CN114994330A - Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof - Google Patents

Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof Download PDF

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CN114994330A
CN114994330A CN202210491401.6A CN202210491401A CN114994330A CN 114994330 A CN114994330 A CN 114994330A CN 202210491401 A CN202210491401 A CN 202210491401A CN 114994330 A CN114994330 A CN 114994330A
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heat shock
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叶青
毛建华
周希
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Zhejiang University ZJU
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Abstract

The invention relates to a kit for detecting an anti-HSP 90-beta-IgG autoantibody and application thereof, belonging to the technical field of biology. The invention provides an application of a reagent for detecting an anti-HSP 90-beta-IgG autoantibody in preparing a kit for detecting vascular endothelial injury, which can carry out qualitative or quantitative detection on the anti-HSP 90-beta-IgG antibody of the autoantibody from blood, tissues or body fluid through immune reaction, thereby realizing the detection of all vascular endothelial injuries of the whole body including glomerular vascular endothelium.

Description

Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to a kit for detecting an anti-HSP 90-beta-IgG autoantibody and application thereof.
Background
Blood, blood vessels and heart constitute the blood circulation system of human body, and blood in the blood circulation system flows in the blood vessels and flows through the organs of the whole body such as heart, lung, liver, etc., and the innermost layer of the blood vessels is attached with vascular endothelial cells. The vascular endothelial cells 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 the proliferation of smooth muscle cells, inhibiting the inflammatory reaction of the vascular wall 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.
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.
At present, the underlying pathogenesis of MCD is still poorly understood. One view is that the disease is triggered by the circulating permeability factors produced by immune cells. Since the pathogenesis of primary Focal Segmental Glomerulosclerosis (FSGS) is very similar to that of MCD, many scholars consider MCD and FSGS to be phenotypes of the same disease at different stages. T cells were first suspected to be the source of the circulating permeability factor based on the association between MCD and non-hodgkin's lymphoma, the remission induced by measles infection and prolonged remission 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 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. They used human endothelial cell line ECV 304 and incubated with INS patient serum to perform endothelial cell toxicity test, and found that the FSGS patient serum caused the most obvious endothelial cell damage. Therefore, they speculated that glomerular vascular endothelial cell injury may be responsible for insufficient renal perfusion in INS patients. Purohit S et al found that there was an increase in the endothelial cell injury marker syndecan 1 in the circulatory system of MCD patients, but it was not clear whether there was simultaneous injury to the glomerular endothelial cells. Trachtman H et al observed the co-deposition of IgM with complement components in kidney tissues of FSGS and MCD patients and confirmed that IgM is an antibody against GEC and cardiolipin epitopes. Bauer C et al found in 2022 that the endothelial cell marker in the serum of MCD patients was elevated, and meanwhile, renal histopathology confirmed that the expression of glomerular endothelial cells caveolin-1 was significantly elevated, and further incubation of the serum of patients with human glomerular endothelial cells cultured in vitro significantly increased the expression of thrombomodulin, a marker of glomerular vascular endothelial cell injury, thereby demonstrating that MCD patients had injury to glomerular vascular endothelial cells.
Nevertheless, it is not clear to date what are the causative agents responsible for the damage to glomerular endothelial cells. A series of glomerular vascular endothelial cell autoantibodies were screened and identified by our research team in MCD and FSGS nephrotic syndrome patients from 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 autoantibodies-induced glomerular vascular endothelial cell injury may be the initiating factor of characteristic podocyte injury in MCD, and is one of the important causes of the disease. Therefore, we have proposed the second hit theory of the onset of MCD and FSGS nephrotic syndrome for the first time internationally: that is, pathogenic agents including autoantibodies first damage the glomerular vascular endothelial cells, and then these pathogenic agents further damage the podocytes, eventually causing morbidity to the patient. Because the pathogenic agents in the blood circulation system are unlikely to come into contact with the podocytes from the specific anatomical location of the podocytes unless the integrity of the glomerular vascular endothelial cells has been compromised. Therefore, the research result of the autoantibodies of the endothelial cells of the glomerular vessels is a breakthrough in the theoretical research of the pathogenesis of the nephrotic syndrome. Meanwhile, the autoantibodies also have important clinical value and commercial value and have high value for guiding the diagnosis and treatment of nephrotic syndrome.
