CN114720700A

CN114720700A - Application of reagent for detecting anti-cytoskeleton-associated protein4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

Info

Publication number: CN114720700A
Application number: CN202210491015.7A
Authority: CN
Inventors: 叶青; 毛建华; 刘颖
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2022-05-07
Filing date: 2022-05-07
Publication date: 2022-07-08

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of Cytoskeleton-associatedprotein4 in preparation of a vascular endothelial cell injury detection kit. The invention also provides a kit for detecting the anti-Cytoseleton-associatedprotein 4-IgG antibody, which is used for qualitatively and quantitatively detecting the anti-Cytoseleton-associatedprotein 4-IgG antibody in a biological sample, has higher accuracy and sensitivity, is simple, convenient and quick, and fills the blank of the kit for detecting the anti-Cytoseleton-associatedprotein-IgG antibody in the field.

Description

Application of reagent for detecting anti-cytoskeleton-associated protein4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of Cytoskeleton-associated protein4 in preparation of a vascular endothelial cell injury detection kit.

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 each organ, including nephrotic syndrome.

Endothelial cells, which are a monolayer of cells inside blood vessels, have high metabolic activity and play a key role in many physiological processes, including regulation of vasomotor tone, blood cell trafficking between blood and tissue, maintenance of blood fluidity, permeability, angiogenesis and innate and adaptive immunity, are involved in the pathophysiological processes of most diseases, and are the major determinants or victims of pathophysiology. The kidney has the most abundant and diverse population of endothelial cells compared to the rest of the organs, and this wide diversity includes the different transport capacities of the kidney endothelial cells contributing across the various parts of the kidney and the different endothelial cells being subjected to different oxygen content and osmotic pressure in the environment. Thus, damage to vascular endothelial cells can lead to a variety of visceral diseases including nephrotic syndrome, with serious risks.

Nephrotic Syndrome (NS) is a group of clinical signs manifested by massive proteinuria, hypoproteinemia, high edema, and hyperlipidemia. The primary nephrotic syndrome belongs to primary glomerular diseases and is composed of a plurality of pathological types. The minimal central lesion (MCD) is the main cause of nephrotic syndrome in children and is one of autoimmune nephrotic syndrome, and accounts for 10-15% of the cause 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. Corticosteroids are common drugs for the treatment of MCD and proteinuria after treatment with them is completely relieved, while progressive renal failure caused by MCD is rare. However, MCD can lead to serious complications, and the complications associated with MCD disease observed in adults mainly include venous thrombosis and severe acute kidney injury requiring temporary dialysis. In addition, 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. 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 after 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 effects of corticosteroids and rituximab on podocytes are also considered to have therapeutic effects.

Although podocyte injury as presently observed is a major classical feature of MCD, the disease mechanism may also involve glomerular vascular endothelial cells. As early as 2000, patients with Idiopathic Nephrotic Syndrome (INS) reported by Futrakul N and the like are often accompanied by insufficient renal perfusion, and it is speculated that damage to glomerular vascular endothelial cells may be the cause of 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, the current studies to date do not understand what the causative agent responsible for the damage to glomerular endothelial cells is. The applicant group screened and identified a series of glomerular vascular endothelial cell autoantibodies 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 glomerular vascular endothelial cell autoantibodies are associated with a hypercoagulable state and poor prognosis in patients.

The anti-Cytoseleton-associated protein-IgG 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. However, the current research on the Cytoseleton-associated protein4 and anti-Cytoseleton-associated protein4-IgG antibodies of kidney disease patients at home and abroad is limited to the research on the molecular mechanism level, the level of the antibodies in the serum of the patients is not quantitatively detected, and the market lacks of corresponding clinical detection kits.

Disclosure of Invention

The invention aims to provide application of an antibody specifically bound with Cytoseleton-associated protein4 as a biomarker in preparation of a reagent or a kit for detecting vascular endothelial cell injury, and the antibody specifically bound with Cytoseleton-associated protein4 as the biomarker can be used for detecting diseases related to vascular endothelial cell injury, so that the medical application of the Cytoseleton-associated protein4 is increased.

