CN114910647A

CN114910647A - Application of filamin-A-IgG antibody in preparation of kit for detecting vascular endothelial injury

Info

Publication number: CN114910647A
Application number: CN202210500157.5A
Authority: CN
Inventors: 叶青; 毛建华; 田丹丹
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-08-16

Abstract

The invention provides an application of a filamin-A-IgG antibody in preparing a kit for detecting vascular endothelial injury. The kit of the invention uses IgG antibody of human anti-tag peptide as standard substance, and combines biotin-avidin amplification system and magnetic particle chemiluminescence immunoassay to greatly improve the accuracy, sensitivity, specificity and detection speed of detection. The invention takes the IgG antibody of human anti-tag peptide as the standard substance, thus greatly improving the detection accuracy. Meanwhile, 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. The kit for qualitatively detecting the anti-filaggrin-A-IgG antibody in human serum by solid-phase membrane immunization is introduced into a biotin-avidin amplification system, so that the detection sensitivity is greatly improved, the high-efficiency detection of vascular endothelial injury can be realized, and the vascular endothelial injury is judged to exist when the anti-filaggrin-A-IgG autoantibody is detected.

Description

Application of filamin-A-IgG antibody in preparation of kit for detecting vascular endothelial injury

Technical Field

The invention belongs to the technical field of biomedicine, and relates to application of a filamin-A-IgG antibody in preparing a kit for detecting vascular endothelial injury, in particular to application of a reagent for detecting an anti-filamin-A-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.

Background

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, are a layer of mononuclear cells between blood flow and vascular wall tissues, and can secrete a series of vasoactive substances such as NO, PGI2, ET-1 and the like through three ways of autocrine, endocrine and paracrine to play the functions of regulating the blood vessel tone, resisting thrombosis, inhibiting smooth muscle cell proliferation, inhibiting vascular wall inflammatory reaction and the like. NO is the most important vasodilator factor produced by endothelial cells, and is generated by the action of NO synthase (eNOS) of the endothelial cells on L-arginine, and the NO can diffuse to vascular wall smooth muscle cells to activate ornithine cyclase and mediate cGMP-regulated vasodilation. Moreover, NO also has the effects of inhibiting platelet aggregation, inhibiting monocyte adhesion to endothelial cells, and inhibiting smooth muscle cell proliferation. However, when the vascular endothelium is affected by a series of harmful factors, the release of the vasomotor factors by endothelial cells is reduced, the vasomotor factors are increased, the vascular equilibrium is broken, and finally a series of cardiovascular events are caused. The vascular endothelial cell autoantibody can cause vascular endothelial cell damage and induce dysfunction of blood circulation system, thereby causing damage to organs such as heart, lung, liver and the like and causing diseases related to organs, including nephrotic syndrome.

Minimal disease (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, the underlying pathogenesis of MCD is still poorly understood. One of the views 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 Futrakul N 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 unclear whether there was simultaneous damage 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 internationally for the first time: 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.

Filamin-a (Filamin-a) is the first actin-crosslinking protein found in non-myogenic cells. It can be combined with more than 90 kinds of ligands, including pathway protein, intracellular signal molecule and transcription factor, etc., and has the functions of integrating cell mechanics and signal transduction. Filamin-A is widely researched in the field of tumors, such as breast cancer, prostate cancer, pancreatic cancer, lung cancer and the like. For example, in some studies, it is found that Filamin-A can continuously monitor the migration and infiltration of breast cancer in breast cancer cells by regulating the dynamic change of focal adhesion, and Filamin-A is expected to be used as a specific and sensitive detection index for sorting metastatic breast cancer patients for prognosis evaluation. Filamin-A is expressed in benign prostate disease, prostate epithelial tumors and clinically localized cancer tissues higher than metastatic prostate cancer. In addition, Filamin-A has been shown to be involved in some autoimmune diseases. anti-Filamin-A antibodies are found in large amounts in the serum of patients with myasthenia gravis and in the serum of mice with chronic graft versus host disease leading to glomerulonephritis. However, the expression of Filamin-A and the presence of autoantibodies to Filamin-A have not been reported in nephrotic syndrome. In addition, the prior art does not relate to the application of the target-based Filamin-A or the autoantibody thereof as a serological marker in nephrotic syndrome. The Filamin-A-IgG 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. However, there is currently a lack of corresponding clinical test kits on the market.

