CN114910649A - Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury - Google Patents

Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury Download PDF

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CN114910649A
CN114910649A CN202210491431.7A CN202210491431A CN114910649A CN 114910649 A CN114910649 A CN 114910649A CN 202210491431 A CN202210491431 A CN 202210491431A CN 114910649 A CN114910649 A CN 114910649A
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enolase
alpha
antibody
kit
tag
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叶青
毛建华
张俊峰
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Zhejiang University ZJU
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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    • G01—MEASURING; TESTING
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    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders

Abstract

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

Description

Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury
Technical Field
The invention relates to the technical field of diagnostic kits, in particular to application of a reagent for detecting an anti-alpha-enolase-IgG antibody in preparation of a kit for detecting vascular endothelial injury.
Background
Blood, blood vessels, and the heart constitute the blood circulation system of the human body. Blood in the blood circulation system flows through blood vessels and flows through the whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of the blood vessel, are a layer of mononuclear cells between blood flow and vessel wall tissues, can secrete a series of vasoactive substances such as NO, PGI2, ET-1 and the like through three ways of autocrine, endocrine and paracrine, and have the functions of regulating the blood vessel tone, resisting thrombosis, inhibiting the proliferation of smooth muscle cells, inhibiting the inflammatory reaction of the blood vessel 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 central lesion (MCD) is the leading cause of nephrotic syndrome in children, accounting for 10-15% of nephrotic syndrome in adults. Glomeruli of patients with minimal disease appeared essentially normal under light microscopy, and the only histopathological abnormality seen under electron microscopy was the disappearance of diffuse podocyte foot process fusion. Thus, MCD is considered to be a primary podocyte disease. Complete remission of proteinuria after corticosteroid treatment is a marker of MCD and, in general, progressive renal failure is rare. However, MCD can lead to serious complications. Complications associated with the disease observed in adults include mainly venous thrombosis and severe acute kidney injury requiring temporary dialysis. Furthermore, because MCD is characterized by a chronic, recurrent course, prolonged immunosuppressive therapy is often required to maintain proteinuria remission. However, long-term immunosuppressive therapy increases the risk of serious infection and carries a long-term risk of malignancy.
Currently, little is known about the underlying pathogenesis of MCD. One of the views is that the disease is triggered by the circulating permeability factors produced by immune cells. Since the pathogenesis of primary Focal Segmental Glomerulosclerosis (FSGS) is very similar to that of MCD, many scholars consider MCD and FSGS to be phenotypes of the same disease at different stages. T cells were first suspected to be the source of the circulating permeability factor based on the association between MCD and non-hodgkin's lymphoma, the remission induced by measles infection and prolonged remission following cyclophosphamide treatment. However, the therapeutic effects of rituximab and other specific B cell depleting drugs have presented challenges to T cell sources in recent years. Notably, the direct effect of corticosteroids and rituximab on podocytes is also considered to have therapeutic effect. The screening and identification of many podocyte autoantibodies in MCD and FSGS nephrotic syndrome patients by our team provides a potential link between podocyte injury, autoimmunity and proteinuria response to anti-B cell therapy, and therefore, the concept of 'Autoimmune podocytosis' (Autoimmune podocytopathies) is first proposed internationally and gradually recognized by the same lines at home and abroad. Recently, the Harvard medical college team Watts et al found that anti-Nephrin autoantibodies also exist in the serum of children and adults with minimal change nephrotic syndrome, which provides a powerful evidence for our innovative theory.
Although the observed podocyte injury is a major classical feature of MCD, the disease mechanism may also involve glomerular vascular endothelial cells. Idiopathic Nephrotic Syndrome (INS) reported as early as 2000 by Futrakul N et al is often accompanied by renal hypoperfusion. 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 patients with MCD and FSGS nephrotic syndrome through previous studies. Animal experiments prove that the glomerular vascular endothelial cell autoantibodies can cause severe damage to the glomerular vascular endothelial cells of mice. In vitro cell culture experiments also show that these autoantibodies can affect the morphology and function of vascular endothelial cells. Clinical studies have shown that these autoantibodies to glomerular vascular endothelial cells are associated with a high coagulation status and poor prognosis in patients. In addition, our findings suggest that glomerular vascular endothelial cell injury caused by autoantibodies to glomerular vascular endothelial cells may be the initiating factor of characteristic podocyte injury in MCD, and is one of the important causes of the disease. Therefore, we have proposed the second hit theory of the onset of MCD and FSGS nephrotic syndrome for the first time internationally: that is, pathogenic agents including autoantibodies first damage the glomerular vascular endothelial cells, and then these pathogenic agents further damage the podocytes, eventually causing morbidity to the patient. Because the pathogenic agents in the blood circulation system are unlikely to come into contact with the podocytes from the specific anatomical location of the podocytes unless the integrity of the glomerular vascular endothelial cells has been compromised. Therefore, the research result of the autoantibodies of the endothelial cells of the glomerular vessels is a breakthrough in the theoretical research of the pathogenesis of the nephrotic syndrome. However, a kit for detecting vascular endothelial injury with high efficiency is still lacking at present.
