CN114895023A

CN114895023A - Application of reagent for detecting anti-Talin-1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

Info

Publication number: CN114895023A
Application number: CN202210496640.0A
Authority: CN
Inventors: 叶青; 毛建华; 韩秀翠
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-08-12

Abstract

The invention relates to an application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparing a kit for detecting vascular endothelial injury, belonging to the technical field of diagnostic kits. The invention provides an application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The detection of the anti-Talin-1-IgG autoantibody can realize the detection of vascular endothelial injury.

Description

Application of reagent for detecting anti-Talin-1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

Technical Field

The invention relates to the technical field of diagnostic kits, in particular to application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparation of a kit for detecting vascular endothelial injury.

Background

Minimal central lesion (MCD) is the leading cause of nephrotic syndrome in children, accounting for 10-15% of nephrotic syndrome in adults. Glomeruli of patients with minimal disease appeared essentially normal under light microscopy, and the only histopathological abnormality seen under electron microscopy was the disappearance of diffuse podocyte foot process fusion. Thus, MCD is considered to be a primary podocyte disease. Complete remission of proteinuria after corticosteroid treatment is a marker of MCD and, in general, progressive renal failure is rare. However, MCD can lead to serious complications. Complications associated with the disease observed in adults include mainly venous thrombosis and severe acute kidney injury requiring temporary dialysis. Furthermore, because MCD is characterized by a chronic, recurrent course, prolonged immunosuppressive therapy is often required to maintain proteinuria remission. However, long-term immunosuppressive therapy increases the risk of serious infection and carries a long-term risk of malignancy.

Currently, little is known about the underlying pathogenesis of MCD. One view is that the disease is triggered by the circulating permeability factors produced by immune cells. Since the pathogenesis of primary Focal Segmental Glomerulosclerosis (FSGS) is very similar to that of MCD, many scholars consider MCD and FSGS to be phenotypes of the same disease at different stages. T cells were first suspected to be the source of the circulating permeability factor based on the association between MCD and non-hodgkin's lymphoma, the remission induced by measles infection and prolonged remission following cyclophosphamide treatment. However, the therapeutic effects of rituximab and other specific B-cell depleting drugs have presented challenges to T-cell sources in recent years. Notably, the direct effect of corticosteroids and rituximab on podocytes is also considered to have therapeutic effect. The screening and identification of many podocyte autoantibodies in MCD and FSGS nephrotic syndrome patients by our team provides a potential link between podocyte injury, autoimmunity and proteinuria response to anti-B cell therapy, and therefore, the concept of 'Autoimmune podocytosis' (Autoimmune podocytopathies) is first proposed internationally and gradually recognized by the same lines at home and abroad. Recently, the Harvard medical college team Watts et al found that anti-Nephrin autoantibodies also exist in the serum of children and adults with minimal change nephrotic syndrome, which provides a powerful evidence for our innovative theory.

Although the observed podocyte injury is a major classical feature of MCD, the disease mechanism may also involve glomerular vascular endothelial cells. Idiopathic Nephrotic Syndrome (INS) reported as early as 2000 by futrakun et al is often accompanied by renal hypoperfusion. The human endothelial cell line ECV 304 is used by the patients and incubated with INS patient serum to carry out endothelial cell toxicity tests, and the results show that the FSGS patient serum causes the most obvious endothelial cell damage. Therefore, they speculate that glomerular vascular endothelial cell injury may be responsible for insufficient renal perfusion in INS patients. Purohit S et al found that there was an increase in the endothelial cell injury marker syndecan 1 in the circulatory system of MCD patients, but it was not clear whether there was simultaneous injury to the glomerular endothelial cells. Trachtman H et al observed the co-deposition of IgM with complement components in kidney tissues of FSGS and MCD patients and confirmed that IgM is an antibody against GEC and cardiolipin epitopes. Bauer C et al found in 2022 that the endothelial cell marker in the serum of MCD patients was elevated, and meanwhile, renal histopathology confirmed that the expression of glomerular endothelial cells caveolin-1 was significantly elevated, and further incubation of the serum of patients with human glomerular endothelial cells cultured in vitro significantly increased the expression of thrombomodulin, a marker of glomerular vascular endothelial cell injury, thereby demonstrating that MCD patients had injury to glomerular vascular endothelial cells.

Nevertheless, it is not clear to date what are the causative agents responsible for the damage to glomerular endothelial cells. A series of glomerular vascular endothelial cell autoantibodies were screened and identified by our research team in patients with MCD and FSGS nephrotic syndrome through previous studies. Animal experiments prove that the glomerular vascular endothelial cell self-antibody can cause severe damage to the glomerular vascular endothelial cells of the mice. In vitro cell culture experiments also indicate that these autoantibodies affect the morphology and function of vascular endothelial cells. Clinical studies have shown that these autoantibodies to glomerular vascular endothelial cells are associated with a high coagulation status and poor prognosis in patients. In addition, our findings suggest that glomerular vascular endothelial cell injury caused by autoantibodies to glomerular vascular endothelial cells may be the initiating factor of characteristic podocyte injury in MCD, and is one of the important causes of the disease. Therefore, we have proposed the second hit theory of the onset of MCD and FSGS nephrotic syndrome for the first time internationally: that is, pathogenic agents including autoantibodies first damage the glomerular vascular endothelial cells, and then these pathogenic agents further damage the podocytes, eventually causing morbidity to the patient. Because the pathogenic agents in the blood circulation system are unlikely to come into contact with the podocytes from the specific anatomical location of the podocytes unless the integrity of the glomerular vascular endothelial cells has been compromised. Therefore, the research result of the autoantibodies of the endothelial cells of the glomerular vessels is a breakthrough in the theoretical research of the pathogenesis of the nephrotic syndrome. However, a kit for detecting vascular endothelial injury with high efficiency is still lacking at present.