The invention provides an application of a reagent for detecting an anti-Cytoskeleton-associated protein4-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.

Preferably, the reagent for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody comprises a Cytoseleton-associated protein4 protein, a Cytoseleton-associated protein4 recombinant protein or a polypeptide containing a tag.

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 Cytoskeleton-associated protein4 protein containing the tag comprises SEQ ID NO. 1.

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

The invention also provides a kit for detecting the anti-Cytoseleton-associated protein4-IgG antibody, which comprises the following components in percentage by weight: the reagent for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody, the solid phase carrier and the labeled antibody in the application of any one of the technical schemes.

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;

preferably, the secondary antibody comprises an anti-human IgG antibody.

Preferably, the enzyme-labeled secondary antibody 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.

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

Has the advantages that:

the invention provides application of an antibody specifically bound with Cytoseleton-associated protein4 as a biomarker in preparation of a reagent or a kit for detecting vascular endothelial cell injury, and the antibody specifically bound with Cytoseleton-associated protein4 as the biomarker can be used for detecting diseases related to vascular endothelial cell injury. In addition, the invention firstly detects an autoantibody against Cytoskeleton-associated protein4-IgG in the bodies of partial patients with nephrotic syndrome, and determines that the target antigen to which the autoantibody aims is Cytoskeleton-associated protein4 on endothelial cells of glomerular vessels. The invention finds that the Cytoskeleton-associated protein4 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 autoantibody of the anti-Cytoseleton-associated protein4-IgG can realize the detection of the injury of the vascular endothelium, and particularly provides a basis for researching the molecular mechanism of the nephrotic syndrome and clinical diagnosis and treatment. The kit for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody provided by the invention can qualitatively and quantitatively detect the anti-Cytoseleton-associated protein4-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 the high-efficiency detection of the vascular endothelial injury, and judges that the vascular endothelial injury exists when detecting the autoantibody of the anti-Cytoskeleton-associated protein 4-IgG.

2. At present, related Cytoseleton-associated protein4 and anti-Cytoseleton-associated protein4-IgG antibodies of 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 Cytoskeleton-associated protein4 for the first time, invents a detection kit aiming at the autoantibody and fills the blank at home and abroad. The kit is used for detecting the anti-Cytoseleton-associated protein4-IgG antibody in the serum of 298 nephrotic syndrome patients, and the result shows that the anti-Cytoseleton-associated protein4-IgG antibody of 116 patients is positive, namely the positive detection rate is 38.93%. The invention can provide a basis for researching the molecular mechanism and clinical diagnosis and treatment of nephrotic syndrome after detecting the anti-Cytoseleton-associated protein4-IgG antibody.

3. The kit of the invention relates to a solid-phase membrane qualitative immunoassay for anti-Cytoseleton-associated protein4-IgG antibody in human serum, and the IgG antibody of human anti-tag peptide is used as a standard substance, thereby greatly improving the detection accuracy. 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-Cytoseleton-associated protein4-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-Cytoseleton-associated protein4-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 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)₂O₂NaOH)0.4s later, the emission intensity can reach the maximum, halfThe decay period is 0.9s, and the decay period is basically finished within 2s, so that the rapid detection is convenient.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.

FIG. 1-1 shows that the Cytoskeleton-associated protein4 protein on the endothelial cells of glomerular vessels is the main target antigen for the autoantibody in patients with nephrotic syndrome, wherein A shows the two-dimensional electrophoresis protein spot of the primary antibody on the serum of healthy human and B shows the two-dimensional electrophoresis protein spot of the primary antibody on the serum of patients with nephrotic syndrome;

FIGS. 1-2 are mass spectrometric identification diagrams of the Cytoskeleton-associated protein4 protein on glomerular vascular endothelial cells;

FIG. 2 is an SDS-PAGE identification of the expressed recombinant protein Cytoskeleton-associated protein4, wherein lane A is the supernatant of the cell lysate induced at 15 ℃ for 16 hours and lane B is the supernatant of the cell lysate induced at 37 ℃ for 4 hours;