The anti-Heat shock protein90-beta antibody (namely the anti-HSP 90-beta 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. Heat shock protein90-beta has been studied and found to be essential in tumorigenesis and malignant progression. Unlike the study and application of Heat shock protein90-beta antigen in tumor diseases, our study is directed to the study of anti-Heat shock protein90-beta antibody in the damage of vascular endothelial cells including glomerular vascular endothelial cells in patients with nephrotic syndrome. However, due to the novel frontier of the research, the corresponding clinical detection kit is lacking in the market.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting a Heat shock protein90-beta-IgG autoantibody in preparing a kit for detecting vascular endothelial injury, and qualitative or quantitative detection of the Heat shock protein90-beta-IgG antibody of the autoantibody from blood, tissues or body fluid can be carried out through immune reaction, so that the detection of all vascular endothelial injuries of the whole body including glomerular vascular endothelium is realized.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides application of a reagent for detecting a Heat shock protein90-beta-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.
Preferably, the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody comprises a Heat shock protein90-beta protein or a Heat shock protein90-beta recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the Heat shock protein90-beta protein is BC 014485.
More preferably, the tag is modified at the N-terminal or C-terminal of the Heat shock protein90-beta recombinant protein, and the tag is selected from any one of His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, C-Myc tag, Flag tag and biotin tag.
The invention also provides a kit for detecting vascular endothelial injury, which comprises a reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody.
Preferably, the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody is fixed on a solid phase carrier, and the solid phase carrier is selected from any one of a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic microparticle and an enzyme labeling micropore plate.
Preferably, the kit further comprises a labeled antibody, wherein the labeled antibody is any one selected from an enzyme-labeled anti-human IgG antibody, a chemiluminescent substance-labeled anti-human IgG antibody and a biotin-labeled anti-human IgG antibody.
Preferably, the test sample of the kit is any one selected from whole blood, serum, plasma, urine, lymph fluid or pleural effusion of a mammal.
The invention provides an application of a reagent for detecting a Heat shock protein90-beta-IgG autoantibody in preparing a kit for detecting vascular endothelial injury, which can carry out qualitative or quantitative detection on the Heat shock protein90-beta-IgG antibody of the autoantibody from blood, tissues or body fluid through immune reaction, thereby realizing the detection of all vascular endothelial injuries of the whole body including glomerular vascular endothelium. Based on the above, the kit for detecting the vascular endothelial injury provided by the invention greatly improves the detection accuracy, is simple to operate, has good stability and small reagent dosage, and is saved by about 10 times compared with the traditional ELISA. Experiments prove that the kit disclosed by the invention is used for detecting the anti-Heat shock protein90-beta-IgG antibody in the serum of 298 nephrotic syndrome patients, the positive detection rate of the anti-Heat shock protein90-beta-IgG antibody is 40.27%, and the kit suitable for detecting the anti-Heat shock protein90-beta-IgG antibody of the vascular endothelial cell autoantibody is obtained for the first time.
Drawings
FIG. 1 shows that Heat shock protein90-beta protein on endothelial cells of glomerular vessels is the main target antigen for autoantibodies in patients with nephrotic syndrome; wherein, A is a two-dimensional electrophoresis protein point of which the primary antibody is the serum of a healthy human, B is a two-dimensional electrophoresis protein point of which the primary antibody is the serum of a nephrotic syndrome patient, and C is the mass spectrum identification of a target antigen Heat shock protein90-beta protein.