Disclosure of Invention

The invention aims to provide application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparation of a kit for detecting vascular endothelial injury. The detection of the anti-Talin-1-IgG autoantibody can realize the effective detection of vascular endothelial injury.

The invention provides an application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.

Preferably, the reagent for detecting the anti-Talin-1-IgG autoantibody comprises a Talin-1 protein or a Talin-1 recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the Talin-1 protein is BC 042923.

Preferably, the tag comprises a His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag or biotin tag.

Preferably, when the tag is a His tag, the amino acid sequence of the tag-containing Talin-1 recombinant protein is shown in SEQ ID NO. 1.

Preferably, the vascular endothelial injury comprises glomerular vascular endothelial cell injury.

The invention also provides a kit for detecting anti-Talin-1-IgG autoantibody, comprising: the reagent for detecting the anti-Talin-1-IgG autoantibody, the solid phase carrier and the labeled antibody in the application of the technical scheme.

Preferably, the solid phase carrier comprises a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle and/or an enzyme labeling micropore plate.

Preferably, the labeled antibody comprises an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody; the secondary antibody comprises an anti-human IgG antibody.

Preferably, the enzyme-labeled secondary antibody comprises a horseradish peroxidase-labeled anti-human IgG antibody; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.

The invention provides an application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The invention firstly detects an anti-Talin-1-IgG autoantibody in a part of patients with nephrotic syndrome, and determines that a target antigen aimed by the autoantibody is Talin-1 on glomerular vascular endothelial cells. The invention finds that the Talin-1 protein antibody is an important glomerular vascular endothelial cell autoantibody, is closely related to the occurrence and development of MCD and FSGS nephrotic syndrome, and can guide clinical diagnosis and treatment. The detection of the anti-Talin-1-IgG autoantibody can realize the detection of vascular endothelial injury, and particularly provides a basis for researching the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment. The kit for detecting the anti-Talin-1-IgG autoantibody provided by the invention can qualitatively and quantitatively detect the anti-Talin-1-IgG antibody in serum of a patient with nephrotic syndrome, and the kit provided by the invention utilizes the IgG antibody of human anti-tag peptide as a standard substance and greatly improves the detection accuracy, sensitivity, specificity and detection speed by combining a biotin-avidin amplification system and magnetic particle chemiluminescence immunoassay. Specifically, compared with the prior art, the kit has the following benefits:

1. the kit can realize high-efficiency detection of vascular endothelial injury, and judges that the vascular endothelial injury exists when the anti-Talin-1-IgG autoantibody is detected.

2. At present, the Talin-1 and anti-Talin-1-IgG antibodies related to kidney disease patients at home and abroad are only limited to molecular mechanism research, and the level of the antibodies in the serum of the patients is not quantitatively detected. The invention identifies the IgG autoantibody aiming at the Talin-1 for the first time, invents a detection kit aiming at the Talin-1-IgG autoantibody and fills the blank at home and abroad. The kit provided by the invention is used for detecting anti-Talin-1-IgG antibodies in the serum of 298 nephrotic syndrome patients, and the result shows that the anti-Talin-1-IgG antibodies of 150 patients are positive, namely the positive detection rate of the anti-Talin-1-IgG antibodies is 50.34%. The invention can provide a basis for researching the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment by detecting the anti-Talin-1-IgG antibody.

3. The kit of the invention relates to a solid-phase membrane immunoassay qualitative analysis of anti-Talin-1-IgG antibody in human serum, and the detection accuracy is greatly improved by taking the human anti-labeled peptide IgG antibody as a standard substance. The solid-phase membrane immunoassay qualitative detection is simple to operate, the reagent dosage is less, and the solid-phase membrane immunoassay qualitative detection is saved by about 10 times compared with the traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely close to 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane; the NC membrane with adsorbed antigen or antibody or existing result can be preserved for a long time (half a year at-20 ℃), and the activity of the NC membrane is not influenced; in addition, the kit for qualitatively detecting the anti-Talin-1-IgG antibody in the human serum by the solid-phase membrane immunoassay is introduced into a biotin-avidin amplification system, so that the detection sensitivity is greatly improved.