Disclosure of Invention
The invention aims to provide application of a reagent for detecting an anti-alpha-enolase-IgG antibody in preparation of a kit for detecting vascular endothelial injury. The detection of the alpha-enolase-IgG resisting antibody can realize the effective detection of vascular endothelial damage.
The invention provides an application of a reagent for detecting an anti-alpha-enolase-IgG antibody in preparing a kit for detecting vascular endothelial injury.
Preferably, the reagent for detecting the anti-alpha-enolase-IgG antibody comprises alpha-enolase or alpha-enolase recombinant protein or polypeptide containing a label; the NCBI protein accession number of the α -enolase is BC 015641.
Preferably, the tag comprises a His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag or biotin tag.
Preferably, when the tag is a His tag, the amino acid sequence of the tag-containing alpha-enolase recombinant protein is shown as SEQ ID No. 1.
Preferably, the vascular endothelial cell injury comprises glomerular vascular endothelial cell injury.
The invention also provides a kit for detecting the anti-alpha-enolase-IgG antibody, which comprises: the reagent for detecting the anti-alpha-enolase-IgG antibody, the solid phase carrier and the labeled antibody in the application of the technical scheme.
Preferably, the labeled antibody comprises an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody; the secondary antibody comprises an anti-human IgG antibody.
Preferably, the enzyme-labeled secondary antibody comprises a horseradish peroxidase-labeled anti-human IgG antibody; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.
Preferably, the solid phase carrier comprises a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle or an enzyme labeling micropore plate.
The invention provides an application of a reagent for detecting an anti-alpha-enolase-IgG antibody in preparing a kit for detecting vascular endothelial injury. The invention firstly detects an anti-alpha-enolase-IgG antibody in a part of patients with nephrotic syndrome, and determines that the target antigen aimed by the autoantibody is alpha-enolase on glomerular vascular endothelial cells. The invention finds that the alpha-enolase 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 alpha-enolase-IgG resisting antibody can realize the detection of vascular endothelial injury, and provides a basis for the research of the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment. The kit for detecting the anti-alpha-enolase-IgG antibody can qualitatively and quantitatively detect the anti-alpha-enolase-IgG antibody in serum of patients with nephrotic syndrome, and the kit provided by the invention utilizes the IgG antibody of human anti-tag peptide as a standard substance and greatly improves the detection accuracy, sensitivity, specificity and detection speed by combining a biotin-avidin amplification system and magnetic particle chemiluminescence immunoassay. Specifically, compared with the prior art, the kit has the following beneficial effects:
1. the kit can realize high-efficiency detection of vascular endothelial injury, and judges that the vascular endothelial injury exists when the anti-alpha-enolase-IgG antibody is detected.
2. At present, alpha-enolase and anti-alpha-enolase-IgG antibodies related to kidney disease patients at home and abroad are only limited to molecular mechanism research, and the level of the antibodies in serum of the patients is not quantitatively detected. The invention identifies the IgG autoantibody aiming at the alpha-enolase for the first time, and invents a detection kit aiming at the alpha-enolase-IgG autoantibody, thereby filling the blank at home and abroad. The kit is used for detecting the anti-alpha-enolase-IgG antibody in the serum of 298 nephrotic syndrome patients, and the result shows that the anti-alpha-enolase-IgG antibody of 152 patients is positive, namely the positive detection rate of the anti-alpha-enolase-IgG antibody is 51.01%. The invention can provide a basis for researching the molecular mechanism and clinical diagnosis and treatment of nephrotic syndrome by detecting the anti-alpha-enolase-IgG antibody.
3. The kit of the invention relates to a solid-phase membrane immunoassay qualitative analysis of an anti-alpha-enolase-IgG antibody in human serum, and the detection accuracy is greatly improved by taking the human anti-tag peptide IgG antibody as a standard substance. The solid-phase membrane immunoassay qualitative detection is simple to operate, the reagent dosage is less, and the solid-phase membrane immunoassay qualitative detection is saved by about 10 times compared with the traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely 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 thereof is not influenced; in addition, the kit for qualitatively detecting the anti-alpha-enolase-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-alpha-enolase-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 surface area of the coating is greatly increased, the adsorption quantity of the antigen is increased, the reaction speed is improved, the cleaning and the separation are simpler and more convenient, the pollution is reduced, and the probability of cross infection is reduced. On the other hand, the acridine ester luminescent agent is adopted to directly mark the antihuman IgG, the chemical reaction is simple and quick, and no catalyst is needed; the acridinium ester chemiluminescence is of the scintillation type by initiating the luminescent reagent (H) 2 O 2 NaOH) can reach the maximum after 0.4s, the half-life period is 0.9s, the detection is basically finished within 2s, and the rapid detection is convenient.