FIG. 2 is an SDS-PAGE identification of the expressed recombinant protein Heat shock protein 90-beta.
FIG. 3 is a solid-phase membrane immunoassay kit for detecting anti-Heat shock protein90-beta-IgG antibody in serum of patients with nephrotic syndrome.
FIG. 4 is a schematic diagram of the magnetic particle chemiluminescence immunoassay kit for detecting anti-Heat shock protein90-beta-IgG antibody.
FIG. 5 is a schematic diagram of an antigenic protein Heat shock protein90-beta coated carboxyl magnetic particle.
FIG. 6 shows the detection of anti-Heat shock protein90-beta-IgG antibody in various patients with renal diseases; wherein NS is nephrotic syndrome, HP is anaphylactoid purpura, HPN is purpura nephritis, KD is Kawasaki disease, and NC is healthy child.
FIG. 7 is a linear correlation of anti-Heat shock protein90-beta-IgG antibody with a marker of glomerular vascular endothelial cell injury.
Detailed Description
At present, related Heat shock protein90-beta and anti-Heat shock protein90-beta-IgG antibodies of kidney disease patients at home and abroad are only limited to molecular mechanism research, and the level of the antibodies in serum of the patients is not quantitatively detected. The invention discovers a blood vessel endothelial cell autoantibody-anti-Heat shock protein90-beta-IgG autoantibody for the first time, and invents a detection kit aiming at the Heat shock protein90-beta-IgG autoantibody, and the autoantibody can be used for realizing the detection of all the blood vessel endothelium injuries of the whole body including glomerular blood vessel endothelium, thereby filling the blank at home and abroad.
The invention provides application of a reagent for detecting a Heat shock protein90-beta-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.
In the present invention, the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody is not particularly limited, and all fragments of the Heat shock protein90-beta protein or the whole protein are within the scope of the present invention. As a specific embodiment, the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody of the invention preferably comprises a Heat shock protein90-beta protein or a tagged Heat shock protein90-beta recombinant protein or polypeptide; the NCBI protein accession number of the Heat shock protein90-beta protein is preferably BC 014485. In the present invention, the tag is a sequence or a domain capable of specifically binding to a ligand, and is preferably modified at the N-terminus or the C-terminus of the Heat shock protein90-beta recombinant protein, and is preferably selected from any one of a His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, C-Myc tag, Flag tag, and biotin tag. In the present invention, the tag facilitates purification, immobilization and precipitation of the antigenic protein.
The invention also provides a kit for detecting vascular endothelial injury, which comprises a reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody.
In the invention, the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody is preferably fixed on a solid phase carrier, the solid phase carrier is preferably selected from any one of a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle and an enzyme labeling micropore plate, and more preferably the magnetic particle. As a specific embodiment, when the solid phase carrier is a magnetic particle, the diameter of the magnetic particle is preferably 1.0 μm, which can greatly increase the coated surface area, increase the adsorption amount of the antigen, improve the reaction speed, and simultaneously make the cleaning and separation simpler, thereby reducing pollution and reducing the probability of cross infection.
In the present invention, the Heat shock protein90-beta protein is preferably expressed in bacteria such as Escherichia coli, yeast, mammalian cells. The Heat shock protein90-beta protein is preferably purified before being immobilized on a solid phase carrier; the purification method preferably comprises Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography and hydrophobic column purification. In the invention, the accuracy and the detection rate of the Heat shock protein90-beta protein can be improved through optimization before the protein is fixed on the solid phase carrier.