4. The kit for quantitatively detecting the anti-Talin-1-IgG antibody in human serum by magnetic particle chemiluminescence immunoassay utilizes magnetic particles as solid phase carriers, the diameter of the magnetic particles is only 1.0 mu m, so that the coating surface area is greatly increased, the adsorption quantity of antigens is increased, the reaction speed is improved, the cleaning and the separation are simpler and more convenient, the pollution is reduced, and the probability of cross infection is reduced. On the other hand, the acridine ester luminescent agent is adopted to directly mark the anti-human IgG, the chemical reaction is simple and quick, and no catalyst is needed; the acridinium ester chemiluminescence is of the scintillation type by initiating the luminescent reagent (H) ₂ O ₂ NaOH) can reach the maximum after 0.4s, the half-life period is 0.9s, the detection is basically finished within 2s, and the rapid detection is convenient.

Drawings

FIG. 1 is a graph showing the results that the Talin-1 protein on the endothelial cells of glomerular vessels provided by the present invention is the main target antigen for the autoantibodies in patients with nephrotic syndrome; wherein, A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; b: the first antibody is a two-dimensional electrophoresis protein spot of serum of a nephrotic syndrome patient; c: mass spectrum identification of target antigen Talin-1 protein;

FIG. 2 is an SDS-PAGE identification picture of the expressed recombinant protein Talin-1 provided by the present invention;

FIG. 3 is a graph showing the results of detecting anti-Talin-1-IgG antibody in serum of a nephrotic syndrome patient by using the solid-phase membrane immunoassay kit provided by the present invention;

FIG. 4 is a schematic diagram of the principle of detecting anti-Talin-1-IgG antibody by the magnetic particle chemiluminescence immunoassay kit provided by the invention;

FIG. 5 is a schematic diagram of an antigenic protein Talin-1 coated carboxyl magnetic particle provided by the invention;

FIG. 6 is a diagram showing the detection of anti-Talin-1-IgG antibodies in various patients with renal diseases according to the present invention; wherein, NS: nephrotic syndrome, HSP: allergic purpura, HSPN: purpuric nephritis, NC: a healthy child;

FIG. 7 is a graph showing the linear correlation between the anti-Talin-1-IgG antibody provided by the present invention and a vascular endothelial injury marker.

Detailed Description

The invention provides an application of a reagent for detecting anti-Talin-1-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The Talin-1 protein is a protein existing in cytoplasm, is composed of a head (talinhead) part and a body (talinrod) part, and is an important molecule for regulating the function of integrin protein on cell membrane. Blood, blood vessels, and the heart constitute the blood circulation system of the human body. Blood in the blood circulation system flows through blood vessels and flows through the whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of the blood vessel, and antibodies of the vascular endothelial cells can cause damage to the vascular endothelial cells and induce dysfunction of a blood circulation system, so that the heart, the lung, the liver and other organs are damaged, and diseases related to the organs are caused, including nephrotic syndrome. Therefore, the detection of vascular endothelial cell autoantibodies in the blood circulation system can be clinically used to indicate the presence of vascular endothelial cell damage. Because the vascular endothelial cells of different organs are the same, the invention firstly discovers the vascular endothelial cell autoantibody-anti-Talin-1-IgG autoantibody, and the application of the invention can realize the detection of all vascular endothelial injuries of the whole body including glomerular vascular endothelium.

The reagent for detecting the anti-Talin-1-IgG autoantibody provided by the invention takes the Talin-1 protein as a target spot to detect the Talin-1 autoantibody (namely, the anti-Talin-1-IgG autoantibody is a biomarker for detecting vascular endothelial cell injury), and the reagent can realize high-efficiency detection of vascular endothelial injury. In the present invention, the reagent is capable of immunoreacting with an autoantibody to the Talin-1 protein derived from a tissue (kidney biopsy) or a body fluid (in particular, blood, plasma, serum). In the present invention, the reagent for detecting an anti-Talin-1-IgG autoantibody preferably comprises a Talin-1 protein or a Talin-1 recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the Talin-1 protein is BC 042923. In the present invention, the tag is preferably a tag having some biological or physical function, in particular an N-terminus or a C-terminus; the existence of the tags is beneficial to the purification, fixation and precipitation of antigen protein; more preferably, the tag is a sequence or domain capable of specifically binding to a ligand, such as a tag peptide, preferably selected from the group consisting of: his tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag or biotin tag. In the present invention, when the tag is a His tag, the amino acid sequence of the tagin-1 recombinant protein containing the tag is preferably as shown in SEQ ID No. 1: MGNSCRQEDVIATANLSRRAIADMLRACKEAAYHPEVAPDVRLRALHYG RECANGYLELLDHVLLTLQKPSPELKQQLTGHSKRVAGSVTELIQAAEAM KGTEWVDPEDPTVIAENELLGAAAAIEAAAKKLEQLKPRAKPKEADESL NFEEQILEAAKSIAAATSALVKAASAAQRELVAQGKVGAIPANALDDGQW SQGLISAARMVAAATNNLCEAANAAVQGHASQEKLISSAKQVAASTAQLL VACKVKADQDSEAMKRLQAAGNAVKRASDNLVKAAQKAAAFEEQENE TVVVKEKMVGGIAQIIAAQEEMLRKERELEEARKKLAQIRQQQYKFLPSE LRDEHHHHHHH are provided.