Disclosure of Invention

The invention aims to provide application of a Filamin-A-IgG antibody in preparing a kit for detecting vascular endothelial injury, and particularly relates to application of a reagent for detecting an anti-Filamin-A-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The detection of the anti-Filamin-A-IgG autoantibody can realize the effective detection of the vascular endothelial injury.

Preferably, the reagent for detecting the anti-Filamin-A-IgG autoantibody comprises Filamin-A protein or Filamin-A recombinant protein or polypeptide containing a label, wherein NCBI protein accession number of the Filamin-A protein is BC 014654.

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

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

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

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

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 an anti-Filamin-A-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The invention firstly detects an anti-Filamin-A-IgG autoantibody in a part of patients with nephrotic syndrome, and determines that a target antigen aimed by the autoantibody is Filamin-A on glomerular vascular endothelial cells. The invention finds that the Filamin-A 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-Filamin-A-IgG autoantibody can realize the detection of the 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-Filamin-A-IgG autoantibody provided by the invention can qualitatively and quantitatively detect the anti-Filamin-A-IgG antibody in serum of a nephrotic syndrome patient, 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 beneficial effects:

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

2. At present, the Filamin-A and anti-Filamin-A-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 Filamin-A for the first time, invents a detection kit aiming at the Filamin-A-IgG autoantibody and fills the blank at home and abroad. The kit disclosed by the invention is used for detecting the anti-Filamin-A-IgG antibodies in the serum of 298 nephrotic syndrome patients, and the result shows that the anti-Filamin-A-IgG antibodies of 144 patients are positive, namely the positive detection rate of the anti-Filamin-A-IgG antibodies is 48.32%. The invention can provide a basis for researching the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment after detecting the anti-Filamin-A-IgG antibody.

3. The kit of the invention relates to a solid-phase membrane immunoassay qualitative analysis of an anti-Filamin-A-IgG antibody in human serum, and the detection accuracy is greatly improved by taking the human anti-tag 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 strong and 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-Filamin-A-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 invention relates to a magnetic particle chemiluminescence immunoassay quantitative detection kit for anti-Filamin-A-IgG antibody in human serumThe magnetic particle is used as solid phase carrier, and its diameter is only 1.0 micrometer, so that it can greatly increase coating surface area, increase adsorption quantity of antigen, raise reaction speed and make cleaning and separation more simple and convenient so as to reduce pollution and reduce cross infection probability. 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: the Filamin-A protein on the endothelial cell of glomerular blood vessel is the main target antigen for the autoantibody in the body of the patient with nephrotic syndrome. FIG. 1A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; FIG. 1B: the first antibody is a two-dimensional electrophoresis protein spot of serum of a nephrotic syndrome patient; FIG. 1C: and (3) identifying the mass spectrum of the target antigen Filamin-A protein.

FIG. 2 shows the expression and identification of recombinant protein Filamin-A, FIG. 2A: SDS-PAGE identification picture of recombinant protein Filamin-A expressed under different induction conditions; FIG. 2B: SDS-PAGE identification chart of the recombinant protein Filamin-A obtained by different purification schemes.

FIG. 3: the solid-phase membrane immunoassay kit is used for detecting an anti-Filamin-A-IgG antibody in serum of a patient with nephrotic syndrome.

FIG. 4: schematic diagram of the principle of detecting the anti-Filamin-A-IgG antibody by the magnetic particle chemiluminescence immunoassay kit.

FIG. 5: schematic diagram of protein antigen Filamin-A coated carboxyl magnetic particles.