In the present invention, the immobilization refers to the binding of Heat shock protein90-beta protein to a water-insoluble solid phase carrier, preferably by covalent bonding, electrostatic interaction, aqueous interaction or disulfide bond interaction, more preferably by one or more covalent bonds. In the present invention, the fixing method preferably includes a direct fixing mode and a reversible/irreversible mode. The direct immobilization means preferably separates the immobilized molecules from the aqueous solution together with the insoluble support by filtration, centrifugation or chromatography; the reversible/irreversible mode is that the Heat shock protein90-beta protein is fixed by utilizing the reversible or irreversible mode; the reversible mode preferably comprises: proteins are immobilized to a carrier by a cleavable covalent bond (e.g., a disulfide bond that can be cleaved by addition of a thiol-containing reagent); said irreversible means preferably comprise: the antigenic protein is immobilized to the carrier by covalent bonds that are not cleaved in aqueous solution (e.g., disulfide bonds that can be cleaved by thiol-containing reagents).
As another specific embodiment, the immobilization method of the present invention includes an indirect method and a direct coating method. The indirect means is preferably immobilization of an antibody with specific affinity for the protein followed by formation of a protein antigen-antibody complex for the purpose of immobilization. The direct coating method is preferably as follows: (1) the protein Heat shock protein90-beta is combined on a nitrocellulose membrane or a polystyrene microporous plate in a physical adsorption mode or a non-covalent bond; or (2) the magnetic particle with the carboxyl functional group is combined with the amino group of the protein Heat shock protein90-beta, and the protein Heat shock protein90-beta is combined on the magnetic particle by a chemical coupling mode.
In the present invention, the kit preferably further comprises: the kit comprises a labeled antibody, an antigen diluent, a sample diluent buffer, an antibody diluent, a substrate color developing agent, a washing solution, a standard substance, a positive quality control substance and a negative quality control substance.
In the present invention, the labeled antibody is preferably selected from any one of an enzyme-labeled anti-human IgG antibody, a chemiluminescent substance-labeled anti-human IgG antibody, and a biotin-labeled anti-human IgG antibody, and more preferably a chemiluminescent substance-labeled anti-human IgG antibody or a biotin-labeled anti-human IgG antibody; the chemiluminescent substance-labeled anti-human IgG antibody is preferably an acridinium ester-labeled anti-human IgG antibody. As a specific implementation mode, when the labeled antibody is an acridinium ester labeled anti-human IgG antibody, the chemical reaction is simple and rapid, and a catalyst is not needed; the chemiluminescence of acridinium ester is of the scintillation type, whichBy activating the luminescent agent (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. As another specific embodiment, when the labeled antibody is a biotin-labeled anti-human IgG antibody, the signal can be amplified, and the detection sensitivity can be greatly improved.
In the present invention, the standard and the positive quality control substance are preferably recombinant human anti-tag peptide immunoglobulin G or a fragment thereof, or an anti-Heat shock protein90-beta-IgG antibody extracted from the serum of a patient. In the invention, the negative quality control product is serum of healthy people. As a specific embodiment, when the IgG antibody of human anti-tag peptide is used as a standard substance, the detection accuracy can be greatly improved.
In the present invention, the detection sample of the kit is preferably a sample containing autoantibodies, and the sample is preferably any one selected from whole blood, serum, plasma, urine, lymph fluid or pleural effusion of a mammal, and more preferably serum of a mammal.
In the present invention, the antigen diluent is preferably 1 XPBS containing 0.15mol/LNaCl and 1% (W/V) TritonX-100, and the pH is preferably 7.35. The sample dilution buffer is preferably 0.01M PBS containing 10% (W/V) BSA, at a pH of preferably 7.35. The antibody dilution is preferably 0.01M PBS containing 1M D-glucose, 2% (W/V) glycerol, 0.3% (W/V) Tween20, and preferably at a pH of 7.35. The substrate color developing agent is preferably selected from one of TMB, hydrogen peroxide, AMPPD, 4-MUP and BCIP. The detergent is preferably 1 XPBS containing 0.15mol/LNaCl, 10% (W/V) glycerol, 1% (W/V) TritonX-100, and the pH is preferably 7.35.