In the present invention, the vascular endothelial injury preferably comprises glomerular vascular endothelial cell injury. More specifically, the vascular endothelial injury of the present invention preferably includes vascular endothelial injury of nephrotic syndrome. In the present invention, the nephrotic syndrome preferably includes a morbid disease or primary focal segmental glomerulosclerosis.

In the present invention, the reagent for detecting an anti-Talin-1-IgG autoantibody (Talin-1 protein or Talin-1 recombinant protein containing a tag) is preferably immobilized on a solid support. As used herein, "immobilized" refers to binding to a solid support insoluble in water of the Talin-1 antigen protein, the solid support or support being insoluble in water, more preferably by covalent bonding, electrostatic interaction, hydrophobic interaction, or interaction by disulfide bonding, most preferably by one or more covalent bonds. The immobilization may be by direct immobilization, e.g. by filtration, centrifugation or chromatography, and the immobilized molecules are separated from the aqueous solution together with the insoluble support. Also included is the immobilization of the Talin-1 antigenic protein in a reversible or irreversible manner. For example, the antigenic protein is immobilized to the carrier by a cleavable covalent bond (e.g., a disulfide bond that can be cleaved by a thiol-containing reagent), which is reversible. In addition, if the antigenic protein is immobilized to the support by a covalent bond that does not cleave in aqueous solution (bond formed by reaction of epoxide group with amine group coupling lysine side chain to affinity column), the immobilization is irreversible. Fixation may also be indirect: such as fixing an antibody having a specific affinity for the antigen protein, and then forming an antigen protein-antibody complex for the purpose of fixing. The antigen protein Talin-1 fixing method is preferably a direct coating method: (1) the antigen protein Talin-1 is combined on a nitrocellulose membrane or a polystyrene microporous plate in a physical adsorption mode or a non-covalent bond; (2) the magnetic particles with carboxyl functional groups are combined with the amino group of the antigen protein Talin-1, and the antigen protein Talin-1 is combined on the magnetic particles in a chemical coupling mode. In the invention, the solid phase carrier preferably comprises a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle or an enzyme labeling micropore plate.

The invention preferably adopts a gene recombination prokaryotic expression method to successfully express and purify the recombinant protein Talin-1, and uses the recombinant protein Talin-1 as an antigen protein in a kit to develop a set of kits suitable for detecting the anti-Talin-1-IgG antibody of the glomerular vascular endothelial cell autoantibody of a patient with nephrotic syndrome, and the kit comprises a detection kit for qualitatively or quantitatively analyzing and detecting the anti-Talin-1-IgG antibody in human serum.

In the present invention, the Talin-1 protein is preferably expressed in bacterial (e.g., E.coli), yeast, insect or mammalian cells. After the Talin-1 protein is obtained through expression, the Talin-1 protein is preferably purified by using methods such as Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, hydrophobic column purification and the like.

In the present invention, the labeled antibody preferably includes an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody; the secondary antibody comprises an anti-human IgG antibody.

In the present invention, the enzyme-labeled secondary antibody preferably comprises an anti-human IgG antibody labeled with horseradish peroxidase; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.

In the present invention, the types of the kit preferably include a solid-phase membrane immunoassay kit or a magnetic particle chemiluminescence immunoassay kit; when the kit is a solid-phase membrane immunoassay kit, the kit preferably further comprises an antigen diluent, a sample diluent buffer, an antibody diluent, a substrate developing solution, a washing solution, an enzyme working solution, a standard substance, a positive quality control substance and a negative quality control substance; when the kit is a magnetic particle chemiluminescence immunoassay kit, the kit preferably further comprises chemiluminescence pre-excitation liquid A, chemiluminescence excitation liquid B, a standard substance and a cleaning solution. In the present invention, the standard substance and the positive quality control substance are preferably both recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-Talin-1-IgG autoantibodies extracted from patient serum; the negative quality control product is preferably serum of a healthy physical examiner.

Specifically, when the kit is a solid-phase membrane immunoassay kit, in the kit, the antigen, which is the reagent for detecting the anti-Talin-1-IgG autoantibody, is preferably recombinant protein Talin-1 (the amino acid sequence is shown in SEQ ID No. 1); the solid phase carrier is preferably a cellulose nitrate membrane of Sataurus CN 140; the positive quality control product (standard product) is preferably human anti-His tag immunoglobulin G (purchased from English Chuang, Huzhou); the negative quality control product is preferably serum of a healthy physical examiner; the labeled antibody is preferably a biotin-labeled anti-human IgG antibody; the enzyme working solution is preferably alkaline phosphatase-streptavidin; the substrate color developing agent is preferably TMB, hydrogen peroxide, AMPPD, 4-MUP or BCIP; the antigen diluent is preferably 1 XPBS pH7.4 containing 163mM NaCl and 1% TritonX-100; the sample dilution buffer is preferably 0.01M PBS containing 10% BSA, pH 7.4; the antibody diluent is preferably 0.01M PBS pH7.4 containing 1M D-glucose, 2% glycerol, 0.35% Tween 20; the washing liquid is preferably: 1 XPBS pH7.4 containing 163mM NaCl, 10% glycerol, 1% TritonX-100.