FIG. 6: detection of anti-Filamin-A-IgG antibodies in various renal disease patients, wherein NS: nephrotic syndrome, HP: allergic purpura, HPN: purpuric nephritis, KD: kawasaki disease, IgAN: IgA nephropathy, NC: healthy children.

FIG. 7: graph showing the results of linear correlation analysis of the anti-Filamin-A-IgG antibody level and the glomerular vascular endothelial cell injury marker Plvap.

Detailed Description

The invention provides application of a reagent for detecting an anti-Filamin-A-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. Filamin-a (Filamin-a) is the first actin-crosslinking protein found in non-myogenic cells. It can be combined with more than 90 ligands, including pathway protein, intracellular signal molecule and transcription factor, etc., and has the functions of integrating cell mechanics and signal transduction. 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 circulatory 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-Filamin-A-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-Filamin-A-IgG autoantibody provided by the invention takes the Filamin-A protein as a target spot to detect the Filamin-A autoantibody (namely, the anti-Filamin-A-IgG autoantibody is a biomarker for detecting the injury of vascular endothelial cells), and the reagent can realize high-efficiency detection of the injury of the vascular endothelial cells. In the present invention, the agent is capable of immunoreacting with the Filamin-A protein autoantibody from tissue (kidney biopsy) or body fluids (in particular blood, plasma, serum). In the invention, the reagent for detecting the anti-Filamin-A-IgG autoantibody preferably comprises Filamin-A protein or Filamin-A recombinant protein or polypeptide containing a label; the NCBI protein accession number of the Filamin-A 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, c-Myc tag, Flag tag, or biotin tag. In the invention, when the tag is a His tag, the amino acid sequence of the Filamin-A recombinant protein containing the tag is preferably as shown in SEQ ID NO. 1:

MNQPASFAVSLNGAKGAIDAKVHSPSGALEECYVTEIDQDKYAVRFIPRENGVYLIDVKFNGTHIPGSPFKIRVGEPGHGGDPGLVSAYGAGLEGGVTGNPAEFVVNTSNAGAGALSVTIDGPSKVKMDCQECPEGYRVTYTPMAPGSYLISIKYGGPYHIGGSPFKAKVTGPRLVSNHSLHETSSVFVDSLTKATCAPQHGAPGPGPADASKVVAKGLGLSKAYVGQKSSFTVDCSKAGNNMLLVGVHGPRTPCEEILVKHVGSRLYSVSYLLKDKGEYTLVVKWGDEHIPGSPYRVVVPHHHHHH。

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 minimal disease or primary focal segmental glomerulosclerosis.

In the present invention, the reagent for detecting an anti-Filamin-A-IgG autoantibody (Filamin-A protein or Filamin-A recombinant protein containing a tag) is preferably immobilized on a solid phase carrier. By "immobilized" herein is meant bound to a solid support that is insoluble in water of the Filamin-a antigenic 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 comprises fixing the Filamin-A antigen protein in a reversible or irreversible mode. 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 addition of 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 Filamin-A fixing method is preferably a direct coating method: (1) the antigen protein Filamin-A is combined on a nitrocellulose membrane or a polystyrene microporous plate in a physical adsorption mode or by non-covalent bonds; (2) the magnetic particles with the carboxyl functional groups are combined with the amino group of the antigen protein Filamin-A, and the antigen protein Filamin-A is combined on the magnetic particles in a chemical coupling mode. In the present invention, the solid phase carrier preferably comprises a nitrocellulose membrane, a magnetic microparticle or an enzyme-labeled microplate.