According to the kit, an indirect method reaction principle is utilized, firstly, a Heat shock protein90-beta antigen is adsorbed on a solid phase carrier to serve as a coating antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled secondary antibody is added for reaction, if the serum to be detected contains an anti-Heat shock protein90-beta-IgG antibody, a coating antigen Heat shock protein 90-beta-serum to be detected anti-Heat shock protein90-beta-IgG antibody-labeled anti-human IgG antibody ternary complex is formed, and finally, a light color development method, a chemical luminescence method and a fluorescence method are utilized to detect a light signal, so that the purpose of qualitatively or quantitatively analyzing the anti-Heat shock protein90-beta-IgG antibody in human serum is achieved.
In the present invention, unless otherwise specified, all chemical reagents used are conventional commercially available reagents, and all technical means used are conventional technical means well known to those skilled in the art.
In order to further illustrate the present invention, the following embodiments are described in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Identification of Heat shock protein90-beta protein on vascular endothelial cells as the major target antigen for autoantibody targeting in nephrotic syndrome patients
(1) Extraction of total protein of vascular endothelial cells: the vascular endothelial cell strain EAhy926 was cultured, washed 2-3 times with PBS, then sufficiently lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (# ab 65621; Abcam, 1: 200 dilution), and then the sample was centrifuged at 12000g at 4 ℃ for 30 min. Collecting the supernatant, namely the total protein of the vascular endothelial cells. The total protein concentration of the collected vascular endothelial cells was measured using the BCA protein concentration measurement kit.
(2) Two-dimensional electrophoresis: the total protein of vascular endothelial cells was extracted, subjected to two-dimensional electrophoresis, transferred to a nitrocellulose membrane, incubated with serum of healthy persons and patients with nephrotic syndrome as primary antibodies, respectively, and then developed with secondary antibodies, the results are shown in fig. 1A and fig. 1B.
(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 then peptides were extracted from the gel with 0.1% trifluoroacetic acid. 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 Heat shock protein90-beta protein, and the results are shown in FIG. 1C.
Example 2
Recombinant antigen protein Heat shock protein90-beta expression and purification
The cDNA sequence of the Heat shock protein90-beta human gene is obtained from GenBank (NC-000012.12), and the partial sequence of the gene coding for Heat shock protein90-beta is amplified by PCR and ligated with the vector. Then cloning into a prokaryotic expression vector pET30a (+) to express Heat shock protein90-beta protein. The antigen protein expressed by the invention contains a tag peptide of His tag. The expressed recombinant protein is purified by nickel column affinity chromatography, and finally Westernblot is used for identifying the molecular weight of the recombinant protein Heat shock protein90-beta to be 45KDa, and the result is shown in figure 2.
Example 3
Reaction conditions of the kit are optimized
Orthogonal tables were selected based on the antigen Heat shock protein90-beta coating concentration (five coating concentrations of 20. mu.g, 40. mu.g, 60. mu.g, 80. mu.g, 100. mu.g), each reaction time (10min, 20min, 30min, 40min) and temperature (25 ℃, 37 ℃), enzyme-labeled secondary antibody optimal dilutions (four dilutions of 1: 200, 1:400, 1: 800, 1: 1600), 4 factors, each factor was repeatedly assayed at 2 levels for standard positive sera and standard negative sera, and the ratio (P/N) of the highest light signal value (P) of positive sera to the lowest light signal value (N) of negative sera was selected. The optimal antigen Heat shock protein90-beta coating concentration of the kit is 60 mu g/mL, the optimal antigen antibody reaction temperature of the kit for detecting the anti-Heat shock protein90-beta-IgG antibody by solid-phase membrane immunoassay is 25 ℃, the optimal antigen antibody reaction time is 20min, and the optimal work dilution of the biotin-labeled anti-human IgG antibody is 1:400, respectively; the optimal antigen-antibody reaction temperature of the kit for detecting the anti-Heat shock protein90-beta-IgG antibody by magnetic particle chemiluminescence immunoassay is 37 ℃, the optimal antigen-antibody reaction time is 20min, and the optimal working dilution of the optimal acridinium ester labeled anti-human IgG antibody is 1: 400.
