSP type-1 1+TSP type-1 2+TSP type-1 3+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-1 21( is SEQ ID NO. 17, and the nucleotide sequence is SEQ ID NO. 18);

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

5. Recombinant protein comprising full-length THSD7A antigen (amino acid sequence SEQ ID NO:21, nucleotide sequence SEQ ID NO: 22)

50 Healthy human samples and 30 THSD7A positive samples were detected respectively, and the detection results of the kit prepared from 5 different THSD7A antigen recombinant proteins were compared, as shown in Table 9. Wherein, sensitivity: the proportion of positive samples is detected; specificity: detecting the proportion of negative in the sample which is actually negative; accuracy: the ratio of the sum of the number of true positive and true negative samples to the total number of samples; each replicate was tested 5 times.

TABLE 9 detection results of recombinant protein THSD7A before and after sequence optimization

THSD7A antibody detection	SEQ ID NO:4	SEQ ID NO:16	SEQ ID NO:18	SEQ ID NO:20	SEQ ID NO:22
						Sensitivity of	93.3％	83.3％	83.3％	80％	73.3％
Specificity (specificity)	98％	94％	94％	92％	90％
						Accuracy of	96.3％	90％	90％	87.5％	83.6％

As can be seen from table 9, there are significant differences in experimental results between THSD7A recombinant proteins using different sequences. As can be seen from a comparison of items 1 to 3, the four domains of TSP type-1 1/TSP type-1 2/TSP type-1 9/TSP type-1 10 are sufficient to allow the THSD7A recombinant protein to be recognized by antibodies in almost all serum samples, and the addition of more domains instead reduces sensitivity and specificity, probably because the four domains of THSD7A are affected by the excess domains, and therefore the THSD7A antigen recombinant protein constructed using the four domains of TSP type-1 1/TSP type-1 2/TSP type-1 9/TSP type-1 is preferred. As can be seen from comparing the 1 st and 4 th, although the same four domains are used, the GS linker and the alpha helix polypeptide are not used for connection between the domains TSP type-1 1/TSP type-1 2 and between the TSP type-1 9/TSP type-1 10, otherwise the sensitivity, specificity and accuracy of detection are affected, probably because the connecting peptide in the natural sequence is more favorable for the protein to form a correct folding structure than the artificial connecting peptide.

Example 3: screening of first and second antigen dialysate

1. Screening of first antigen dialysate

In this example, a kit for detecting relevant indexes of human membranous nephropathy is prepared according to the method provided in example 1, wherein 6 different antigen dialysates shown in table 10-1 and the first antigen dialysates adopted in example 1 are respectively adopted in the preparation process of PLA2R antigen, naOH (1M) and concentrated hydrochloric acid are used for adjusting the pH to 7.5 and respectively dialyzing to prepare PLA2R antigen, then microsphere-coupled antigen solutions are prepared according to the method provided in example 1, a corresponding kit for detecting relevant indexes of human membranous nephropathy is prepared, the kit is used for detecting PLA2R, a sample to be detected is positive serum (the concentration of PLA2R is 26.15RU/mL through mass spectrometry), the prepared kit is placed for 0 day, 1 month, 3 months, 6 months and 12 months at 2-8 ℃, the detection is performed according to the method provided in example 1, the influence of different first antigen dialysates on the PLA2R detection result is examined, 10 times of repeated detection is performed, an average value is obtained, and a CV value is calculated.

Table 10-1, composition of different first antigen dialysate (proportional relationship according to example 1)

TABLE 10-2 influence of different first antigen dialysate on PLA2R stability

It can be seen from table 10-2 that after optimizing the PLA2R antigen sequence, the formulation of the PLA2R antigen dialysate also needs to be further screened to achieve better detection sensitivity, and the PLA2R antigen prepared by using different first antigen dialysate not only affects the stability of the PLA2R antigen, but also affects the detection result of the PLA2R, which may be due to different dialysis effects of different antigen dialysate, resulting in different purities of the prepared PLA2R antigen, thereby affecting the stability in the preservation process. There is therefore a need to screen for optimal first antigen dialysate formulations.

Comparison of PLA2R antigens prepared using different antigen dialysate, with different sensitivities for PLA2R detection, and also different detection values after a period of time, the most preferred first antigen dialysate formulation was formulated as in example 1: the detection sensitivity and the antigen stability are obviously better at the moment of the combination of the trimethylol aminomethane, the bovine serum albumin, the mannitol, the L-histidine, the sodium chloride, the disodium ethylenediamine tetraacetate dihydrate, the dithiothreitol and the 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride.

