CN114910650A - Application of reagent for detecting anti-moesin-IgG antibody in preparation of kit for detecting vascular endothelial injury - Google Patents

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

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CN114910650A
CN114910650A CN202210496945.1A CN202210496945A CN114910650A CN 114910650 A CN114910650 A CN 114910650A CN 202210496945 A CN202210496945 A CN 202210496945A CN 114910650 A CN114910650 A CN 114910650A
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moesin
antibody
tag
igg antibody
kit
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叶青
毛建华
张俊峰
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Zhejiang University ZJU
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Abstract

The invention relates to an application of a reagent for detecting an anti-moesin-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-moesin-IgG antibody in preparing a kit for detecting vascular endothelial injury. The detection of the anti-moesin-IgG antibody can realize the detection of vascular endothelial injury.

Description

Application of reagent for detecting anti-moesin-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-moesin-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 vascular wall tissues, and can secrete a series of vasoactive substances such as NO, PGI2, ET-1 and the like through three ways of autocrine, endocrine and paracrine to play the functions of regulating the blood vessel tone, resisting thrombosis, inhibiting smooth muscle cell proliferation, inhibiting vascular wall inflammatory reaction and the like. NO is the most important vasodilator factor produced by endothelial cells, and is generated by the action of NO synthase (eNOS) of the endothelial cells on L-arginine, and the NO can diffuse to vascular wall smooth muscle cells to activate ornithine cyclase and mediate cGMP-regulated vasodilation. Moreover, NO also has the effects of inhibiting platelet aggregation, inhibiting monocyte adhesion to endothelial cells, and inhibiting smooth muscle cell proliferation. However, when the vascular endothelium is affected by a series of harmful factors, the release of the vasomotor factors by endothelial cells is reduced, the vasomotor factors are increased, the vascular equilibrium is broken, and finally a series of cardiovascular events are caused. The vascular endothelial cell autoantibody can cause vascular endothelial cell damage and induce dysfunction of blood circulation system, thereby causing damage to organs such as heart, lung, liver and the like and causing diseases related to each organ, including nephrotic syndrome.
Minimal Change Disease (MCD) is the main cause of nephrotic syndrome in children and accounts 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. The human endothelial cell line ECV 304 is used by the patients and incubated with INS patient serum to carry out endothelial cell toxicity tests, and the results show that the FSGS patient serum causes the most obvious endothelial cell damage. Therefore, they 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 self-antibody can cause severe damage to the glomerular vascular endothelial cells of the 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-moesin-IgG antibody in preparation of a kit for detecting vascular endothelial injury. The detection of the anti-moesin-IgG antibody can realize the effective detection of the vascular endothelial injury.
The invention provides an application of a reagent for detecting an anti-moesin-IgG antibody in preparing a kit for detecting vascular endothelial injury.
Preferably, the reagent for detecting anti-moesin-IgG antibody comprises moesin protein or a moesin recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the moesin protein is BC 017293.
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 moesin recombinant protein is shown in SEQ ID No. 1.
Preferably, the vascular endothelial injury comprises glomerular vascular endothelial cell injury.
The invention also provides a kit for detecting the anti-moesin-IgG antibody, which comprises: the reagent for detecting the anti-moesin-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-moesin-IgG antibody in preparing a kit for detecting vascular endothelial injury. The invention firstly detects an anti-moesin-IgG antibody in the body of part of patients with nephrotic syndrome, and determines that the target antigen aimed by the autoantibody is moesin on endothelial cells of glomerular vessels. The invention finds that the moesin antibody is an important glomerular vascular endothelial cell autoantibody, is closely related to the occurrence and development of MCD and FSGS nephrotic syndrome, and can guide clinical diagnosis and treatment. The detection of the anti-moesin-IgG antibody can realize the detection of vascular endothelial injury, and particularly provides a basis for researching the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment. The kit for detecting the anti-moesin-IgG antibody provided by the invention can qualitatively and quantitatively detect the anti-moesin-IgG antibody in serum of a nephrotic syndrome patient, and the kit provided by the invention utilizes the IgG antibody of human anti-tag peptide as a standard substance, and greatly improves the detection accuracy, sensitivity, specificity and detection speed by combining a biotin-avidin amplification system and magnetic particle chemiluminescence immunoassay. Specifically, compared with the prior art, the kit has the following benefits:
1. the kit can realize high-efficiency detection of vascular endothelial injury, and judges that the vascular endothelial injury exists when the anti-moesin-IgG antibody is detected.