FIG. 3 is a solid-phase membrane immunoassay kit for detecting antibodies against Cytoskeleton-associated protein 41-IgG in serum of patients with nephrotic syndrome;

FIG. 4 is a schematic diagram of the principle of detecting anti-Cytoseleton-associated protein4-IgG antibody by the magnetic particle chemiluminescence immunoassay kit;

FIG. 5 is a schematic diagram of a carboxyl magnetic microparticle coated with an antigenic protein Cytoskeleton-associated protein 4;

FIG. 6 shows the detection of antibodies against Cytoskeleton-associated protein4-IgG in various renal patients, in which NS: nephrotic syndrome, HSP: allergic purpura, HSPN: purpuric nephritis, KD: kawasaki disease, NC: a healthy child;

FIG. 7 is a graph showing the linear correlation of an anti-Cytoseleton-associated protein4-IgG antibody with a vascular endothelial injury marker.

Detailed Description

The invention provides an application of a reagent for detecting an anti-Cytoskeleton-associated protein4-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The reagent for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody preferably comprises a Cytoseleton-associated protein4 protein, a Cytoseleton-associated protein4 recombinant protein or a polypeptide containing a label. The accession number of the Cytoskeleton-associated protein4 in NCBI is BC 082972. The vascular endothelial cell damage according to the present invention preferably includes nephrotic syndrome, and more preferably, autoimmune nephrotic syndrome. Blood, blood vessels and the heart constitute the blood circulation system of the human body, and blood in the blood circulation system flows through the blood vessels and flows through the organs of the whole body such as the heart, the lung, the liver and the like. The innermost layer of the blood vessel is attached with vascular endothelial cells, and the antibodies of the vascular endothelial cells can cause the damage of the vascular endothelial cells, induce the dysfunction of the blood circulation system, further cause the damage of the organs such as the heart, the lung and the liver, and cause the related diseases of various organs including nephrotic syndrome, but the vascular endothelial cells of different organs are the same. Therefore, the method can be used for clinical vascular endothelial cell injury based on the detection of vascular endothelial cell autoantibodies in the blood circulation system. The invention can realize the detection of the vascular endothelial cell injury by taking the antibody specifically combined with the Cytoskeleton-associated protein4 as a biomarker.

The reagent for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody provided by the invention takes the Cytoseleton-associated protein4 protein as a target spot to detect the Cytoseleton-associated protein4 autoantibody (namely, the anti-Cytoseleton-associated protein4-IgG autoantibody is a biomarker for detecting vascular endothelial cell injury), and the reagent can realize high-efficiency detection of the vascular endothelial injury. In the present invention, the agent is capable of immunoreacting with an autoantibody to the Cytoskeleton-associated protein4 protein from tissue (kidney biopsy) or body fluid (in particular blood, plasma, serum). In the present invention, the reagent for detecting an anti-Cytoseleton-associated protein4-IgG autoantibody preferably comprises a Cytoseleton-associated protein4 protein or a Cytoseleton-associated protein4 recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the Cytoskeleton-associated protein4 protein is BC 082972. 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 antigen protein purification, immobilization and precipitation; 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 cytosketon-associated protein4 recombinant protein containing the tag is preferably as shown in SEQ ID No. 1: MIFTEVQKRSQKEINDMKAKVASLEESEGNKQDLKALKEAVKEIQTSAKSRE WDMEALRSTLQTMESDIYTEVRELVSLKQEQQAFKEAADTERLALQALTEKL LRSEESVSRLPEEIRRLEEELRQLKSDSHGPKEDGGFRHSEAFEALQQKSQGL DSRLQHVEDGVLSMQVASARQTESLESLLSKSQEHEQRLAALQGRLEGLGSS EADQDGLASTVRSLGETQLVLYGDVEELKRSVGELPSTVESLQKVQEQVHTL LSQDQAQAARLPPQDFLDRLSSLDNLKASVSQVEADLKMLRTAVDSLVAYSV KIETNENNLESAKGLLDDLRNDLDRLFVKVEKIHEKVHHHHHH are provided. The vascular endothelial cell damage according to the present invention preferably includes nephrotic syndrome, and more preferably, autoimmune nephrotic syndrome. The invention can detect the damage of the vascular endothelial cells, particularly nephrotic syndrome and the like by taking the Cytoskeleton-associated protein4 as a detection target spot, and further can be used for preparing a detection kit related to the damage of the vascular endothelial cells, thereby increasing the medical application of Cytoskeleton-related protein 4.