2. Screening of second antigen dialysate

In this example, a human membranous nephropathy related index detection kit is prepared according to the method provided in example 1, wherein 6 different antigen dialyzates shown in table 11-1 and the second antigen dialyzate adopted in example 1 are respectively adopted in the preparation process of THSD7A antigen, naOH (1M) and concentrated hydrochloric acid are used for adjusting the pH to purify and prepare THSD7A antigen, microsphere-coupled antigen solution is prepared according to the method provided in example 1, a corresponding human membranous nephropathy related index detection kit is prepared for the detection of THSD7A, the sample to be detected is positive serum (the concentration detected by mass spectrometry is 8.52 ng/mL), the influence of different second antigen dialyzates on the detection result of THSD7A is examined according to the method provided in example 1, and 10 times of repeated detection and average value is taken.

Table 11-1, composition of different second antigen dialysate (proportional relationship according to example 1)

TABLE 11-2 influence of different second antigen dialysis solutions on THSD7A stability

As can be seen from Table 11-2, THSD7A antigen prepared using different second antigen dialysate not only affects the stability of THSD7A antigen, but also affects the detection result of THSD 7A. There is therefore a need to optimize the second antigen dialysate formulation to increase the detection sensitivity of THSD7A, while at the same time increasing the stability of THSD7A antigen. As can be seen from table 11-2, the use of a different formulation from the first antigen dialysate, and a different pH, was only suitable for stable preservation of THSD7A antigen, while improving detection sensitivity, and the most preferred second antigen dialysate formulation was formulated as in example 1: sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediamine tetraacetic acid disodium salt dihydrate, dithiothreitol and 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and the pH is adjusted to 8.0, the detection sensitivity and antigen stability are obviously better at this time.

Example 4: effect of antigen preservation solution

1. Stability Effect of antigen preservation solution on PLA2R antigen and THSD7A antigen

The study in this example proves that the PLA2R antigen and THSD7A antigen have a problem of poor stability, so that the PLA2R antigen and THSD7A antigen need to be stored in an antigen storage solution after preparation is completed, and in the initial stage of the study, the same storage solution (directly using the first antigen dialysate as the storage solution of the two antigens) is used for both PLA2R antigen and THSD7A antigen, and the following three cases are examined respectively:

the first group is directly replaced by water without adopting antigen preservation solution;

a second group in which Tris buffer was used as an antigen-preserving solution (pH 8.0);

And a third group, adopting: tris buffer, bovine serum albumin, mannitol, L-histidine, sodium chloride, disodium edetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, ph=7.5;

Respectively placing the prepared antigen at 2-8deg.C for 0 days, 1 day, 10 days, 30 days, 60 days, and 90 days, preparing kit according to the method provided in example 1, and detecting PLA2R antigen activity by using PLA2R calibrator (20 RU/mL); the results of activity assays for THSD7A antigen using THSD7A calibrator (10 ng/mL) are shown in Table 12.

Table 12, stability study of PLA2R antigen and THSD7A antigen

As can be seen from table 12, the stability of both the PLA2R antigen and THSD7A antigen of the first/second group was poor, and the activity was significantly reduced after 1 day of standing, mainly due to oxidation of the side chains of the protein molecules in the solution, and the stability was significantly improved by the reducing agent added to the antigen storage solution.

2. Stability Effect of antigen preservation solution on PLA2R antigen solution of coupled microspheres and THSD7A antigen solution of coupled microspheres

Since PLA2R antigen and THSD7A antigen had stability problems, this example also attempted to immediately prepare PLA2R antigen solution of coupled microspheres and THSD7A antigen solution of coupled microspheres on the day of preparing PLA2R antigen and THSD7A antigen, examined whether stability problems still exist after coupling microspheres, and the effect of adding an antigen preservation solution on the antigen solution for preparing coupled microspheres.

The following two groups of cases were examined separately:

the first group, not adopting antigen preservation solution, the antigen is used for preparing antigen solution of the coupling microsphere immediately on the day of antigen preparation;

The second group, prepared antigen, was added to the antigen stock solution (third group of this example) and used immediately the day to prepare an antigen solution of coupled microspheres;

the antigen solutions of the prepared conjugate microspheres were placed at 2-8℃for 0, 1, 5, 10, 60, and 90 days, respectively, and the fluorescence signal values (MFI) of PLA2R and THSD7A were measured, and the measurement results are shown in Table 13.