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

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

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

In the present invention, the enzyme-labeled secondary antibody preferably 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 comprises 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 invention, the standard substance and the positive quality control substance are preferably recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-Filamin-A-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 reagent for detecting the anti-Filamin-A-IgG autoantibody, namely the antigen, is preferably recombinant protein Filamin-A (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; substrate color developing agent, antigen diluent, sample diluent buffer, antibody diluent and washing liquid. The antigen diluent is 1x PBS pH7.4 containing 163mM NaCl and 1% TritonX-100; the sample dilution buffer was 0.01M PBS ph7.4 containing 5% BSA; the antibody diluent is 0.01M PBS pH7.4 containing 1M D-glucose, 2% glycerol, 0.5% Tween 20; the washing solution is as follows: 1 XPBS pH7.4 containing 163mM NaCl, 5% glycerol, 1% TritonX-100; the substrate color developing agent is hydrogen peroxide, TMB, 4-MUP, AMPPD or BCIP.

When the kit is a magnetic particle chemiluminescence immunoassay kit, in the kit, the antigen is preferably recombinant protein Filamin-A (an 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-Filamin-A-IgG autoantibody with different concentrations; the cleaning solution is preferably a Tris-HCl solution at pH 7.2, 25mmol/L, 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-Filamin-A-IgG antibody in serum is preferably as follows: by utilizing the indirect method reaction principle, firstly, Filamin-A antigen is adsorbed on a solid phase carrier 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, then a labeled antibody (labeled secondary antibody) is added for reaction, if the serum to be detected contains an anti-Filamin-A-IgG antibody, a ternary complex of the coating antigen Filamin-A-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 aim of qualitatively or quantitatively analyzing the anti-human serum Filamin-A-IgG antibody is fulfilled.

The application of the reagent for detecting anti-Filamin-A-IgG autoantibody in the preparation of a kit for detecting vascular endothelial injury according to the present invention will be described in further detail with reference to the following specific examples, which include, but are not limited to, the following examples.

Example 1Filamin-A protein on the endothelial cells of glomerular vessels is the main target antigen for autoantibodies in patients with nephrotic syndrome

According to the invention, a large number of clinical and molecular mechanism researches at the early stage are carried out, the serum IgG level of a patient with nephrotic syndrome is found to be high for the first time, and the Filamin-A on the vascular endothelial cells is proved to be a main target antigen for the autoantibody in the patient with nephrotic syndrome. It would therefore be advantageous to detect the presence and quantitative levels of anti-Filamin-A-IgG antibodies in serum to aid in the early identification of nephrotic syndrome, particularly in screening patients for symptoms of interest. Specifically, the following (1) extraction of total protein of vascular endothelial cells is carried out: 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 then 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: extracting total protein of vascular endothelial cell, performing two-dimensional electrophoresis, transferring to nitrocellulose membrane, incubating with serum of healthy person and nephrotic syndrome patient as primary antibody, and developing with secondary antibody (see fig. 1A and 1B, wherein the primary antibody is two-dimensional electrophoresis protein spot of serum of healthy person, and the primary antibody is two-dimensional electrophoresis protein spot of serum of nephrotic syndrome patient). (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 with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain peptide mass spectrum, and identifying as Filamin-A protein, shown in FIG. 1C.

Example 2 recombinant protein antigen Filamin-A expression and purification

The gene of Filamin-A protein is used as a template to carry out PCR amplification by using a genetic engineering method, and then an expression vector pET-30a is constructed to carry out protein expression. In order to express high-concentration recombinant protein, the invention respectively optimizes the expression conditions of the target protein, including different induction time of an inducer, culture temperature of the recombinant bacteria, and broken bacteria supernatant and broken bacteria sediment of the induced expression recombinant bacteria. The broken cell supernatants and precipitates of the recombinant bacteria induced at 37 ℃ for 4 hours and 15 ℃ for 16 hours are respectively collected and subjected to SDS-PAGE electrophoresis, and the result shows that the expression level of the recombinant protein Filamin-A in the cell lysate supernatant of the recombinant bacteria collected at 15 ℃ for 16 hours is the maximum (strip 2), and the expression level accounts for 30% of the total protein amount of the bacteria, which is shown in figure 2A. The protein antigen expressed by the invention contains His-tagged tag peptide, then the recombinant protein purification scheme expressed in the collected jejunum supernatant is optimized, the scheme 1 is to carry out purification by nickel column affinity chromatography, molecular sieve, hydrophobic column and ion affinity chromatography, the scheme 2 is to carry out purification by nickel column affinity chromatography, molecular sieve chromatography, gel filtration chromatography, ion exchange column chromatography and hydrophobic column purification, and the protein antigen expressed by the invention contains His-tagged tag peptide. The expressed recombinant protein is purified by nickel column affinity chromatography, molecular sieve, hydrophobic column, ion affinity chromatography and the like, and finally SDS-PAGE is used to identify the molecular weight of the recombinant protein Filamin-A to be 32.34KDa, and the result shows that the concentration of the recombinant protein obtained by purifying the scheme 1 (the purification scheme of the invention) is the highest (a band 1), and the figure is 2B.