The invention also provides a kit for detecting the anti-Cytoseleton-associated protein4-IgG antibody, which comprises the reagent for detecting the anti-Cytoseleton-associated protein4-IgG autoantibody, a solid phase carrier and a labeled antibody in the application of the technical scheme.

In the present invention, the reagent for detecting an anti-Cytoseleton-associated protein4-IgG autoantibody (Cytoseleton-associated protein4 protein or Cytoseleton-associated protein4 recombinant protein containing a tag) is preferably immobilized on a solid phase carrier. As used herein, "immobilized" refers to binding to a water-insoluble solid support of a Cytoskeleton-associated protein4 antigenic protein, the solid support or supports being insoluble in water, more preferably by covalent bonding, electrostatic interaction, hydrophobic interaction, or by disulfide bond interaction, 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 are methods for immobilizing the Cytoskeleton-associated protein4 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 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 method for fixing the antigen protein Cytoskeleton-associated protein4 is preferably a direct coating method: (1) the antigen protein Cytoskeleton-associated protein4 is combined on a nitrocellulose membrane or a polystyrene micropore plate by 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 Cytoskeleton-associated protein4, and the antigen protein Cytoskeleton-associated protein4 is combined on the magnetic particles by a chemical coupling mode. In the invention, the solid phase carrier comprises one or more of a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle and an enzyme labeling micropore plate.

The invention preferably adopts a gene recombination prokaryotic expression method to successfully express and purify the recombinant protein Cytoseleton-associated protein4, and uses the recombinant protein as the antigen protein in a kit to develop a set of kits suitable for detecting the anti-Cytoseleton-associated protein4-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-Cytoseleton-associated protein4-IgG antibody in human serum.

In the present invention, the Cytoskeleton-associated protein4 protein is preferably expressed in bacterial (e.g., E.coli), yeast, insect or mammalian cells. After the Cytoseleton-associated protein4 protein is obtained through expression, the Cytoseleton-associated protein4 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 reagent for detecting the autoantibody against Cytoseleton-associated protein4-IgG, i.e., the antigen is preferably recombinant protein Cytoseleton-associated protein4 (the amino acid sequence of which comprises 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 Cytoskeleton-associated protein4 (the amino acid sequence comprises 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-Cytoseleton-associated protein4-IgG autoantibody with different concentrations; the washing 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 anti-Cytoseleton-associated protein4-IgG antibody in serum is preferably as follows: by utilizing the indirect method reaction principle, firstly, the Cytoskeleton-associated protein4 antigen is adsorbed to a solid phase carrier to be used 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, if the serum to be detected contains the anti-Cytoskeleton-associated protein4-IgG antibody, a ternary complex of the coating antigen Cytoskeleton-associated protein 4-serum anti-Cytoskeleton-associated protein4-IgG antibody-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 as to achieve the purpose of qualitatively or quantitatively analyzing the anti-Cytoskeleton-associated protein4-IgG antibody.

The application of the reagent for detecting anti-Cytoskeleton-associated protein4-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, and the technical scheme of the present invention includes, but is not limited to, the following examples.

Example 1

The Cytoskeleton-associated protein4 protein on vascular endothelial cells is a main target antigen for autoantibody 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 Cytoskeleton-associated protein4 on vascular endothelial cells is proved to be a main target antigen for the autoantibody in the patient with autoimmune nephrotic syndrome. Thus, detection of the presence and quantitative levels of antibodies against Cytoshieton-associated protein4-IgG in serum is helpful in defining vascular endothelial cell damage.