Drawings
FIG. 1 shows that Alpha-enolase on glomerular vascular endothelial cells is the primary target antigen for autoantibodies in patients with nephrotic syndrome; wherein A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; b: the first antibody is a two-dimensional electrophoresis protein spot of serum of a nephrotic syndrome patient; c: mass spectrometric identification of the target antigen Alpha-enolase protein;
FIG. 2 shows the optimization of Alpha-enolase expression conditions, in which lane M 1 : protein marker; lane PC 1 : BSA (1. mu.g); lane PC 2 : BSA (2 μ g); lane NC: no induced cell lysate; lane 1: induction of cell lysate for 16h at 15 ℃; lane 2: induction of cell lysates for 4h at 37 ℃; lane NC 1 : cell lysate supernatant without induction; lane 3: inducing cell lysate supernatant for 16h at 15 ℃; lane 4: inducing the cell lysate supernatant for 4h at 37 ℃; lane NC 2 : non-induced inclusion of cell lysate pelletsA body; lane 5: inducing the cell lysate for 16h at 15 ℃ to precipitate the inclusion bodies; lane 6: inducing the cell lysate for 4h at 37 ℃ to precipitate the inclusion bodies;
FIG. 3 is an SDS-PAGE identification of the expressed recombinant protein Alpha-enolase;
FIG. 4 is a solid-phase membrane immunoassay kit for detecting anti-alpha-enolase-IgG antibody in serum of patients with nephrotic syndrome;
FIG. 5 is a schematic diagram of the principle of detecting anti-alpha-enolase-IgG antibody by the magnetic particle chemiluminescence immunoassay kit.
FIG. 6 is a schematic diagram of an antigen protein Alpha-enolase coated carboxyl magnetic microparticle;
FIG. 7 shows the detection of anti- α -enolase-IgG antibodies in various renal patients, where NC: a healthy child; HP: allergic purpura; HPN: purpuric nephritis; KD: kawasaki disease; and NS: nephrotic syndrome;
FIG. 8 is a linear correlation of anti-alpha-enolase-IgG antibody with vascular endothelial injury markers.
Detailed Description
The invention provides an application of a reagent for detecting an anti-alpha-enolase-IgG antibody in preparing a kit for detecting vascular endothelial injury. Alpha-enolase (Alpha-enolase) is a glycolytic enzyme whose multiple functions far exceed its main metabolic role as an energy supplier in the glycolytic process. Blood, blood vessels, and the heart constitute the blood circulation system of the human body. Blood in the blood circulation system flows through blood vessels and flows through the whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of the blood vessel, and antibodies of the vascular endothelial cells can cause damage to the vascular endothelial cells and induce dysfunction of a blood circulation system, so that the heart, the lung, the liver and other organs are damaged, and diseases related to the organs are caused, including nephrotic syndrome. Therefore, the detection of vascular endothelial cell autoantibodies in the blood circulation system can be clinically used to indicate the presence of vascular endothelial cell damage. Because the vascular endothelial cells of different organs are the same, the invention firstly discovers the vascular endothelial cell autoantibody-anti-alpha-enolase-IgG antibody, and the application of the invention can realize the detection of all vascular endothelial injuries of the whole body including glomerular vascular endothelium.
The reagent for detecting the anti-alpha-enolase-IgG antibody provided by the invention takes alpha-enolase as a target spot to detect the alpha-enolase autoantibody (namely, the anti-alpha-enolase-IgG antibody is a biomarker for detecting vascular endothelial cell damage), and the reagent can realize high-efficiency detection of vascular endothelial damage. In the present invention, the agent is capable of immunoreacting with alpha-enolase autoantibodies derived from tissue (kidney biopsy) or body fluids (in particular blood, plasma, serum). In the present invention, the reagent for detecting an anti- α -enolase-IgG antibody preferably comprises α -enolase or a tag-containing α -enolase recombinant protein or polypeptide; the NCBI protein accession number of the alpha-enolase is BC 015641. In the present invention, the tag is preferably a tag having some biological or physical function, in particular an N-terminus or a C-terminus; the existence of the tags is beneficial to the purification, fixation and precipitation of antigen protein; more preferably, the tag is a sequence or domain capable of specifically binding to a ligand, such as a tag peptide, preferably selected from the group consisting of: his tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag or biotin tag. In the present invention, when the tag is a His tag, the amino acid sequence of the tag-containing α -enolase recombinant protein is preferably as shown in SEQ ID No. 1: MSILKIHAREIFDSRGNPTVEVDLFTSKGLFRAAVPSGASTGIYEALELRDNDKTRYMGKGVSKAVEHINKTIAPALVSKKLNVTEQEKIDKLMIEMDGTENKSKFGANAILGVSLAVCKAGAVEKGVPLYRHIADLAGNSEVILPVPAFNVINGGSHAGNKLAMQEFMILPVGAANFREAMRIGAEVYHNLKNVIKEKYGKDATNVGDEGGFAPNILENKEGLELLKTAIGKAGYTDKVVIGMDVAASEFFRSGKYDLDFKSPDDPSRYISPDQLADLYKSFIKDYPVVSIEDPFDQDDWGAWQKFTASAGIQVVGDDLTVTNPKRIAKAVNEKSCNCLLLKVNQIGSVTESLQACKLAQANGWGVMVSHRSGETEDTFIADLVVGLCTGQIKTGAPCRSERLAKYNQLLRIEEELGSKAKFAGRNFRNPLAKHHHHHH are provided.