When the kit is a magnetic particle chemiluminescence immunoassay kit, in the kit, the antigen is preferably recombinant protein Talin-1 (the amino acid sequence is shown as SEQ ID NO. 1); the solid phase carrier is preferably carboxyl magnetic beads; the labeled antibody is preferably an acridinium ester labeled anti-human IgG antibody; the chemiluminescence pre-excitation liquid A and the chemiluminescence excitation liquid B are preferably conventional commercial products, and the standard substance is preferably anti-Talin-1-IgG autoantibody with different concentrations; the cleaning solution is preferably a pH 7.2, 25mmol/L LTris-HCL solution containing 0.15mol/L NaCL and 0.05% Tween-20.

In the invention, the sample to be tested of the kit is preferably from whole blood, serum, plasma, urine, lymph fluid and hydrothorax and ascites; more preferably mammalian (human) serum.

In the present invention, the principle of the kit for detecting an anti-Talin-1-IgG antibody in serum is preferably as follows: the indirect method reaction principle is utilized, firstly, a Talin-1 antigen is adsorbed to a solid phase carrier to serve as a coating antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled antibody (labeled secondary antibody) is added for reaction, then if the serum to be detected contains an anti-Talin-1-IgG antibody, a ternary complex of the coating antigen Talin-1-IgG antibody of the serum to be detected and a labeled anti-human IgG antibody is formed, and finally, a light signal is detected by utilizing a light color development method, a chemiluminescence method and a fluorescence method, so that the purpose of qualitatively or quantitatively analyzing the anti-Talin-1-IgG antibody of human serum is achieved.

The application of the reagent for detecting anti-Talin-1-IgG autoantibody of the present invention in the preparation of a kit for detecting vascular endothelial injury will be described in further detail with reference to the following specific examples, but the technical solution of the present invention includes, but is not limited to, the following examples.

Example 1

The Talin-1 protein on the vascular endothelial cells is the main target antigen for the autoantibody in patients with nephrotic syndrome

The specific implementation is as follows:

(1) extraction of total protein of vascular endothelial cells: vascular endothelial cell lines (EAhy926) were cultured, washed 2-3 times with PBS, then extensively lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitors (# ab 65621; Abcam, 1: 200 dilution), and the samples were centrifuged at 12000g, 4 ℃ for 30 min. Collecting the supernatant, namely the total protein of the vascular endothelial cells. The total protein concentration of the collected vascular endothelial cells was measured using the BCA protein concentration measurement kit.

(2) Two-dimensional electrophoresis: the total protein of the vascular endothelial cells is extracted, subjected to two-dimensional electrophoresis, transferred to a nitrocellulose membrane, incubated with serum of a healthy person and a patient with nephrotic syndrome as primary antibodies respectively, and then developed by adding secondary antibodies, and the result is shown as A in figure 1 and B in figure 1.

(3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after visualization in step (2), protein spots were selected on two-dimensional electrophoresis gel which were strongly positive for nephrotic syndrome patients and negative or weakly positive for healthy persons, the selected protein spots were removed from the gel, the dried gel was digested with trypsin (0.1. mu.g/. mu.l), 10. mu.l of 25mM ammonium bicarbonate was added to the reaction mixture, incubated overnight at 37 ℃, and peptides were then extracted from the gel with trifluoroacetic acid (0.1%). Analyzing the extracted peptide by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass spectrum, and identifying the peptide mass spectrum as Talin-1 protein, as shown in figure 1C.

FIG. 1 is a graph showing the results that the Talin-1 protein on the endothelial cells of glomerular vessels is the main target antigen for autoantibodies in patients with nephrotic syndrome; wherein, A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of a healthy person; b: the first antibody is a two-dimensional electrophoresis protein spot of serum of a nephrotic syndrome patient; c: mass spectrometric identification of target antigen Talin-1 protein

Example 2

Expression and purification of recombinant antigen protein Talin-1

The gene of coding Talin-1 protein is used as a template by utilizing a genetic engineering method to carry out PCR amplification, and then an expression vector is constructed to carry out protein expression. The antigen protein expressed by the invention contains a tag peptide of His tag. The expressed recombinant protein was purified by nickel column affinity chromatography, and finally the molecular weight of the recombinant protein Talin-1 was identified by SDS-PAGE as 45KDa, as shown in fig. 2 (SDS-PAGE identification of the expressed recombinant protein Talin-1), wherein, lane a: supernatant of cell lysate, induced at 15 ℃ for 16 hours; lane B: cell lysate supernatant, induced at 37 ℃ for 4 hours.