Example 3 the present invention optimizes the reaction conditions of the kit by orthogonal assay design

Orthogonal tables are selected according to 4 factors such as the antigen Filamin-A coating concentration (four coating concentrations of 100. mu.g, 150. mu.g, 250. mu.g and 300 ug), each reaction time (15min, 30min and 45min) and temperature (25 ℃ and 37 ℃), the enzyme-labeled secondary antibody optimal dilution (four dilutions of 1:100, 1:500, 1:750 and 1: 1000), each factor repeatedly measures 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 is selected. The optimal antigen Filamin-A coating concentration of the kit is 250 mu g/ml, the optimal antigen-antibody reaction temperature of the solid-phase membrane immunoassay anti-Filamin-A-IgG antibody kit is 25 ℃, the optimal antigen-antibody reaction time is 30min, and the optimal work dilution of an optimal biotin-labeled anti-human IgG antibody is 1: 750; the optimal antigen-antibody reaction temperature of the kit for detecting the anti-Filamin-A-IgG antibody by 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: 750.

EXAMPLE 4 preparation of solid-phase Membrane immunoassay kit for detecting anti-Filamin-A-IgG antibody

4.1 composition of solid phase membrane immunoassay kit for detecting anti-Filamin-A-IgG antibody:

1. antigen: the recombinant protein Filamin-A is,

2. solid phase carrier: a cellulose nitrate membrane of Satourius CN140,

3. positive quality control (standard): human anti-His tag immunoglobulin G (purchased from invitro lake),

4. negative quality control product: the serum of a healthy physical examination person is obtained,

5. labeling the antibody: the anti-human IgG antibody is marked by biotin,

6. an antigen diluent is added to the antigen-containing solution,

7. the buffer solution is diluted by the sample,

8. the dilution liquid of the antibody is used as the antibody,

9. the washing liquid is used for washing the surface of the workpiece,

10. enzyme working solution: alkaline phosphatase-streptavidin (streptavidin) was added to the alkaline phosphatase,

11. substrate color developing solution: BCIP color developing solution.

4.2 the detection steps of the solid-phase membrane immunoassay kit for detecting the anti-Filamin-A-IgG antibody are as follows:

4.2.1 coating, sealing: placing 8 μ l of Filamin-A antigen direct contact with concentration of 250 μ g/ml on nitrocellulose membrane, drying in 37 deg.C incubator for 30min, placing nitrocellulose membrane in detection plate, adding 200 μ l of 5% BSA, sealing in 37 deg.C incubator for 30min, discarding the sealing solution, and washing with washing solution for 2 times;

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. And (3) taking out the test nitrocellulose membrane strip, drying the membrane strip by using a blower, qualitatively judging by using a colorimetric card by naked eyes, and drawing a standard curve to perform semi-quantitative analysis on the antibody level of the anti-Filamin-A-IgG in the serum by using the analysis software carried by the developing instrument and taking the concentration of the reference standard as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate, wherein the positive spot is shown in figure 3, or the membrane strip is placed on the developing instrument for scanning.