In the present invention, the vascular endothelial injury preferably comprises glomerular vascular endothelial cell injury. More specifically, the vascular endothelial injury of the present invention preferably includes vascular endothelial injury of nephrotic syndrome. In the present invention, the nephrotic syndrome preferably includes a morbid disease or primary focal segmental glomerulosclerosis.
The invention also provides a kit for detecting the anti-alpha-enolase-IgG antibody, which comprises: the reagent for detecting the anti-alpha-enolase-IgG antibody, the solid phase carrier and the labeled antibody in the application of the technical scheme.
In the present invention, the reagent for detecting an anti- α -enolase-IgG antibody (α -enolase or α -enolase recombinant protein containing a tag) is preferably immobilized on a solid support. By "immobilized" herein is meant bound to a solid support that is insoluble in water of the alpha-enolase antigenic protein, the solid support or support being insoluble in water, more preferably by covalent bonding, electrostatic interaction, hydrophobic interaction, or interaction by disulfide bond, 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 of immobilizing alpha-enolase antigen proteins 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 immobilization of an antibody having a specific affinity for the antigenic protein, followed by formation of an antigenic protein-antibody complex for the purpose of immobilization. The antigen protein alpha-enolase immobilization method is preferably a direct coating method: (1) the antigen protein alpha-enolase is combined on the nitrocellulose membrane or a polystyrene microporous plate in a physical adsorption mode or by non-covalent bonds; (2) the magnetic particles with carboxyl functional groups are combined with amino groups of the antigen protein alpha-enolase, and the antigen protein alpha-enolase is combined on the magnetic particles in a chemical coupling mode. In the invention, the solid phase carrier preferably comprises a nitrocellulose membrane, a fluorescence coding microsphere, a magnetic stripe chip, a magnetic particle or an enzyme labeling micropore plate.
The invention preferably adopts a gene recombination prokaryotic expression method to successfully express and purify the recombinant protein alpha-enolase, and uses the recombinant protein alpha-enolase as the antigen protein in a kit to develop a set of kits suitable for detecting the glomerular vascular endothelial cell autoantibody anti-alpha-enolase-IgG antibody of a nephrotic syndrome patient, including a detection kit for qualitatively or quantitatively analyzing and detecting the anti-alpha-enolase-IgG antibody in human serum.
In the present invention, the α -enolase is preferably expressed in bacterial (e.g., E.coli), yeast, insect or mammalian cells. After the alpha-enolase is obtained by expression, the alpha-enolase 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 type of the kit preferably includes a solid phase membrane immunoassay kit or a magnetic microparticle chemiluminescence immunoassay kit; when the kit is a solid-phase membrane immunoassay kit, the kit preferably further comprises an antigen diluent, a sample diluent buffer, an antibody diluent, a substrate developing solution, a washing solution, an enzyme working solution, a standard substance, a positive quality control substance and a negative quality control substance; when the kit is a magnetic particle chemiluminescence immunoassay kit, the kit preferably further comprises chemiluminescence pre-excitation liquid A, chemiluminescence excitation liquid B, a standard substance and a cleaning solution. In the invention, the standard substance and the positive quality control substance are preferably recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-alpha-enolase-IgG antibody extracted from patient serum; the negative quality control product is preferably serum of a healthy physical examiner.
Specifically, when the kit is a solid-phase membrane immunoassay kit, in the kit, the antigen, which is the reagent for detecting the anti-alpha-enolase-IgG antibody, is preferably recombinant protein alpha-enolase (the amino acid sequence is shown in SEQ ID No. 1); the solid phase carrier is preferably a cellulose nitrate membrane of Sataurus CN 140; the positive quality control product (standard product) is preferably human anti-His tag immunoglobulin G (purchased from Yingchu 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 alpha-enolase (the amino acid sequence is shown as SEQ ID NO. 1); the solid phase carrier is preferably carboxyl magnetic beads; the labeled antibody is preferably an acridinium ester labeled anti-human IgG antibody; the chemiluminescence pre-excitation liquid A and the chemiluminescence excitation liquid B are preferably conventional commercial products, and the standard substance is preferably anti-alpha-enolase-IgG antibody with different concentrations; the cleaning solution is preferably a Tris-HCl solution at pH 7.2, 25mmol/L, containing 0.15mol/L NaCL and 0.05% Tween-20.
In the invention, the sample to be tested of the kit is preferably from whole blood, serum, plasma, urine, lymph fluid and hydrothorax and ascites; more preferably mammalian (human) serum.