Example 3

Optimization of kit reaction conditions by orthogonal experimental design

An orthogonal table was selected based on 4 factors such as the coating concentration of the antigen Talin-1 (four coating concentrations of 50. mu.g, 80. mu.g, 100. mu.g, 150. mu.g), the respective reaction times (15min, 30min, 45min) and temperatures (25 ℃ C., 37 ℃), the optimal dilution of the enzyme-labeled secondary antibody (four dilutions of 1:100, 1:500, 1:1000, 1: 1500), and the like, each factor repeatedly determining standard positive serum and standard negative serum at 2 levels, and the ratio (P/N) of the highest light signal value (P) of the positive serum to the lowest light signal value (N) of the negative serum was selected. The optimal antigen Talin-1 coating concentration of the kit is obtained through orthogonal design and is 80 mu g/ml, the optimal antigen-antibody reaction temperature of the anti-Talin-1-IgG antibody kit for solid-phase membrane immunodetection is 25 ℃, the optimal antigen-antibody reaction time is 30min, and the optimal work dilution of the anti-human IgG antibody of the optimal biotin labeling is 1: 500; the optimal antigen-antibody reaction temperature of the kit for detecting the anti-Talin-1-IgG antibody through magnetic particle chemiluminescence immunoassay is 37 ℃, the optimal antigen-antibody reaction time is 15min, and the optimal working dilution of the optimal acridinium ester labeled anti-human IgG antibody is 1: 500.

example 4

Preparation of solid-phase membrane immunoassay kit for detecting anti-Talin-1-IgG antibody

4.1 composition of solid-phase membrane immunoassay kit for detecting anti-Talin-1-IgG antibodies:

antigen: recombinant protein Talin-1

Solid phase carrier: sataurus CN140 nitrocellulose membrane

Positive quality control (standard): human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang)

Negative quality control product: serum for health physical examination person

Labeling the antibody: biotin-labeled anti-human IgG antibody

Antigen diluent

Sample dilution buffer

Antibody diluent

Cleaning solution

Enzyme working solution: alkaline phosphatase-streptavidin

Substrate color developing solution: BCIP color developing solution.

4.2 detection procedure of the solid-phase membrane immunoassay kit for detecting anti-Talin-1-IgG antibody is as follows:

4.2.1 coating, sealing: placing 8 μ l of Talin-1 antigen direct contact with the concentration of 80 μ g/ml on a nitrocellulose membrane in a 37 ℃ incubator for drying for 30min, placing the nitrocellulose membrane in a detection plate, adding 200 μ l of 5% BSA in a 37 ℃ incubator for sealing for 30min, discarding the sealing solution, and washing for 2 times with a washing solution;

4.2.2 antigen incubation: adding 10 μ l of antibody standard or serum to be detected diluted with diluent into the detection plate, performing negative control and positive control, incubating at 25 deg.C for 30min, and arranging 3 parallel holes for each sample;

4.2.3 Secondary antibody incubation: discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding 20 μ l of 1:500 biotin-labeled anti-human IgG antibody, and incubating at 25 deg.C for 30 min;

4.2.4 color development: discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding 500 μ l alkaline phosphatase-streptavidin, incubating at room temperature for 20min, discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding BCIP color developing solution, reacting at room temperature for 20min, washing the detection plate with running water, and terminating the enzyme reaction. Taking out the test nitrocellulose membrane strip, drying the membrane strip by using a blower, qualitatively judging by using a colorimetric card with naked eyes, wherein the result is shown in figure 3 (the result of detecting the anti-Talin-1-IgG antibody in the serum of the nephrotic syndrome patient by using a solid-phase membrane immunoassay kit), or placing the membrane strip on a developing instrument for scanning, and drawing a standard curve to perform semi-quantitative analysis on the anti-Talin-1-IgG antibody level in the serum by using analysis software carried by the developing instrument by using the reference standard substance concentration as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate.

Example 5

Preparation of magnetic particle chemiluminescence immunoassay kit for detecting anti-Talin-1-IgG antibody (FIG. 4 is a schematic diagram of the principle of detecting anti-Talin-1-IgG antibody by the magnetic particle chemiluminescence immunoassay kit)

5.1 anti-Talin-1-IgG antibody chemiluminescence detection kit, comprising the following components:

(1) acridinium ester labeled anti-human IgG;

(2) carboxyl magnetic beads coupled with Talin-1 antigen;

(3) chemiluminescent pre-excitation liquid A (H) ₂ O ₂ ) And chemiluminescent excitation liquid B (NaOH);

(4) anti-Talin-1-IgG antibody series standard solutions, standard concentrations: 0 mu g/ml, 2 mu g/ml, 4 mu g/ml, 8 mu g/ml, 16 mu g/ml and 20.0 mu g/ml, and the buffer solution is 5.0 percent BSA and 0.1 to 0.5 percent PC300 containing 0.5mol/L Tris-HCL;

(5) cleaning solutions, especially 25mmol/L Tris-HCl, pH 7.2, containing 0.15mol/L NaCl and 0.05% Tween-20.

5.2 preparation of magnetic bead coupled antigen (FIG. 5, schematic diagram of antigen protein Talin-1 coated carboxyl magnetic particles)

(1) 1mg of carboxyl magnetic particles are put into a 0.5mL centrifuge tube, 200 mu L of 0.1mol/L MES buffer solution is added, the mixture is evenly mixed by vortex, the mixture is placed on a magnetic frame and is kept stand for 5min, the magnetic particles are separated from liquid, and supernatant liquid is discarded. Wash 3 times, then add 200 μ Ι _ MES (pH 5.0) buffer and vortex;

(2) adding 18 μ l (18 μ g) of Talin-1 antigen, vortexing, rotating the reaction tube, and incubating at room temperature for 30 min;

(3) adding 10 mu l of 10mg/mL coupling reagent EDC, vortexing, rotating the reaction tube, and incubating for 2h at room temperature;

(4) the supernatant was removed and washed 3 times with 200. mu.l of washing buffer (TBS + 0.05% Tween-20);

(5) blocking with 1% BSA in buffer was repeated 4 times for 10min each. The magnetic particle suspension is stored at 2-8 ℃.