The specific implementation is as follows:

(1) extraction of total protein of vascular endothelial cells: the vascular endothelial cell strain (ECV 304) was cultured, washed 2-3 times with PBS, then sufficiently lysed on ice in a lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and a protease inhibitor (# ab 65621; Abcam, 1: 200 dilution) using a focused ultrasound machine (Covaris S220, Gene), and then the sample was 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 people and autoimmune nephrotic syndrome patients as primary antibody, and developing with secondary antibody, as shown in A and B of FIG. 1.

(3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after imaging 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%). The extracted peptides were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass spectrum, which was identified as the Cytoskeleton-associated protein4 protein, as shown in panel C of FIG. 1.

Example 2

Expression and purification of recombinant antigen protein Cytoskeleton-associated protein4

The gene of the coded Cytoskeleton-associated protein4 protein is taken 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 Cytoskeleton-associated protein4 was identified by SDS-PAGE as 40kDa, 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

The invention adopts orthogonal test design to optimize the reaction conditions of the kit

An orthogonal table was selected based on 4 factors such as the antigen cytosketon-associated protein4 coating concentration (four coating concentrations of 50. mu.g, 80. mu.g, 100. mu.g, 150. mu.g), each reaction time (15min, 30min, 45min) and temperature (25 ℃, 37 ℃), the enzyme-labeled secondary antibody optimal dilution (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 positive serum to the lowest light signal value (N) of negative serum was selected. The optimal antigen Cytoseleton-associated protein4 coating concentration of the kit obtained by the orthogonal design is 250ug/mL, the optimal antigen-antibody reaction temperature of the anti-Cytoseleton-associated protein4-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 optimal biotin-labeled anti-human IgG antibody is 1: 500, a step of; the kit for detecting the anti-Cytoskeleton-associated protein4-IgG antibody by magnetic particle chemiluminescence immunoassay has the optimal antigen-antibody reaction temperature of 37 ℃, the optimal antigen-antibody reaction time of 15min and the optimal working dilution of an acridinium ester labeled anti-human IgG antibody of 1: 500.

example 4

Preparation of a solid-phase membrane immunoassay kit for detecting anti-Cytoskeleton-associated protein4-IgG antibody:

antigen: recombinant protein Cytoskeleton-associated protein4

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-Cytoskeleton-associated protein4-IgG antibody is as follows:

4.2.1 coating, sealing: placing 8 mu L of Cytoskeleton-associated protein4 antigen direct contact with the concentration of 250 mu g/mL on a nitrocellulose membrane in a 37 ℃ incubator for drying for 30min, placing the nitrocellulose membrane in a detection plate, adding 200 mu L of 5% BSA into a 37 ℃ incubator for sealing for 30min, discarding the sealing solution, and washing for 2 times by using 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: the liquid in the assay plate was discarded, the wash was washed 5 times × 1min, and 20 μ L of 1: labeling anti-human IgG antibody with 500 biotin, 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, and if the test nitrocellulose membrane strip is positive, the result chart is shown in figure 3 (the result chart of the solid-phase membrane immunoassay kit for detecting the anti-cytopaeton-associated protein4-IgG antibody in the serum of a nephrotic syndrome patient), or placing the membrane strip on a developing instrument for scanning, wherein analysis software carried by the developing instrument takes the concentration of a reference standard substance as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate, and drawing a standard curve to perform semi-quantitative analysis on the anti-cytopaeton-associated protein4-IgG antibody level in the serum.

Example 5

Preparation of magnetic particle chemiluminescence immunoassay kit for detecting anti-Cytoseleton-associated protein4-IgG antibody (FIG. 4 is a schematic diagram of the principle of detecting anti-Cytoseleton-associated protein4-IgG antibody by the magnetic particle chemiluminescence immunoassay kit)

5.1 anti-Cytoseleton-associated protein4-IgG antibody chemiluminescence detection kit, including 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)₂O₂) 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, 20.0 mu g/mL, and the buffer solution is Tris-HCl 5.0% BSA containing 0.5mol/L and 0.1-0.5% PC 300;

(5) cleaning solutions, in particular a 25mmol/L Tris-HCl solution at 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 carboxyl-based magnetic particle coated with antigen protein Cytoskeleton-associated protein 4)

(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 Cytoskeleton-associated protein4 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 ℃.