2. At present, relevant moesin and anti-moesin-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 identifies the IgG autoantibody aiming at the moesin for the first time, invents a detection kit aiming at the moesin-IgG autoantibody and fills the blank at home and abroad. The kit provided by the invention is used for detecting moesin-IgG antibodies in the serum of 298 nephrotic syndrome patients, and the result shows that 94 patients have positive moesin-IgG antibodies, namely the positive detection rate of the moesin-IgG antibodies is 31.54%. The invention can provide a basis for researching the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment after detecting the moesin-IgG antibody.
3. The kit of the invention relates to a solid-phase membrane immunoassay qualitative analysis of an anti-moesin-IgG antibody in human serum, and the IgG antibody of human anti-tag peptide is used as a standard substance, so that the detection accuracy is greatly improved. The solid-phase membrane immunoassay qualitative detection is simple to operate, the reagent dosage is less, and the solid-phase membrane immunoassay qualitative detection is saved by about 10 times compared with the traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely close to 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane; the NC membrane with adsorbed antigen or antibody or existing result can be preserved for a long time (half a year at-20 ℃), and the activity of the NC membrane is not influenced; in addition, the kit for qualitatively detecting the anti-moesin-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-moesin-IgG antibody in human serum by magnetic particle chemiluminescence immunoassay utilizes magnetic particles as solid phase carriers, the diameter of the magnetic particles is only 1.0 mu m, so that the coating surface area is greatly increased, the adsorption quantity of antigens is increased, the reaction speed is improved, the cleaning and the separation are simpler and more convenient, the pollution is reduced, and the probability of cross infection is reduced. On the other hand, the acridine ester luminescent agent is adopted to directly mark the anti-human IgG, the chemical reaction is simple and quick, and no catalyst is needed; acridinium chemiluminescence is of the flash type, by initiating a luminescent reagent (H) 2 O 2 NaOH) can reach the maximum after 0.4s, the half-life period is 0.9s, the detection is basically finished within 2s, and the rapid detection is convenient.
Drawings
FIG. 1 is a graph showing the results of the present invention in which moesin on endothelial cells of glomerular vessels is the main target antigen for autoantibodies in patients with nephrotic syndrome; wherein, A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of 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 moesin;
figure 2 shows the optimization of Moesin expression conditions, where lane M: 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 cell lysate supernatant at 37 ℃ for 4 h; lane NC 2: precipitating inclusion bodies without induced cell lysate; 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 Moesin;
FIG. 4 is a solid-phase membrane immunoassay kit for detecting moesin-IgG antibodies in serum of patients with nephrotic syndrome;
FIG. 5 is a schematic diagram of the detection of anti-moesin-IgG antibody by the magnetic particle chemiluminescence immunoassay kit.
FIG. 6 is a schematic diagram of the antigen protein Moesin coated carboxyl magnetic particle;
FIG. 7 shows the detection of anti-moesin-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-moesin-IgG antibodies to markers of vascular endothelial injury.