5.3 preparation of acridinium ester-labeled antibody

(1) Putting 100 μ L of anti-human IgG antibody into dialysis bag, putting the dialysis bag into not less than 1L of labeled buffer solution, dialyzing for at least 3 times, and dialyzing overnight for the last time, wherein the labeled buffer solution is Na ₂ CO ₃ -NaHCO ₃ A buffer solution with the pH of 10.1 and the concentration of 0.1 mol/L;

(2) weighing 1.7mg of acridinium ester NSP-DMAE-NHS and dissolving in 447 mu.l of anhydrous dimethylformamide DMF to form 6.5mmol/LNSP-DMAE-NHS DMF solution;

(3) placing the dialyzed antibody solution into a 500-mu-L centrifuge tube, adding 100-mu-L of 6.5mmol/LNSP-DMAE-NHS DMF solution, wherein the molar ratio of the acridinium ester to the antibody is 7.4:1, adding 200-mu-L of labeling buffer solution, reacting at room temperature for 45min, adding 10-mu-L of lysine for 10-mu-L, and continuing to react for 15min to terminate the labeling reaction;

(4) the marker NSP-DMAE-NHS-Ab was separated from free NSP-DMAE-NHS by Sephadex G-50 column (1X 25cm) with a purification buffer pH 6.3 and concentration 0.1 mol/L;

(5) during the separation process, detecting protein peaks by using a chromatograph, and respectively measuring the chemiluminescence intensity of effluent and the absorbance at 430 nm;

(6) the high-light, high-absorbance eluate was collected, 1% BSA (by volume) was added, and stored on ice.

5.4 sample preparation-dilution of the samples in a 1:10 ratio

5.5 detection procedures of the chemiluminescence kit for detecting anti-Talin-1-IgG antibody are as follows:

(1) sequentially adding 100 mu l of sample to be detected, 150 mu l of coupled magnetic powder suspension and 150 mu l of acridine ester labeled secondary antibody into a reaction tube, shaking up and mixing, and keeping the temperature at 37 ℃ for 15 min;

(2) washing for 5 times in an isolation way;

(3) fully shaking the washed reaction container to uniformly disperse the magnetic particles;

(4) the relative luminescence intensity was measured by adding 100. mu.l of the chemiluminescent pre-excitation liquid A followed by 100. mu.l of the chemiluminescent excitation liquid B. The content of anti-Talin-1-IgG antibody in the sample is directly proportional to the intensity of luminescence.

Example 6

Clinical application of kit for detecting anti-Talin-1-IgG antibody of serum

6.1 subject Admission

Patients diagnosed with various types of nephropathies from 6 months in 2018 to 6 months in 2020, including 298 Nephrotic Syndrome (NS), 100 anaphylactoid purpura (HP), 100 purpura nephritis (HPN), 100 Kawasaki Disease (KD), and 100 healthy children (NC) at the same time. Serum samples were taken from various renal patients and healthy controls. All subjects received a first serum sample collection prior to no immunosuppressive treatment.

6.2 detection of anti-Talin-1-IgG antibodies in various renal disease patients

The kit of the invention is used for detecting the anti-Talin-1-IgG antibody levels in the serum of patients diagnosed with various nephropathies from 6 months in 2018 to 6 months in 2020, including 298 nephrotic syndrome, 100 anaphylactoid purpura, 100 purpura nephritis, 100 Kawasaki disease and 100 healthy children in the same period, the result shows that anti-Talin-1-IgG antibody in part of nephrotic syndrome patients is positive (anti-Talin-1-IgG antibody in 150 patients is positive, namely the positive detection rate of anti-Talin-1-IgG antibody is 50.34%), while anti-Talin-1-IgG antibody in purpura nephritis, anaphylactoid purpura, Kawasaki disease and healthy children is negative, as shown in figure 6 (a detection condition chart of anti-Talin-1-IgG antibody in various nephrotic patients, wherein NS is nephrotic syndrome, HP is anaphylactoid purpura, HPN is purpura nephritis, KD: kawasaki disease, NC: a healthy child). The detection of the presence of anti-Talin-1-IgG antibodies in the serum contributes to the determination of the vascular endothelial lesions of nephrotic syndrome.