In the present invention, the principle of the kit for detecting anti- α -enolase-IgG antibodies in serum is preferably as follows: the indirect method reaction principle is utilized, firstly, alpha-enolase antigen is adsorbed on a solid phase carrier to serve as coating antigen, then positive quality control products or standard products or serum samples to be detected are added for incubation, then labeled antibodies (labeled secondary antibodies) are added for reaction, if anti-alpha-enolase-IgG antibodies are contained in the serum to be detected, a ternary complex of the coating antigen alpha-enolase-anti-alpha-enolase-IgG antibodies in the serum to be detected and labeled anti-human IgG antibodies is formed, and finally, light signals are detected by a photogenesis method, a chemiluminescence method and a fluorescence method, so that the aim of qualitatively or quantitatively analyzing the anti-alpha-enolase-IgG antibodies in human serum is fulfilled.
The application of the reagent for detecting anti- α -enolase-IgG antibody in the preparation of a kit for detecting vascular endothelial injury according to the present invention will be described in further detail with reference to the following specific examples, which include, but are not limited to, the following examples.
Example 1 Alpha-enolase on vascular endothelial cells is the major target antigen for autoantibodies in nephrotic syndrome patients
According to the invention, through a large number of early clinical and molecular mechanism researches, the serum IgG level of patients with nephrotic syndrome is found to be high for the first time, and Alpha-enolase on vascular endothelial cells is proved to be a main target antigen for the autoantibody in patients with nephrotic syndrome. It would therefore be advantageous to detect the presence and quantitative levels of anti- α -enolase-IgG antibodies in serum to aid in the early identification of nephrotic syndrome, particularly in screening patients for symptoms of interest. The specific implementation is as follows:
1. extraction of total protein of vascular endothelial cells: vascular endothelial cell lines (EAhy926) were cultured, washed 2-3 times with PBS, then extensively lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitors (# ab 65621; Abcam, 1: 200 dilution), and the samples were then centrifuged at 12000g, 4 ℃ for 30 min. Collecting the supernatant, namely the total protein of the vascular endothelial cells. The total protein concentration of the collected vascular endothelial cells was measured using the BCA protein concentration measurement kit.
2. Two-dimensional electrophoresis: extracting total protein of vascular endothelial cell, performing two-dimensional electrophoresis, transferring to nitrocellulose membrane, incubating with serum of healthy person and nephrotic syndrome patient as primary antibody, and developing with secondary antibody, shown as A in figure 1 and B in figure 1.
3. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after imaging in step 2, and protein spots strongly positive for nephrotic syndrome patients and negative or weakly positive for healthy persons were selected on two-dimensional electrophoresis gel, 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, identified as Alpha-enolase protein, see C in fig. 1.
Example 2 expression and purification of recombinant Alpha-enolase antigen protein
Performing PCR amplification by using a gene encoding Alpha-enolase protein as a template by using a genetic engineering method, and then constructing an expression vector with a His label for protein expression and optimization, wherein the optimization conditions are as follows: inducing cell lysate for 16h at 15 ℃, inducing cell lysate for 4h at 37 ℃, inducing cell lysate supernatant for 16h at 15 ℃, inducing cell lysate supernatant for 4h at 37 ℃, inducing cell lysate precipitate inclusion bodies for 16h at 15 ℃ and inducing cell lysate precipitate inclusion bodies for 4h at 37 ℃. The results show that the Alpha-enolase protein expression in the cell lysate supernatant induced for 16h at 15 ℃ is optimal, see FIG. 2. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve and the like, and finally the molecular weight of the recombinant protein Alpha-enolase is identified by SDS-PAGE, and the result is shown in figure 3.
Example 3
Standard positive serum and standard negative serum were repeatedly measured at 2 levels for each of 4 factors, such as antigen Alpha-enolase coating concentration (100. mu.g/mL, 200. mu.g/mL, 300. mu.g/mL, 400. mu.g/mL), each reaction time (15min, 30min, 45min) and temperature (20 ℃ C., 25 ℃ C.), optimal dilution of enzyme-labeled secondary antibody (1: 100, 1: 500, 1: 1000, 1: 1500). The ratio (P/N) of the highest luminescence (P) of the positive sera to the lowest luminescence (N) of the negative sera was selected. The average P/N value of repeated determination is tested by determining the optimal coating condition and the optimal dilution of the secondary antibody through statistical treatment, and the positive detection rate of the standard positive serum is obviously improved. The optimal antigen coating concentration of the kit is 200 mug/mL, the optimal antigen-antibody reaction temperature of solid-phase membrane immunization 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: 1000. the optimal antigen-antibody reaction temperature of the magnetic particle chemiluminescence immunoassay is 37 ℃, the optimal antigen-antibody reaction time is 15min, and the optimal working dilution of the optimal acridinium ester labeled anti-human IgG antibody is 1: 500.