TABLE 13 stability study of microsphere-coupled PLA2R antigen and microsphere-coupled THSD7A antigen

As can be seen from table 13, the PLA2R antigen and THSD7A antigen still have stability problems after being coupled to the microsphere, and are difficult to store stably, so that the shelf life of the kit is seriously affected, so that an antigen storage solution must be added immediately after the PLA2R antigen and THSD7A antigen are prepared to improve the stability of the PLA2R antigen and THSD7A antigen, so that the stability of the antigen solution of the coupled microsphere in the kit is also improved, and the shelf life of the kit is remarkably prolonged.

Example 5: screening of first antigen-preserving fluid and second antigen-preserving fluid

The study of the embodiment proves that the PLA2R antigen and the THSD7A antigen are required to be stored by different antigen storage solutions respectively, so that the stability of the PLA2R antigen and the THSD7A antigen can be improved more effectively, and the shelf life of the kit is prolonged.

1. Screening of first antigen-preserving fluid

In this example, a kit for detecting relevant indexes of human membranous nephropathy was prepared according to the method provided in example 1, wherein 6 different first antigen preservation solutions as shown in table 14-1 and the first antigen preservation solution adopted in example 1 were used to preserve the prepared PLA2R antigens, naOH (1M) and concentrated hydrochloric acid were used to adjust pH to 7.5, the THSD7A antigen was preserved according to the second antigen preservation solution provided in example 1, other microsphere-conjugated antigen solutions were prepared according to the method provided in example 1, the corresponding kit for detecting relevant indexes of human membranous nephropathy was prepared, and the PLA2R antigens were subjected to detection for 0 day, 30 day, 90 day, 180 day, 360 day at 2-8 ℃ respectively, while PLA2R and THSD7A were examined, the samples to be tested were PLA2R positive serum (detected by mass spectrometry, the content was 23.35/mL) and THSD7A positive serum (detected by mass spectrometry, the content was 11.76 ng/mL), the different first antigen solutions provided in example 1 were examined, and the results of the detection were repeated as shown in table 14, and the results of the detection were obtained.

Table 14-1, composition of different first antigen-preserving liquids (proportional relationship according to example 1)

TABLE 14-2 influence of different first antigen-preserving fluids on detection results

It can be seen from table 14-2 that the formulation of the antigen preservation solution also needs to be further optimized to achieve better detection sensitivity, and that the use of different first antigen preservation solutions not only affects the stability of PLA2R antigen, but also affects the detection result of PLA2R, and also affects the detection result of THSD 7A. The reason for this may be that during the detection process, the two antigen solutions of the coupled microspheres need to be mixed together and thus there is a mutual influence. The most preferred first antigen preservation solution formulation is: tris (hydroxymethyl) aminomethane + bovine serum albumin + mannitol + L-histidine + sodium chloride + disodium ethylenediamine tetraacetate dihydrate + dithiothreitol +4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride + glycerol + ProClin300,300, pH was adjusted to 7.5.

2. Screening of second antigen preservation solution

In this example, a human membranous nephropathy related index detection kit was prepared according to the method provided in example 1, wherein for THSD7A antigen, 6 different second antigen preservation solutions as shown in table 15-1 and the second antigen preservation solution adopted in example 1 were used to preserve the prepared THSD7A antigen, naOH (1M) and concentrated hydrochloric acid were used to adjust pH to 8.0, the PLA2R antigen was preserved according to the first antigen preservation solution provided in example 1, then microsphere-coupled antigen solution was prepared according to the method provided in example 1, a corresponding human membranous nephropathy related index detection kit was prepared, and the activities of the THSD7A antigen of the coupled microspheres were examined for 0 day, 30 day, 90 day, 180 day, 360 day, simultaneously for detection of PLA2R and THSD7A, the samples to be tested were PLA2R positive serum (detected by mass spectrometry, content was 23.35/mL) and sd7A positive serum (detected by mass spectrometry, content was 11.76/mL), the method was performed according to example 1, different detection results were provided for the second sample, and the average value of the THSD7A was measured, and the results were repeated as shown in table 15 ng.

Table 15-1, composition of different second antigen-conserving solutions (proportional relation according to example 1)

TABLE 15-2 influence of different second antigen-preserving fluids on detection results

As can be seen from table 15, the formulation of the antigen preservation solution needs to be further optimized to achieve better detection sensitivity, and the use of a different second antigen preservation solution can affect not only the stability of THSD7A antigen, but also the detection result of THSD7A, and also the detection result of PLA 2R. The most preferred second antigen preservation solution formulation is therefore: sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, disodium ethylenediamine tetraacetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol, proClin, 300, and adjusting the pH to 8.0.