Preparation of 5.3 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 finally dialyzing overnight, wherein the labeled buffer solution is Na₂CO₃-NaHCO₃A 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, dissolving in 447 mu L of anhydrous dimethylformamide DMF to form 6.5mmol/L of NSP-DMAE-NHS DMF solution;

(3) the dialyzed antibody solution was placed in a 500. mu.L centrifuge tube, and 100. mu.L of 6.5mmol/L NSP-DMAE-NHS DMF solution was added, with a molar ratio of acridinium ester to antibody of 7.4: 1, adding 200 mu L of marking buffer solution, reacting for 45min at room temperature, adding 10 mu L of lysine 10 mu L, and continuing to react for 15min to terminate the marking 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: samples were prepared as follows: dilution by ratio of 10

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 acridinium 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) 100. mu.L of the chemiluminescent pre-excitation liquid A was added, followed by 100. mu.L of the chemiluminescent excitation liquid B, and the relative luminescence intensity was measured. The content of anti-Cytoseleton-associated protein4-IgG antibody in the sample is proportional to the luminous intensity thereof.

Example 6

Clinical application of kit for detecting serum anti-Cytoskeleton-associated protein4-IgG antibody

6.1 subjects included: patients diagnosed with various types of nephropathy from 6 months in 2018 to 6 months in 2020, including 298 Nephrotic Syndrome (NS), 100 anaphylactoid purpura (HSP), 100 purpura nephritis (HSPN), 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-Cytoseleton-associated protein4-IgG antibodies in various nephrotic patients the kit of the present invention was used to detect the anti-Cytoseleton-associated protein4-IgG antibody levels in the serum of patients diagnosed with various nephropathies, including 298 nephrotic syndrome, 100 allergic purpuras, 100 purpuric nephritis, 100 Kawasaki disease and 100 healthy children in the same period, and the results showed that some patients with nephrotic syndrome were positive for the anti-Cytoseleton-associated protein4-IgG antibody (116 patients were positive for the anti-Cytoseleton-associated protein4-IgG antibody, namely the anti-Talin-1-IgG antibody positive detection was 38.93%), while the anti-Henschel nephritis, allergic purpura, Kawasaki disease and healthy children were negative for the anti-Talin-1-IgG antibody, and the anti-Cytoseleton-associated protein4-IgG antibody in various nephrotic patients was shown in FIG. 6 (see the anti-Cytoseleton-Octadin-allergic purpura-IgG antibody detection in patients, wherein NS: nephrotic syndrome, HP: allergic purpura, HPN: purpuric nephritis, KD: kawasaki disease, NC: a healthy child). The determination of the vascular endothelial damage in nephrotic syndrome is facilitated by the presence of antibodies against Cytoseleton-associated protein4-IgG in the serum. .

6.3 serum anti-Cytoskeleton-associated protein4-IgG antibody of nephrotic syndrome patient is linearly related to the expression level of vascular endothelial injury marker

The kit is used for detecting the expression quantity of the anti-cytokine-associated protein4-IgG antibody in the serum of a patient with nephrotic syndrome diagnosed from 6 months 2018 to 6 months 2020, and detecting the expression quantity of the vascular endothelial injury marker Plvap in the serum of the patient, and the result shows that the expression quantity of the anti-cytokine-associated protein4-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-cytokine-associated protein4-IgG antibody can be used for judging the vascular endothelial injury, namely detecting the anti-cytokine-associated protein4-IgG antibody, and judging the vascular endothelial injury, and the figure 7 (a linear correlation result graph of the anti-cytokine-associated protein4-IgG antibody and the vascular endothelial injury marker).