EXAMPLE 4 preparation of solid phase Membrane immunoassay kit for detecting anti-alpha-enolase-IgG antibody
4.1 composition of solid-phase membrane immunoassay kit for the detection of anti-Alpha-enolase-IgG:
1. a nitrocellulose membrane coated with Alpha-enolase antigen protein;
2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro, hu);
3. negative quality control product: serum of healthy examiners;
4. a biotin-labeled anti-human IgG antibody;
5. antibody diluent;
6. a sample diluent;
7. enzyme working solution: alkaline phosphatase-streptavidin;
8. washing liquid;
9. BCIP color developing agent;
10. and (4) stopping the solution.
4.2 the detection steps are as follows:
4.2.1 blocking, the nitrocellulose membrane coated with the Alpha-enolase antigen protein was placed in a plate bath, 150. mu.l of 3% BSA was added and the membrane was blocked in a 37 ℃ incubator for 15min, and after the blocking solution was aspirated, the membrane was washed 2 times with a washing solution.
4.2.2 serum incubation: diluting the standard substance with the antibody diluent according to a certain proportion, diluting the serum specimen to be detected with the sample diluent according to a certain proportion, adding 50 mul of diluted standard substance and the sample to be detected into a reaction tank, simultaneously performing negative control and positive control, paying attention to the fact that the surface of the nitrocellulose membrane is not scraped when the sample is added, and immediately replacing a new sample adding nozzle after adding one sample. After all the samples were added, the reaction tank was placed on a shaker and incubated at room temperature (20-25 ℃) for 30 min.
4.2.3 cleaning: and diluting the washing liquid for later use, pouring the liquid in the reaction tank after incubation is finished, and washing the reaction tank by using the diluted washing liquid to ensure that the washing liquid is fully immersed in the reaction tank. The cleaning is repeated for 5 times, 10s each time, and the liquid flows down along the reaction tank when the cleaning is carried out, so that the cross contamination is avoided. And (5) drying the reaction tank after cleaning.
4.2.4 incubation with secondary antibody working solution: dilution with antibody 1: the biotin-labeled anti-human IgG antibody was diluted at 1000 ℃ and 6 drops (300. mu.l) were added to the reaction tank, and the mixture was incubated on a shaker at room temperature (20-25 ℃) for 30 min.
4.2.5 cleaning: the process is the same as step 3.
4.2.6. Color development and incubation: adding 200 μ l of alkaline phosphatase-streptavidin enzyme working solution, incubating at room temperature for 20min, discarding the liquid in the detection plate, washing with washing solution for 5 times, adding 6 drops (300 μ l) of BCIP color developing solution into the reaction tank, and incubating at room temperature (20-25 deg.C) on a shaking table for 20 min.
4.2.7. And (3) terminating the reaction: the reaction vessel was flushed with running water to terminate the reaction.
4.2.8. Interpretation results: taking out the test strip, and drying the test strip by air blowing (about 5min) or placing the test strip in a drying oven at 37-50 ℃ for more than 20 min. And (3) carrying out naked eye qualitative judgment, wherein the person with obvious brown spots is positive (see figure 4) or placing the membrane strip on a developing instrument for scanning, and drawing a standard curve by analysis software carried by the developing instrument by taking the concentration of a reference standard substance as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate to carry out semi-quantitative analysis on the anti-Alpha-enolase-IgG level in the serum.
EXAMPLE 5 preparation of chemiluminescent immunoassay kit for the detection of anti-alpha-enolase-IgG antibodies
5.1 composition of the chemiluminescent immunoassay kit for the detection of anti-Alpha-enolase-IgG:
1. magnetic particle solution coated with Alpha-enolase antigen protein,
2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro lake),
3. negative quality control product: serum for health physical examination person
4. The dilution liquid of the sample is used for diluting the sample,
5. the acridinium ester is marked on the anti-human IgG solution,
6. the pre-excitation liquid is mixed with the water,
7. the exciting liquid is used for exciting the reaction liquid,
8. and (4) washing liquid.
5.2 detection principle: the kit adopts an indirect method to detect the anti-alpha-enolase-IgG antibody in human serum, and the whole process comprises two steps of reactions: firstly, mixing the magnetic bead solution with a diluted sample, binding a specific anti-alpha-enolase-IgG antibody on the magnetic bead, and washing to remove residual solution. Secondly, acridinium ester labeled anti-human IgG antibody is added to form a magnetic bead-antigen-anti-alpha-enolase-IgG antibody-acridinium ester antibody compound, unbound residual liquid is washed away, and then pre-excitation liquid (H) is added 2 O 2 ) And performing luminescence reaction with exciting solution (NaOH), recording luminescence value, wherein the antibody concentration is in direct proportion to the luminescence value, and calculating the concentration value through a calibration curve, as shown in FIG. 5.
5.3 the solid phase carrier of the kit is magnetic particles containing carboxyl functional groups.