Detailed Description
The invention provides an application of a reagent for detecting an anti-moesin-IgG antibody in preparing a kit for detecting vascular endothelial injury. Moesin (Moesin) is a member of ezrin-radixin-Moesin family proteins, has the function of connecting a plasma membrane with an actin-based cytoskeleton, regulates cell movement and a membrane protein internalization process, is critical to vascular endothelial function, and plays an important role in regulating cell adhesion, migration and morphogenesis. 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-moesin-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-moesin-IgG antibody provided by the invention takes the moesin as a target spot to detect the moesin autoantibody (namely, the anti-moesin-IgG antibody is a biomarker for detecting the damage of the vascular endothelial cells), and the reagent can realize high-efficiency detection of the vascular endothelial damage. In the present invention, the agent is capable of immunoreacting with moesin autoantibodies from tissues (kidney biopsy) or body fluids (in particular blood, plasma, serum). In the present invention, the reagent for detecting an anti-moesin-IgG antibody preferably comprises a moesin protein or a moesin recombinant protein or polypeptide containing a tag; the NCBI protein accession number of the moesin protein is BC 017293. 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 moesin recombinant protein containing the tag is preferably as shown in SEQ ID No. 1: MEAEKLAKERQEAEEAKEALLQASRDQKKTQEQLALEMAELTARISQLEMARQKKESEAVEWQQKAQMVQEDLEKTRAELKTAMSTPHVAEPAENEQDEQDENGAEASADLRADAMAKDHHHHHH 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-moesin-IgG antibody, which comprises: the reagent for detecting the anti-moesin-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-moesin-IgG antibody (moesin or recombinant protein containing tag) is preferably immobilized on a solid support. By "immobilized" as used herein is meant bound to a moesin antigen protein water-insoluble solid support, which solid support or support is water-insoluble, 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 moesin antigenic 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 a thiol-containing reagent), which is reversible. In addition, if the antigenic protein is immobilized to the support by a covalent bond that does not cleave in aqueous solution (bond formed by reaction of epoxide group with amine group coupling lysine side chain to affinity column), the immobilization is irreversible. Fixation may also be indirect: such as fixing an antibody having a specific affinity for the antigen protein, and then forming an antigen protein-antibody complex for the purpose of fixing. The antigen protein moesin fixing method of the invention is preferably a direct coating method: (1) the antigen protein moesin is bonded to a nitrocellulose membrane or a polystyrene microporous plate in a physical adsorption mode or a non-covalent bond; (2) the magnetic particle with carboxyl functional group is combined with the amino group of antigen protein moesin protein, and the antigen protein moesin protein is combined on the magnetic particle by means of chemical coupling. In the invention, the solid phase carrier preferably comprises a nitrocellulose membrane, a fluorescence encoding microsphere, a magnetic strip chip, a magnetic particle or an enzyme labeling micropore plate.
The invention preferably adopts a gene recombination prokaryotic expression method to successfully express and purify recombinant protein moesin, and uses the recombinant protein moesin as an antigen protein in a kit to develop a kit suitable for detecting the anti-moesin-IgG antibody of the glomerular vascular endothelial cell autoantibody of a nephrotic syndrome patient, and the kit comprises a detection kit for qualitatively or quantitatively analyzing and detecting the anti-moesin-IgG antibody in human serum.
In the present invention, the moesin protein is preferably expressed in bacterial (e.g., E.coli), yeast, insect or mammalian cells. After the moesin is obtained by expression, the moesin is preferably purified by using methods such as Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, hydrophobic column purification and the like.
In the present invention, the labeled antibody preferably includes an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody; the secondary antibody comprises an anti-human IgG antibody.
In the present invention, the enzyme-labeled secondary antibody preferably comprises an anti-human IgG antibody labeled with horseradish peroxidase; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.
In the present invention, the types of the kit preferably include a solid-phase membrane immunoassay kit or a magnetic particle chemiluminescence immunoassay kit; when the kit is a solid-phase membrane immunoassay kit, the kit preferably further comprises an antigen diluent, a sample diluent buffer, an antibody diluent, a substrate developing solution, a washing solution, an enzyme working solution, a standard substance, a positive quality control substance and a negative quality control substance; when the kit is a magnetic particle chemiluminescence immunoassay kit, the kit preferably further comprises chemiluminescence pre-excitation liquid A, chemiluminescence excitation liquid B, a standard substance and a cleaning solution. In the present invention, the standard substance and the positive quality control substance are preferably both recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-moesin-IgG antibodies 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-moesin-IgG antibody, is preferably recombinant protein moesin (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 moesin (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-moesin-IgG antibody with different concentrations; the cleaning solution is preferably a pH 7.2, 25mmol/L LTris-HCL solution containing 0.15mol/L NaCL and 0.05% Tween-20.
In the invention, the sample to be tested of the kit is preferably from whole blood, serum, plasma, urine, lymph fluid and hydrothorax and ascites; more preferably mammalian (human) serum.
In the present invention, the principle of the kit for detecting anti-moesin-IgG antibody in serum is preferably as follows: the indirect method reaction principle is utilized, firstly, moesin antigen is adsorbed to a solid phase carrier to be used as a coating antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled antibody (labeled secondary antibody) is added for reaction, then a ternary complex of the coating antigen moesin, the serum to be detected, the anti-moesin-IgG antibody and a labeled anti-human IgG antibody is formed if the serum to be detected contains the anti-moesin-IgG antibody, and finally, an optical signal is detected by utilizing a chromogenic method, a chemiluminescence method and a fluorescence method, so that the aim of qualitatively or quantitatively analyzing the anti-moesin-IgG antibody in human serum is fulfilled.