example 4
Preparation of solid-phase membrane immunoassay kit for detecting anti-Heat shock protein90-beta-IgG antibody
1. The solid-phase membrane immunoassay kit for detecting the anti-Heat shock protein90-beta-IgG antibody comprises the following components:
1) antigen: recombinant protein Heat shock protein 90-beta;
2) solid phase carrier: satourius CN140 nitrocellulose membrane;
3) positive quality control and standard: human anti-His tag immunoglobulin G (purchased from invitro, hu);
4) negative quality control product: serum of healthy examiners;
5) labeling the antibody: biotin-labeled anti-human IgG antibodies;
6) antigen dilution: 1 XPBS with 0.15mol/LNaCl, 1% (W/V) TritonX-100, pH7.35;
7) sample dilution buffer: 0.01M PBS containing 10% (W/V) BSA, pH 7.35;
8) antibody dilution: 0.01M PBS containing 1M D-glucose, 2% (W/V) glycerol, 0.3% (W/V) Tween20, pH7.35;
9) washing liquid: 1 XPBS with 0.15mol/LNaCl, 10% (W/V) glycerol, 1% (W/V) TritonX-100, pH7.35;
10) enzyme working solution: alkaline phosphatase-streptavidin
11) Substrate color developing solution: BCIP color developing solution.
2. The detection steps of the solid-phase membrane immunoassay kit for detecting the anti-Heat shock protein90-beta-IgG antibody are as follows:
2.1 coating and sealing: placing 10 mu L of Heat shock protein90-beta antigen direct contact with the concentration of 60 mu g/mL on a nitrocellulose membrane in an incubator at 37 ℃ for drying for 20min, placing the nitrocellulose membrane in a detection plate, adding 200 mu L of 5% (W/V) BSA in the incubator at 37 ℃ for sealing for 20min, discarding the sealing solution, and washing for 2 times by using a washing solution;
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 20min, and arranging 3 parallel holes for each sample;
2.3 incubation with secondary antibody: discarding the liquid in the detection plate, washing with washing solution for 5 times × 1min, adding 20 μ L of 1:400 biotin-labeled anti-human IgG antibody, and incubating at 25 deg.C for 20 min;
2.4 color development: discarding the liquid in the detection plate, washing with washing solution for 3 times × 3min, 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 3 times × 3min, adding BCIP color developing solution, reacting at room temperature for 20min, washing the detection plate with flowing water, and terminating the enzyme reaction.
2.6 results: the test nitrocellulose membrane strip is taken out and dried by a blower, and qualitatively determined by naked eyes by using a colorimetric card, and the result is shown in figure 3 if the test nitrocellulose membrane strip is positive if obvious brown spots appear.
And (3) placing the membrane strip on a developing instrument for scanning, drawing a standard curve by using analysis software carried by the developing instrument with the concentration of a reference standard substance as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate, and performing semi-quantitative analysis on the anti-Heat shock protein90-beta-IgG antibody level in the serum.
Example 5
Preparation of magnetic particle chemiluminescence immunoassay kit for detecting anti-Heat shock protein90-beta-IgG antibody
1. The chemiluminescence detection kit for the anti-Heat shock protein90-beta-IgG antibody comprises the following components:
(1) acridinium ester labeled anti-human IgG;
(2) carboxyl magnetic beads coupled with Heat shock protein90-beta antigen;
(3) chemiluminescent pre-excitation liquid H 2 O 2 And exciting liquid NaOH;
(4) anti-Heat shock protein90-beta-IgG antibody series standard solution, standard concentration: 0. mu.g/mL, 4. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, 20. mu.g/mL, 40. mu.g/mL;
(5) antigen dilution: 1 XPBS pH7.35 containing 0.15mol/LNaCl, 1% (W/V) TritonX-100;
(6) sample dilution buffer: 0.01M PBS containing 10% (W/V) BSA, pH7.35;
(7) antibody dilution: 0.01M PBS pH7.35 containing 1M D-glucose, 2% (W/V) glycerol, 0.3% (W/V) Tween 20;
(8) washing solution: 1 XPBS pH7.35 containing 0.15mol/LNaCl, 10% (W/V) glycerol, 1% (W/V) TritonX-100.