5.4 the antigen coating mode of the kit is that carboxyl magnetic particles are activated by EDC/Sulfo-NHS and covalently combined with antigen (amino residue) to form a magnetic particle solution. The coating step is as follows:
a) adding 40 μ l of magnetic bead stock solution into 400 μ l of 0.05M phosphate buffer solution, mixing for 8min, performing magnetic separation, and discarding the supernatant;
b) adding 200 μ l of 10mg/mL sodium periodate into 200 μ l of 2% dextran solution to react;
c) after the reaction is finished, adding 10mg/mL EDC solution prepared by phosphate buffer solution and 10mg/mLSulfo-NHS solution, and uniformly mixing for 60 min;
d) magnet separation, discarding supernatant, taking 400. mu.l of 0.05M phosphate buffer solution to wash the magnetic beads, adding 400. mu.l of preservation solution to fix volume for preservation.
Removing supernatant of the activated magnetic beads, adding pre-cooled 1ml of 20mM MES, and continuously washing the magnetic beads for 2 times; adding 200 mu L of 2mg/mL antigen protein Alpha-enolase into the activated magnetic beads, fully and uniformly mixing, and standing at room temperature for reaction for 16 h; after the reaction is finished, adding a PBS buffer solution with the pH of 7.4 and containing 0.2 percent Tween20, and repeatedly washing the magnetic beads for 2 times; then adding PBS buffer solution with pH of 7.4 containing 0.2% Tween20 and 0.2% BSA until the final concentration of the magnetic beads is 10mg/mL, fully and uniformly mixing, and standing at room temperature for reaction for 30 min; after the reaction was completed, the supernatant was discarded, and the magnetic beads were resuspended in a pH7.4 PBS buffer containing 0.2% Tween20 and 0.2% BSA, and the crosslinking of the activated magnetic beads to the antigen protein Alpha-enolase was completed, as shown in FIG. 6.
5.5 acridinium ester labeled anti-human IgG solution, comprising the following steps:
a) preparing 2mg/mL acridinium ester solution by using dimethylformamide;
b) preparing 1mg/mL anti-human IgG antibody by using 0.2M (pH8.0) carbonate buffer solution;
c) uniformly mixing and stirring acridinium ester and an anti-human IgG antibody in a molar ratio of 4:1, and reacting for 40 min;
d) adding 20 μ l of carbonate buffer solution containing 5% lysine for 30min to terminate the reaction;
e) desalting to remove impurities to obtain acridinium ester labeled anti-human IgG solution.
5.6 the detection steps are as follows:
5.6.1 diluting the sample by using the sample diluent according to a certain proportion;
5.6.2 adding 50 μ l diluted sample or anti-His tag IgG standard into magnetic particle solution, reacting at 37 deg.C for 15min, and making negative and positive control;
5.6.3 washing solution for 3 times;
5.6.4 adding acridinium ester labeled anti-human IgG solution, reacting at 37 deg.C for 5 min;
5.6.5 washing solution for 3 times;
5.6.6 adding pre-exciting liquid (H) 2 O 2 ) Carrying out reaction by using an excitation liquid (NaOH), and collecting a luminescence measurement value;
5.6.7 the concentration measurement is calculated from the calibration curve.
Example 6 clinical application of kit for detecting serum anti-alpha-enolase-IgG antibody
6.1 Subjects included patients diagnosed with various types of nephropathies from 6 months in 2018 to 6 months in 2020, including 298 Nephrotic Syndrome (NS), 100 Henoch Schonlein purpura (HP), 100 Henoch Schonlein nephritis (HPN), 100 Kawasaki Disease (KD) and 100 healthy children (NC) at the same time. Serum samples were taken from various renal patients and healthy controls. All subjects received a first serum sample collection prior to no immunosuppressive treatment.
6.2 detection of anti- α -enolase-IgG antibodies in various patients with nephropathy the kit of the present invention was used to detect the anti- α -enolase-IgG antibody levels in the sera of patients diagnosed with various nephropathy from 6 months 2018 to 6 months 2020, including 298 nephrotic syndrome, 100 allergic purpura, 100 purpura nephritis, 100 Kawasaki disease and 100 healthy children at the same time, and the results showed that anti-Alpha-enolase-IgG antibodies were positive in some nephrotic syndrome patients (152 patients were positive in anti-moesin-IgG antibodies, i.e., the anti-moesin-IgG antibody positive detection rate was 51.01%), whereas anti- α -enolase-IgG antibodies were negative in purpura nephritis, allergic purpura, Kawasaki disease and healthy children, as shown in FIG. 7.