6.3 serum anti-Talin-1-IgG antibody of patient with nephrotic syndrome is linearly related to the expression level of vascular endothelial injury marker

The kit is used for detecting the expression quantity of the anti-Talin-1-IgG antibody in the serum of a patient with nephrotic syndrome diagnosed from 6 months in 2018 to 6 months in 2020, and detecting the expression quantity of a vascular endothelial injury marker Plvap in the serum of the patient, and the result shows that the expression quantity of the anti-Talin-1-IgG antibody in the patient with nephrotic syndrome is linearly related to the expression quantity of the vascular endothelial injury marker, the nephrotic syndrome is related to vascular endothelial injury, and the detection of the anti-Talin-1-IgG autoantibody can be used for judging vascular endothelial injury, namely the anti-Talin-1-IgG autoantibody is detected, and then the vascular endothelial injury is judged, and is shown in figure 7 (a result chart of linear correlation of the anti-Talin-1-IgG antibody and the vascular endothelial injury marker).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Zhejiang university

<120> application of reagent for detecting anti-Talin-1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

<160> 1

<170> SIPOSequenceListing 1.0

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<211> 356

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Gly Asn Ser Cys Arg Gln Glu Asp Val Ile Ala Thr Ala Asn Leu

1 5 10 15

Ser Arg Arg Ala Ile Ala Asp Met Leu Arg Ala Cys Lys Glu Ala Ala

20 25 30

Tyr His Pro Glu Val Ala Pro Asp Val Arg Leu Arg Ala Leu His Tyr

35 40 45

Gly Arg Glu Cys Ala Asn Gly Tyr Leu Glu Leu Leu Asp His Val Leu

50 55 60

Leu Thr Leu Gln Lys Pro Ser Pro Glu Leu Lys Gln Gln Leu Thr Gly

65 70 75 80

His Ser Lys Arg Val Ala Gly Ser Val Thr Glu Leu Ile Gln Ala Ala

85 90 95

Glu Ala Met Lys Gly Thr Glu Trp Val Asp Pro Glu Asp Pro Thr Val

100 105 110

Ile Ala Glu Asn Glu Leu Leu Gly Ala Ala Ala Ala Ile Glu Ala Ala

115 120 125

Ala Lys Lys Leu Glu Gln Leu Lys Pro Arg Ala Lys Pro Lys Glu Ala

130 135 140

Asp Glu Ser Leu Asn Phe Glu Glu Gln Ile Leu Glu Ala Ala Lys Ser

145 150 155 160

Ile Ala Ala Ala Thr Ser Ala Leu Val Lys Ala Ala Ser Ala Ala Gln

165 170 175

Arg Glu Leu Val Ala Gln Gly Lys Val Gly Ala Ile Pro Ala Asn Ala

180 185 190

Leu Asp Asp Gly Gln Trp Ser Gln Gly Leu Ile Ser Ala Ala Arg Met

195 200 205

Val Ala Ala Ala Thr Asn Asn Leu Cys Glu Ala Ala Asn Ala Ala Val

210 215 220

Gln Gly His Ala Ser Gln Glu Lys Leu Ile Ser Ser Ala Lys Gln Val

225 230 235 240

Ala Ala Ser Thr Ala Gln Leu Leu Val Ala Cys Lys Val Lys Ala Asp

245 250 255

Gln Asp Ser Glu Ala Met Lys Arg Leu Gln Ala Ala Gly Asn Ala Val

260 265 270

Lys Arg Ala Ser Asp Asn Leu Val Lys Ala Ala Gln Lys Ala Ala Ala

275 280 285

Phe Glu Glu Gln Glu Asn Glu Thr Val Val Val Lys Glu Lys Met Val

290 295 300

Gly Gly Ile Ala Gln Ile Ile Ala Ala Gln Glu Glu Met Leu Arg Lys

305 310 315 320

Glu Arg Glu Leu Glu Glu Ala Arg Lys Lys Leu Ala Gln Ile Arg Gln

325 330 335

Gln Gln Tyr Lys Phe Leu Pro Ser Glu Leu Arg Asp Glu His His His

340 345 350

His His His His

355

Claims

1. The application of the reagent for detecting the anti-Talin-1-IgG autoantibody in the preparation of the kit for detecting the vascular endothelial injury.

2. The use according to claim 1, characterized in that the reagents for the detection of anti-Talin-1-IgG autoantibodies comprise a Talin-1 protein or a Talin-1 recombinant protein or polypeptide comprising a tag; the NCBI protein accession number of the Talin-1 protein is BC 042923.

3. The use of claim 2, wherein the tag comprises a His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag, or biotin tag.

4. The use according to claim 2, wherein the amino acid sequence of said tag-containing Talin-1 recombinant protein comprises SEQ ID No.1, when said tag is a His-tag.

5. The use of claim 1, wherein the vascular endothelial injury comprises glomerular vascular endothelial cell injury.

6. A kit for detecting anti-Talin-1-IgG autoantibodies, comprising: a reagent for detecting an anti-Talin-1-IgG autoantibody, a solid-phase carrier and a labeled antibody for use according to any one of claims 1 to 5.

7. The kit of claim 6, wherein the solid phase carrier comprises a nitrocellulose membrane, a fluorescence-encoded microsphere, a magnetic stripe chip, a magnetic microparticle and/or an enzyme-labeled microplate.

8. The kit of claim 6, wherein the labeled antibody comprises an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody; the secondary antibody comprises an anti-human IgG antibody.

9. The kit of claim 8, wherein the enzyme-labeled secondary antibody comprises a horseradish peroxidase-labeled anti-human IgG antibody; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.