EXAMPLE 5 preparation of magnetic particle chemiluminescence immunoassay kit for detecting anti-Filamin-A-IgG antibody

5.1 magnetic particle chemiluminescence immunoassay kit for detecting anti-Filamin-A-IgG antibody comprises:

1. antigen: the recombinant protein Filamin-A is,

2. solid phase carrier: magnetic fine particles of a carboxyl functional group,

5. labeling the antibody: an acridinium ester-labeled anti-human IgG antibody,

6. an antigen diluent is added to the antigen-containing solution,

7. the buffer solution is diluted by the sample,

8. an antibody diluent is added to the mixture of the antibody and the water,

9. the washing liquid is used for washing the glass fiber,

10. pre-excitation liquid: h ₂ O ₂ ，

11. Excitation liquid: NaOH.

5.2 detection principle of magnetic particle chemiluminescence immunoassay kit for detecting anti-Filamin-A-IgG antibody

The chemiluminescence immunoassay kit is an analysis method combining a magnetic separation technology, an immunoassay technology and a chemiluminescence technology. The kit of the invention adopts an indirect method to quantitatively analyze and detect the anti-Filamin-A-IgG antibody in human serum: firstly, the solution of Filamin-A antigen coated magnetic particles is dilutedMixing samples, binding specific anti-Filamin-A-IgG antibody to the magnetic particles coated by the Filamin-A antigen, washing, adding acridinium ester labeled anti-human IgG antibody to form a composite of the magnetic particles coated by the Filamin-A antigen, the anti-Filamin-A-IgG antibody and the acridinium ester labeled anti-human IgG antibody, separating the unbound substances from the composite formed by the immunoreaction under the action of an external magnetic field, removing the supernatant, washing the deposited composite, and adding a pre-excitation liquid (H) ₂ O ₂ ) Performing luminescence reaction with exciting liquid (NaOH), wherein under alkaline condition, acridine ester molecule is attacked by hydrogen peroxide to generate dioxyethane which is unstable and decomposed into CO ₂ And an electronically excited state of N-methylacridone which emits light having a wavelength of 430nm when it returns to the ground state, and the intensity of the emitted light is collected using a chemiluminescence apparatus. The concentration of the anti-Filamin-A-IgG antibody in the serum to be detected is in direct proportion to the luminous value, and the concentration of the anti-Filamin-A-IgG antibody in the serum to be detected is calculated through a calibration curve, which is shown in figure 4.

5.3 preparation of Filamin-A antigen coated magnetic particle

5.3.1 the principle of the Filamin-A antigen coated magnetic particle: based on the fact that carboxyl functional groups contained on the surface of the magnetic particles react with EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide) solution to generate unstable amino active O-acylurea intermediate, the intermediate reacts with NHS (N-hydroxysuccinimide) to generate semi-stable amino active NHS ester, and the semi-stable amino active NHS ester reacts with amino on protein antigen Filamin-A to form the Filamin-A antigen coated magnetic particles, as shown in figure 5.

5.3.2EDC/NHS activated carboxyl magnetic particles, which comprises the following steps:

a) weighing 10mg of magnetic particles, washing the magnetic particles for 3 times by using 20mM MES, separating by using a magnet, and removing a supernatant;

b) resuspending the washed magnetic particles in 100. mu.l of 20mM MES to give a final magnetic particle concentration of 100 mg/ml;

c) sequentially adding 50 μ l of 20mg/ml EDC and 50 μ l of 24mg/ml Sμ lfo-NHS prepared by phosphate buffer solution into the cleaned magnetic particles, fully mixing, standing and activating at room temperature for 30 min;

d) after the action of the external magnetic field, the supernatant is discarded, 400 mul of 0.05M phosphate buffer solution is taken to wash the magnetic particles, and 400 mul of preservation solution is added for constant volume preservation and standby.