6.3 serum anti-alpha-enolase-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-alpha-enolase-IgG antibody in the serum of the patient with nephrotic syndrome diagnosed from 6 months 2018 to 6 months 2020, and detecting the expression level of the vascular endothelial injury marker Plvap in the serum of the patient, and the result shows that the expression level of the anti-alpha-enolase-IgG antibody of the patient with nephrotic syndrome is linearly related to the expression level of the vascular endothelial injury marker, and the nephrotic syndrome is related to vascular endothelial injury, and the figure 8 shows that the expression level of the anti-alpha-enolase-IgG antibody of the patient with nephrotic syndrome is linearly related to the vascular endothelial injury.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhejiang university
Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 440
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ser Ile Leu Lys Ile His Ala Arg Glu Ile Phe Asp Ser Arg Gly
1 5 10 15
Asn Pro Thr Val Glu Val Asp Leu Phe Thr Ser Lys Gly Leu Phe Arg
20 25 30
Ala Ala Val Pro Ser Gly Ala Ser Thr Gly Ile Tyr Glu Ala Leu Glu
35 40 45
Leu Arg Asp Asn Asp Lys Thr Arg Tyr Met Gly Lys Gly Val Ser Lys
50 55 60
Ala Val Glu His Ile Asn Lys Thr Ile Ala Pro Ala Leu Val Ser Lys
65 70 75 80
Lys Leu Asn Val Thr Glu Gln Glu Lys Ile Asp Lys Leu Met Ile Glu
85 90 95
Met Asp Gly Thr Glu Asn Lys Ser Lys Phe Gly Ala Asn Ala Ile Leu
100 105 110
Gly Val Ser Leu Ala Val Cys Lys Ala Gly Ala Val Glu Lys Gly Val
115 120 125
Pro Leu Tyr Arg His Ile Ala Asp Leu Ala Gly Asn Ser Glu Val Ile
130 135 140
Leu Pro Val Pro Ala Phe Asn Val Ile Asn Gly Gly Ser His Ala Gly
145 150 155 160
Asn Lys Leu Ala Met Gln Glu Phe Met Ile Leu Pro Val Gly Ala Ala
165 170 175
Asn Phe Arg Glu Ala Met Arg Ile Gly Ala Glu Val Tyr His Asn Leu
180 185 190
Lys Asn Val Ile Lys Glu Lys Tyr Gly Lys Asp Ala Thr Asn Val Gly
195 200 205
Asp Glu Gly Gly Phe Ala Pro Asn Ile Leu Glu Asn Lys Glu Gly Leu
210 215 220
Glu Leu Leu Lys Thr Ala Ile Gly Lys Ala Gly Tyr Thr Asp Lys Val
225 230 235 240
Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe Phe Arg Ser Gly Lys
245 250 255
Tyr Asp Leu Asp Phe Lys Ser Pro Asp Asp Pro Ser Arg Tyr Ile Ser
260 265 270
Pro Asp Gln Leu Ala Asp Leu Tyr Lys Ser Phe Ile Lys Asp Tyr Pro
275 280 285
Val Val Ser Ile Glu Asp Pro Phe Asp Gln Asp Asp Trp Gly Ala Trp
290 295 300
Gln Lys Phe Thr Ala Ser Ala Gly Ile Gln Val Val Gly Asp Asp Leu
305 310 315 320
Thr Val Thr Asn Pro Lys Arg Ile Ala Lys Ala Val Asn Glu Lys Ser
325 330 335
Cys Asn Cys Leu Leu Leu Lys Val Asn Gln Ile Gly Ser Val Thr Glu
340 345 350
Ser Leu Gln Ala Cys Lys Leu Ala Gln Ala Asn Gly Trp Gly Val Met
355 360 365
Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu
370 375 380
Val Val Gly Leu Cys Thr Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg
385 390 395 400
Ser Glu Arg Leu Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Glu Glu
405 410 415
Leu Gly Ser Lys Ala Lys Phe Ala Gly Arg Asn Phe Arg Asn Pro Leu
420 425 430
Ala Lys His His His His His His
435 440

Claims (9)

1. The application of the reagent for detecting the alpha-enolase-IgG antibody in preparing the kit for detecting the vascular endothelial injury.
2. The use according to claim 1, wherein the reagent for detecting anti- α -enolase-IgG antibodies comprises α -enolase or a recombinant protein or polypeptide of α -enolase containing a tag; the NCBI protein accession number of the α -enolase is BC 015641.
3. The use of claim 2, wherein the tag comprises a His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag, or biotin tag.
4. The use according to claim 2, wherein when the tag is a His-tag, the amino acid sequence of the tag-containing recombinant α -enolase protein comprises SEQ ID No. 1.
5. The use of claim 1, wherein the vascular endothelial injury comprises glomerular vascular endothelial cell injury.
6. A kit for detecting an anti- α -enolase-IgG antibody, the kit comprising: a reagent for detecting an anti-alpha-enolase-IgG antibody, 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; the secondary antibody comprises an anti-human IgG antibody.
8. The kit of claim 7, wherein the enzyme-labeled secondary antibody comprises a horseradish peroxidase-labeled anti-human IgG antibody; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.
9. The kit of claim 6, wherein the solid support comprises a nitrocellulose membrane, a fluorescent encoded microsphere, a magnetic strip chip, a magnetic microparticle, or an enzyme-labeled microplate.
CN202210491431.7A 2022-05-07 2022-05-07 Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury Pending CN114910649A (en)

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