As can be seen from the above examples, the anti-Cytoseleton-associated protein4-IgG of the invention can be used as a detection target for vascular endothelial injury; the kit prepared by taking the protein as the detection target can be used for detecting the anti-cytoskeleton-related protein4-IgG antibody, and has high sensitivity and accuracy, safety and rapidness.

Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.

Sequence listing

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Met Ile Phe Thr Glu Val Gln Lys Arg Ser Gln Lys Glu Ile Asn Asp

1 5 10 15

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

20 25 30

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

35 40 45

Lys Ser Arg Glu Trp Asp Met Glu Ala Leu Arg Ser Thr Leu Gln Thr

50 55 60

Met Glu Ser Asp Ile Tyr Thr Glu Val Arg Glu Leu Val Ser Leu Lys

65 70 75 80

Gln Glu Gln Gln Ala Phe Lys Glu Ala Ala Asp Thr Glu Arg Leu Ala

85 90 95

Leu Gln Ala Leu Thr Glu Lys Leu Leu Arg Ser Glu Glu Ser Val Ser

100 105 110

Arg Leu Pro Glu Glu Ile Arg Arg Leu Glu Glu Glu Leu Arg Gln Leu

115 120 125

Lys Ser Asp Ser His Gly Pro Lys Glu Asp Gly Gly Phe Arg His Ser

130 135 140

Glu Ala Phe Glu Ala Leu Gln Gln Lys Ser Gln Gly Leu Asp Ser Arg

145 150 155 160

Leu Gln His Val Glu Asp Gly Val Leu Ser Met Gln Val Ala Ser Ala

165 170 175

Arg Gln Thr Glu Ser Leu Glu Ser Leu Leu Ser Lys Ser Gln Glu His

180 185 190

Glu Gln Arg Leu Ala Ala Leu Gln Gly Arg Leu Glu Gly Leu Gly Ser

195 200 205

Ser Glu Ala Asp Gln Asp Gly Leu Ala Ser Thr Val Arg Ser Leu Gly

210 215 220

Glu Thr Gln Leu Val Leu Tyr Gly Asp Val Glu Glu Leu Lys Arg Ser

225 230 235 240

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

245 250 255

Gln Val His Thr Leu Leu Ser Gln Asp Gln Ala Gln Ala Ala Arg Leu

260 265 270

Pro Pro Gln Asp Phe Leu Asp Arg Leu Ser Ser Leu Asp Asn Leu Lys

275 280 285

Ala Ser Val Ser Gln Val Glu Ala Asp Leu Lys Met Leu Arg Thr Ala

290 295 300

Val Asp Ser Leu Val Ala Tyr Ser Val Lys Ile Glu Thr Asn Glu Asn

305 310 315 320

Asn Leu Glu Ser Ala Lys Gly Leu Leu Asp Asp Leu Arg Asn Asp Leu

325 330 335

Asp Arg Leu Phe Val Lys Val Glu Lys Ile His Glu Lys Val His His

340 345 350

His His His His

355

Claims

1. The application of a reagent for detecting the autoantibody of the anti-Cytoseleton-associated protein4-IgG in the preparation of a kit for detecting the vascular endothelial injury.

2. The use according to claim 1, wherein the reagent for detecting autoantibodies against Cytoseleton-associated protein4-IgG comprises a Cytoseleton-associated protein4 protein, a Cytoseleton-associated protein4 recombinant protein or polypeptide comprising a tag.

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 3, wherein the amino acid sequence of the tagged Cytoskeleton-associated protein4 protein comprises SEQ ID No.1 when the 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 an anti-Cytoskeleton-associated protein4-IgG antibody, comprising: a reagent for detecting autoantibodies against Cytoskeleton-associated protein4-IgG, 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 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.

8. The kit of claim 7, wherein the secondary antibody comprises an anti-human IgG antibody.

9. The kit of claim 7 or 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.

10. The kit of claim 6, wherein the solid support comprises one or more of a nitrocellulose membrane, a fluorescently encoded microsphere, a magnetic stripe chip, a magnetic microparticle, and an enzyme-labeled microplate.