The application of the reagent for detecting anti-moesin-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, and the technical solution of the present invention includes, but is not limited to, the following examples.
Example 1 Moesin on vascular endothelial cells is the major target antigen for autoantibodies in nephrotic syndrome patients
According to the invention, through a large number of clinical and molecular mechanism researches at the early stage, the serum IgG level of a patient with nephrotic syndrome is found to be high for the first time, and Moesin on vascular endothelial cells is proved to be a main target antigen for the autoantibody in the patient with nephrotic syndrome. It would therefore be advantageous to detect the presence and quantitative levels of anti-Moesin-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: a vascular endothelial cell strain (EAhy926) was cultured, washed 2 to 3 times with PBS, then sufficiently lysed on ice in a lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and a protease inhibitor (# ab 65621; Abcam, 1: 200 dilution) with a focused ultrasound machine (Covaris S220, Gene), and then the sample was placed in a centrifuge at 12000g, 4 ℃ and centrifuged 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 visualization in step (2), protein spots were selected on two-dimensional electrophoresis gel which were strongly positive for nephrotic syndrome patients and negative or weakly positive for healthy persons, the selected protein spots were removed from the gel, the dried gel was digested with trypsin (0.1. mu.g/. mu.l), 10. mu.l of 25mM ammonium bicarbonate was added to the reaction mixture, incubated overnight at 37 ℃, and peptides were then extracted from the gel with trifluoroacetic acid (0.1%). 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 Moesin protein, see C in fig. 1.
Example 2 expression and purification of recombinant Moesin antigenic proteins
Performing PCR amplification by using a gene encoding Moesin 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: cell lysate induced for 16h at 15 ℃, cell lysate induced for 4h at 37 ℃, cell lysate supernatant induced for 16h at 15 ℃, cell lysate supernatant induced for 4h at 37 ℃, cell lysate precipitation inclusion bodies induced for 16h at 15 ℃ and cell lysate precipitation inclusion bodies induced for 4h at 37 ℃. The results showed that the protein expression in the cell lysate supernatant induced for 4h at 37 ℃ was 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 Moesin is identified by SDS-PAGE to be 14.27KDa, and the result is shown in figure 3.
Example 3
Optimization is carried out according to 4 factors such as antigen Moesin 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 ℃, 25 ℃), enzyme-labeled secondary antibody optimal dilution (1: 100, 1: 500, 1: 1000, 1: 1500) and the like, and each factor repeatedly measures standard positive serum and standard negative serum at 2 levels. 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 statistically processed to determine the optimal coating condition and the optimal dilution of the secondary antibody for optimization, thereby significantly improving the positive detection rate of the standard positive serum. Through tests, the kit is obtained, wherein the optimal antigen coating concentration is 300 mu g/mL, the optimal solid-phase membrane immune antigen-antibody reaction temperature is 25 ℃, the optimal antigen-antibody reaction time is 30min, and the optimal work dilution of an optimal biotin-labeled anti-human IgG antibody is 1: 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-Moesin-IgG antibody
4.1 composition of solid-phase membrane immunoassay kit for detecting anti-Moesin-IgG:
1. a nitrocellulose membrane coated with Moesin antigen protein;
2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro, hu);
3. negative quality control product: serum from healthy examiners;
4. a biotin-labeled anti-human IgG antibody;
5. diluting the antibody;
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, placing the cellulose nitrate membrane coated with Moesin antigen protein in a plate groove, adding 150. mu.l of 3% BSA, placing in a 37 ℃ incubator for blocking for 15min, sucking off blocking liquid, and washing the membrane for 2 times by using washing liquid.
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 mu l of alkaline phosphatase-streptavidin enzyme working solution, incubating at room temperature for 20min, discarding the liquid in the detection plate, washing with washing liquid for 5 times, adding 6 drops (300 mu l) of BCIP color developing solution into the reaction tank, and incubating on a shaking table at room temperature (20-25 ℃) 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. Judging and reading 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 using analysis software carried by the developing instrument to carry out semi-quantitative analysis on the anti-Moesin-IgG level in the serum 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.