5.3.3 activation of magnetic particles and protein antigen Filamin-A crosslinking to the activated magnetic particles solution in addition to pre-cooled 1ml 20mM MES to continue cleaning magnetic particles for 2 times; adding 200 mu l of 2mg/ml protein antigen Filamin-A into the activated magnetic particles, fully and uniformly mixing, and standing at room temperature for reaction for 16 hours; after the reaction is finished, adding a PBS buffer solution with the pH of 7.4 and containing 0.2 percent Tween20, and repeatedly washing the magnetic particles for 2 times; then adding PBS buffer solution with pH of 7.4 containing 0.2% Tween20 and 0.2% BSA to a final concentration of 10mg/ml, mixing, standing at room temperature for 30 min; after the reaction was completed, the supernatant was discarded, and the magnetic microparticles were resuspended in a pH7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA, so that the activated magnetic microparticles were crosslinked with the protein antigen Filamin-A.

The preparation of the acridinium ester labeled anti-human IgG antibody specifically comprises the following steps:

a) preparing 2mg/mL acridinium ester solution by using dimethylformamide;

b) preparing 1mg/mL anti-human IgG antibody by using 0.2M (pH8.0) carbonate buffer solution;

c) taking acridinium ester with a molar ratio of 4:1 and an anti-human IgG antibody, fully and uniformly mixing, and reacting for 40 min;

d) the reaction was stopped by adding 20. mu.l of carbonate buffer containing 5% lysine;

e) and desalting to remove impurities to obtain the acridinium ester labeled anti-human IgG antibody solution with high purity.

5.5 magnetic particle chemiluminescence immunoassay kit for detecting anti-Filamin-A-IgG antibody in serum

5.5.1 adding 100 μ l diluted serum or IgG standard substance resisting His label into 100 μ l Filamin-A antigen coated magnetic particle solution, reacting at 37 deg.C for 15min, and making negative and positive control;

5.5.2 adding 400 u l washing liquid to the labeled antibody and washing 3 times x 1min, adding 100 u l 1:500 diluted acridinium ester labeled anti-human IgG antibody, reacting at 37 ℃ for 15 min;

5.5.3 Signal detection 400. mu.l washing solution was washed 3 times x 1min, 1 was added00 μ l of Pre-excitation liquid (H) ₂ O ₂ ) And 100. mu.l of an excitation solution (NaOH) was reacted. And detecting the luminescence signal by a chemiluminescence instrument, and recording the luminescence value. The concentration of the anti-Filamin-A-IgG antibody in the serum to be detected is in direct proportion to the luminous value, and the concentration of the anti-Filamin-A-IgG antibody in the serum to be detected is calculated through a standard curve.

Example 6 clinical application of kit for detecting serum anti-Filamin-A-IgG antibody

6.1 Subjects included patients diagnosed with various types of nephropathies from 6 months in 2018 to 6 months in 2020, including 298 Nephrotic Syndrome (NS), 100 Henoch-Schonlein purpura (HP), 100 Henoch-Schonlein 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-Filamin-A-IgG antibodies in various nephrotic patients the kit of the present invention was used to detect the levels of anti-Filamin-A-IgG antibodies in the sera of patients diagnosed with various nephropathies, including 298 nephrotic syndrome, 100 Henoch-Schonlein purpura, 100 Henoch-Schonlein nephritis, 100 Kawasaki disease and 100 healthy children at the same time, and the results showed that anti-Filamin-A-IgG antibodies were positive in 144 nephrotic syndrome patients, while anti-Filamin-A-IgG antibodies in Henoch-Schonlein nephritis, Henoch-Schonlein purpura, Kawasaki disease and healthy children were negative, as shown in FIG. 6.