2. Preparation of magnetic bead coupled antigen
The preparation process is shown in figure 4, and the specific steps are as follows:
(1) 1mg of carboxyl magnetic particles are taken into a 0.5mL centrifuge tube, 200 mu L of 0.1mol/L MES buffer solution is added, the mixture is uniformly mixed by vortex, the mixture is placed on a magnetic frame and stands for 5min, and the supernatant is discarded. Wash 3 times, then add 200 μ Ι _ of MES buffer pH 5.0 and vortex;
(2) adding 18 mu L of Heat shock protein90-beta antigen, whirling, rotating the reaction tube, and incubating for 20min at room temperature;
(3) adding 10 mu L of 10mg/mL coupling reagent EDC, vortexing, rotating the reaction tube, and incubating for 1h at room temperature;
(4) the supernatant was removed and washed 3 times with 200. mu.L of washing buffer (TBS + 0.05% (W/V) Tween-20);
(5) blocking with 1% (W/V) BSA in buffer was repeated 4 times for 10min each. The magnetic particle suspension was stored at 2-8 ℃.
3. Preparation of acridinium ester labeled antibody
(1) Putting 100 μ L of anti-human IgG antibody into dialysis bag, putting the dialysis bag into 1L of labeled buffer solution, dialyzing for 3 times, and finally dialyzing overnight, wherein the labeled buffer solution is Na 2 CO 3 -NaHCO 3 A buffer solution with the pH of 10.0 and the concentration of 0.1 mol/L;
(2) weighing 1.7mg of acridinium ester NSP-DMAE-NHS and dissolving in 447 mu L of anhydrous dimethylformamide DMF to form 6.5mmol/LNSP-DMAE-NHS DMF solution;
(3) the dialyzed antibody solution was placed in a 500. mu.L centrifuge tube, 100. mu.L of a 6.5mmol/LNSP-DMAE-NHS DMF solution was added, and the molar ratio of acridinium ester to antibody was 7.4: 1, adding 200 mu L of marking buffer solution, reacting for 45min at room temperature, adding 10 mu L of lysine, 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.
4. Sample preparation: samples were prepared as follows: diluting at a ratio of 10;
5. the detection steps of the chemiluminescence method kit for detecting the anti-Heat shock protein90-beta-IgG antibody are as follows:
(1) sequentially adding 100 μ L of sample to be tested, 100 μ L of coupled magnetic powder suspension and 100 μ L of acridinium ester labeled secondary antibody into a reaction tube, shaking uniformly, mixing, and keeping the temperature at 37 ℃ for 20 min;
(2) washing for 5 times;
(3) fully shaking the washed reaction container to uniformly disperse the magnetic particles;
(4) adding 100 μ L of chemiluminescent pre-excitation liquid H 2 O 2 Then, 100. mu.L of NaOH was added to the chemiluminescence excitation solution, and the relative luminescence intensity was measured, and the results are shown in FIG. 5.
As can be seen, the content of the anti-Heat shock protein90-beta-IgG antibody in the sample is directly proportional to the luminescence intensity thereof.
Example 6
Clinical application of kit for detecting serum anti-Heat shock protein90-beta-IgG antibody
1. Subject inclusion
Patients diagnosed with various types of nephropathies from 6 months in 2018 to 6 months in 2020, including 298 Nephrotic Syndrome (NS), 100 anaphylactoid purpura (HP), 100 purpura nephritis (HPN), 100 Kawasaki Disease (KD), and 100 healthy children (NC) at the same time. Serum samples were taken from various renal patients and healthy controls. All subjects received a first serum sample collection prior to no immunosuppressive treatment.