EXAMPLE 5 preparation of chemiluminescent immunoassay kit for the detection of anti-Moesin-IgG antibodies
5.1 composition of the chemiluminescent immunoassay kit for the detection of anti-Moesin-IgG:
1. magnetic particle solution coated with Moesin 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 resist M in human serumThe whole process comprises two steps of reaction: firstly, mixing the magnetic bead solution with a diluted sample, binding a specific anti-Moesin-IgG antibody to the magnetic beads, and washing to remove residual solution. Secondly, adding acridinium ester labeled anti-human IgG antibody to form a magnetic bead-antigen-anti-Moesin-IgG antibody-acridinium ester antibody compound, washing to remove unbound residual liquid, and adding pre-excitation liquid (H) 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, separating with magnet, and discarding the supernatant;
b) adding 200 μ l10mg/mL sodium periodate into 200 μ l 2% dextran solution for reaction;
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 Moesin 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 PBS buffer solution with pH7.4 and containing 0.2% 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 PBS buffer pH7.4 containing 0.2% Tween20, 0.2% BSA, and the cross-linking of the activated magnetic beads with the antigen protein Moesin 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-Moesin-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 conditions of anti-Moesin-IgG antibodies in various nephrotic patients from 6 months in 2018 to 6 months in 2020 by using the kit disclosed by the invention, the anti-Moesin-IgG antibody levels in serum of patients diagnosed with various nephropathies include 298 nephrotic syndromes, 100 anaphylactoid purpura, 100 purpura nephritis, 100 Kawasaki disease and 100 healthy children in the same period, and the results show that anti-Moesin-IgG antibodies in part of patients with autoimmune nephrotic syndromes are positive (94 patients are positive for anti-Moesin-IgG antibodies, namely the positive detection rate of the anti-Moesin-IgG antibodies is 31.54%), while the anti-Moesin-IgG antibodies in purpura nephritis, anaphylactoid purpura, Kawasaki disease and healthy children are negative, as shown in FIG. 7.
6.3 serum anti-Moesin-IgG antibody of a patient with nephrotic syndrome is linearly related to the expression level of a vascular endothelial injury marker, the kit disclosed by the invention is used for detecting the expression level of the anti-Moesin-IgG antibody in the serum of the patient diagnosed with nephrotic syndrome 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-Moesin-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-Moesin-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 amendments can be made without departing from the principle of the present invention, and these modifications and amendments should also be considered as the protection scope of the present invention.
Sequence listing
<110> Zhejiang university
Application of reagent for detecting anti-moesin-IgG antibody in preparation of kit for detecting vascular endothelial injury
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 125
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Glu Ala Glu Lys Leu Ala Lys Glu Arg Gln Glu Ala Glu Glu Ala
1 5 10 15
Lys Glu Ala Leu Leu Gln Ala Ser Arg Asp Gln Lys Lys Thr Gln Glu
20 25 30
Gln Leu Ala Leu Glu Met Ala Glu Leu Thr Ala Arg Ile Ser Gln Leu
35 40 45
Glu Met Ala Arg Gln Lys Lys Glu Ser Glu Ala Val Glu Trp Gln Gln
50 55 60
Lys Ala Gln Met Val Gln Glu Asp Leu Glu Lys Thr Arg Ala Glu Leu
65 70 75 80
Lys Thr Ala Met Ser Thr Pro His Val Ala Glu Pro Ala Glu Asn Glu
85 90 95
Gln Asp Glu Gln Asp Glu Asn Gly Ala Glu Ala Ser Ala Asp Leu Arg
100 105 110
Ala Asp Ala Met Ala Lys Asp His His His His His His
115 120 125

Claims (9)

1. The application of the reagent for detecting the anti-moesin-IgG antibody in preparing the kit for detecting the vascular endothelial injury.
2. The use of claim 1, wherein the reagent for detecting anti-moesin-IgG antibody comprises a moesin protein or a tag-containing moesin recombinant protein or polypeptide; the NCBI protein accession number of the moesin protein is BC 017293.
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 moesin recombinant 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-moesin-IgG antibody, comprising: the reagent for detecting anti-moesin-IgG antibody for use according to any one of claims 1 to 5, a solid phase carrier, and a labeled antibody.
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.
CN202210496945.1A 2022-05-07 2022-05-07 Application of reagent for detecting anti-moesin-IgG antibody in preparation of kit for detecting vascular endothelial injury Pending CN114910650A (en)

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