Example 6: comparison with the existing kits

The kit provided in example 1 was used to prepare a detection kit related to the existing human membranous nephropathy in the market, and the sample to be detected was positive serum (PLA 2R concentration is 41.05RU/mL, THSD7A concentration is 27.01ng/mL, detected by mass spectrometry) and the detection result is shown in Table 16.

TABLE 16 comparison with the prior art kit

According to the table 16, the detection accuracy and sensitivity of PLA2R and THSD7A can be obviously improved, the batch difference can be eliminated, and the detection result is more stable and reliable.

Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.

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Claims

1. The detection kit for the related indexes of the human membranous nephropathy is characterized by comprising antigen proteins, wherein the antigen proteins comprise PLA2R antigen and THSD7A antigen, the amino acid sequence of the PLA2R antigen is shown as SEQ ID NO. 1, and the amino acid sequence of the THSD7A antigen is shown as SEQ ID NO. 2; the related indicators of human membranous nephropathy include PLA2R antibodies and THSD7A antibodies.

2. The kit for detecting the human membranous nephropathy related index according to claim 1, wherein the coding nucleotide sequence of the PLA2R antigen is shown in SEQ ID NO. 3, and the coding nucleotide sequence of the THSD7A antigen is shown in SEQ ID NO. 4.

3. The kit for detecting an indicator related to human membranous nephropathy according to claim 2, further comprising a detection antibody comprising an anti-human IgG antibody complex, wherein the anti-human IgG antibody complex is an phycoerythrin-labeled anti-human IgG antibody.

4. The kit for detecting the related index of human membranous nephropathy according to claim 3, wherein the PLA2R antigen and the THSD7A antigen are respectively coupled with microspheres to prepare a PLA2R antigen solution coupled with the microspheres and a THSD7A antigen solution coupled with the microspheres; the detection antibody solution is phycoerythrin marked anti-human IgG antibody solution; the kit further comprises a sample diluent.

5. The preparation method of the human membranous nephropathy related index detection kit is characterized by comprising the following steps of:

Step (2), dialyzing the PLA2R antigen with a first antigen dialyzate to obtain a PLA2R antigen solution; dialyzing the THSD7A antigen with a second antigen dialysate to obtain a THSD7A antigen solution;

step (3), preparing PLA2R antigen solution of the coupled microsphere by adopting PLA2R antigen solution containing the first antigen preservation solution;

The THSD7A antigen solution of the coupled microspheres was prepared using a THSD7A antigen solution containing a second antigen preservation solution.

6. The method of claim 5, wherein the first antigen dialysate comprises tris buffer, bovine serum albumin, mannitol, L-histidine, sodium chloride, disodium edetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, PH = 7.5; the first antigen preservation solution comprises a tris buffer solution, bovine serum albumin, mannitol, L-histidine, sodium chloride, disodium ethylenediamine tetraacetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol, proClin, and pH=7.5.

7. The method of claim 6, wherein the second antigen dialysate comprises sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, disodium edetate dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, PH = 8.0; the second antigen preservation solution comprises sodium citrate dihydrate, bovine serum albumin, mannitol, L-glycine, sodium chloride, ethylenediamine tetraacetic acid disodium salt dihydrate, dithiothreitol, 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, glycerol, proClin300,300 and pH=8.0.

8. The method of claim 7, wherein step (1) comprises separately preparing PLA2R antigen and THSD7A antigen: recombinant vectors containing nucleotide sequences for encoding PLA2R antigen and THSD7A antigen are respectively constructed, and the recombinant vectors containing nucleotide sequences for encoding THSD7A antigen are expressed by a eukaryotic expression system to prepare recombinant PLA2R antigen and recombinant THSD7A antigen.

9. A method for detecting an index associated with human membranous nephropathy, wherein the method is used for non-disease diagnosis and is performed using the kit of claim 4, comprising the steps of:

And (d) detecting the complex to be detected by using a flow cytometer.

10. The application of a group of antigen preservation solutions in preparing reagents for improving detection sensitivity of related indexes of human membranous nephropathy is characterized in that the related indexes of human membranous nephropathy comprise PLA2R antibodies and THSD7A antibodies, the antigen preservation solutions comprise a first antigen preservation solution and a second antigen preservation solution, the first antigen preservation solution is used for preserving PLA2R antigen solution of coupled microspheres, and the second antigen preservation solution is used for preserving THSD7A antigen solution of coupled microspheres.