2. Detection condition of anti-Heat shock protein90-beta-IgG antibody in various nephrotic patients
The kit of the invention is used for detecting the anti-Heat shock protein90-beta-IgG antibody level in the serum of patients diagnosed with various nephropathies from 6 months 2018 to 6 months 2020, including 298 nephrotic syndromes, 100 anaphylactoid purpura, 100 purpura nephritis, 100 Kawasaki disease and 100 healthy children at the same period, and the result is shown in figure 6.
As can be seen, the anti-Heat shock protein90-beta-IgG antibody was positive in patients with nephrotic syndrome, while the anti-Heat shock protein90-beta-IgG antibody was negative in purpuric nephritis, Henoch-Schonlein purpura and healthy children.
3. The serum anti-Heat shock protein90-beta-IgG antibody of a patient with nephrotic syndrome is linearly related to the expression level of a vascular endothelial injury marker
The kit provided by the invention is used for detecting the expression level of the anti-Heat shock protein90-beta-IgG antibody in the serum of a patient diagnosed with nephrotic syndrome from 6 months in 2018 to 6 months in 2020, and detecting the expression level of a vascular endothelial injury marker Plvap in the serum of the patient, and the result is shown in figure 7.
As can be seen, the expression level of the anti-Heat shock protein90-beta-IgG antibody in patients with nephrotic syndrome is linearly related to the expression level of the vascular endothelial injury marker, and the nephrotic syndrome is related to the vascular endothelial injury.
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 whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of the blood vessel, and antibodies of the vascular endothelial cells can cause damage to the vascular endothelial cells and induce dysfunction of a blood circulation system, so that the heart, the lung, the liver and other organs are damaged, and diseases related to the organs are caused, including nephrotic syndrome. Therefore, the detection of vascular endothelial cell autoantibodies in the blood circulation system can be clinically used to indicate the presence of vascular endothelial cell damage. Because the vascular endothelial cells of different organs are the same, the invention firstly discovers the vascular endothelial cell autoantibody-anti-Heat shock protein90-beta-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 above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims (7)

1. The application of the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody in the preparation of the kit for detecting the vascular endothelial injury.
2. The use of claim 1, wherein the reagent for detecting anti-Heat shock protein90-beta-IgG autoantibody comprises a Heat shock protein90-beta protein or a tagged Heat shock protein90-beta recombinant protein or polypeptide; the NCBI protein accession number of the Heat shock protein90-beta protein is BC 014485.
3. The use according to claim 2, wherein the tag is modified at the N-terminal or C-terminal of the Heat shock protein90-beta recombinant protein, and the tag is selected from any one of His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, C-Myc tag, Flag tag and biotin tag.
4. A kit for detecting vascular endothelial injury, which is characterized by comprising a reagent for detecting anti-Heat shock protein90-beta-IgG autoantibody.
5. The kit of claim 4, wherein the reagent for detecting the anti-Heat shock protein90-beta-IgG autoantibody is immobilized on a solid support selected from any one of a nitrocellulose membrane, a fluorescence-encoded microsphere, a magnetic stripe chip, a magnetic microparticle, and an enzyme-labeled microplate.
6. The kit according to claim 4, further comprising a labeled antibody selected from any one of an enzyme-labeled anti-human IgG antibody, a chemiluminescent substance-labeled anti-human IgG antibody, and a biotin-labeled anti-human IgG antibody.
7. The kit according to claim 4, wherein the detection sample of the kit is any one selected from whole blood, serum, plasma, urine, lymph fluid and pleural effusion of mammals.
CN202210491401.6A 2022-05-07 2022-05-07 Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof Pending CN114994330A (en)

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