6.3 correlation analysis of anti-Filamin-A-IgG antibody and glomerular vascular endothelial cell injury marker Plvap level Using the kit of the present invention, the expression level of anti-Filamin-A-IgG antibody in the serum of patients diagnosed with nephrotic syndrome from 6 to 6, 2018 and 2020, and the expression level of vascular endothelial injury marker Plvap in the serum of patients were detected, which showed that the expression level of anti-Filamin-A-IgG antibody in nephrotic syndrome patients was linearly correlated with the expression level of vascular endothelial injury marker, nephrotic syndrome was related to vascular endothelial injury, Y ═ 0.442+0.02z70x, r ═ 0.49, P < 0.001, as shown in FIG. 7.

Sequence listing

<110> Zhejiang university

Application of <120> filamin-A-IgG antibody in preparation of vascular endothelial injury detection kit

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 307

<212> PRT

<213> Artificial sequence (Unknow)

<400> 1

Met Asn Gln Pro Ala Ser Phe Ala Val Ser Leu Asn Gly Ala Lys Gly

1 5 10 15

Ala Ile Asp Ala Lys Val His Ser Pro Ser Gly Ala Leu Glu Glu Cys

20 25 30

Tyr Val Thr Glu Ile Asp Gln Asp Lys Tyr Ala Val Arg Phe Ile Pro

35 40 45

Arg Glu Asn Gly Val Tyr Leu Ile Asp Val Lys Phe Asn Gly Thr His

50 55 60

Ile Pro Gly Ser Pro Phe Lys Ile Arg Val Gly Glu Pro Gly His Gly

65 70 75 80

Gly Asp Pro Gly Leu Val Ser Ala Tyr Gly Ala Gly Leu Glu Gly Gly

85 90 95

Val Thr Gly Asn Pro Ala Glu Phe Val Val Asn Thr Ser Asn Ala Gly

100 105 110

Ala Gly Ala Leu Ser Val Thr Ile Asp Gly Pro Ser Lys Val Lys Met

115 120 125

Asp Cys Gln Glu Cys Pro Glu Gly Tyr Arg Val Thr Tyr Thr Pro Met

130 135 140

Ala Pro Gly Ser Tyr Leu Ile Ser Ile Lys Tyr Gly Gly Pro Tyr His

145 150 155 160

Ile Gly Gly Ser Pro Phe Lys Ala Lys Val Thr Gly Pro Arg Leu Val

165 170 175

Ser Asn His Ser Leu His Glu Thr Ser Ser Val Phe Val Asp Ser Leu

180 185 190

Thr Lys Ala Thr Cys Ala Pro Gln His Gly Ala Pro Gly Pro Gly Pro

195 200 205

Ala Asp Ala Ser Lys Val Val Ala Lys Gly Leu Gly Leu Ser Lys Ala

210 215 220

Tyr Val Gly Gln Lys Ser Ser Phe Thr Val Asp Cys Ser Lys Ala Gly

225 230 235 240

Asn Asn Met Leu Leu Val Gly Val His Gly Pro Arg Thr Pro Cys Glu

245 250 255

Glu Ile Leu Val Lys His Val Gly Ser Arg Leu Tyr Ser Val Ser Tyr

260 265 270

Leu Leu Lys Asp Lys Gly Glu Tyr Thr Leu Val Val Lys Trp Gly Asp

275 280 285

Glu His Ile Pro Gly Ser Pro Tyr Arg Val Val Val Pro His His His

290 295 300

His His His

305

Claims

1. An application of a filamin-A-IgG antibody in preparing a kit for detecting vascular endothelial injury is characterized in that the application of a reagent for detecting an anti-filamin-A-IgG autoantibody in preparing the kit for detecting the vascular endothelial injury.

2. The use of claim 1, wherein the reagent for detecting anti-filamin-a-IgG autoantibodies comprises filamin-a protein or a tagged filamin-a recombinant protein or polypeptide; the NCBI protein accession number of the filamin-a protein is BC 014654.

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

4. The use according to claim 2, wherein when the tag is a His-tag, the amino acid sequence of the tagged filamin-a recombinant protein is as set forth in SEQ ID No. 1.

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

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

7. 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.

8. The kit of claim 7, 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.