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

Abstract

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

Description

Application of reagents for detecting anti-cytoskeleton-related protein 4-IgG autoantibodies in the preparation of kits for detecting vascular endothelial damage

technical field

The invention belongs to the technical field of biomedicine, and particularly relates to the application of Cytoskeleton-associated protein 4 in the preparation of a vascular endothelial cell damage detection kit.

Background technique

Blood, blood vessels and the heart make up the body's circulatory system. The blood in the circulatory system flows in the blood vessels and flows through the whole body organs such as the heart, lungs, and liver. Vascular endothelial cells are attached to the innermost layer of blood vessels. Vascular endothelial cells are a layer of mononuclear cells between blood flow and vascular wall tissue. They can secrete a series of NO and PGI2 through autocrine, endocrine and paracrine pathways. , ET-1 and other vasoactive substances play a role in regulating vascular tone, anti-thrombosis, inhibiting smooth muscle cell proliferation and vascular wall inflammation. NO is the most important vasodilatory factor produced by endothelial cells. It is produced by NO synthase (eNOs) of endothelial cells acting on L-arginine. NO can diffuse to smooth muscle cells of the vascular wall to activate ornithine cyclase and mediate cGMP. Regulated vasodilation. Not only that, NO also has the effects of inhibiting platelet aggregation, inhibiting the adhesion of monocytes to endothelial cells, and inhibiting the proliferation of smooth muscle cells. However, when the vascular endothelium is subjected to a series of harmful factors, the vasodilatory factors released by the endothelial cells decrease, and the vasoconstrictor factors increase, breaking the homeostasis of blood vessels, and finally leading to a series of cardiovascular events. Vascular endothelial cell autoantibodies can cause damage to vascular endothelial cells and induce dysfunction of the blood circulatory system, resulting in damage to organs such as the heart, lungs, and liver, resulting in various organ-related diseases, including nephrotic syndrome.

Endothelial cells are the monolayer of cells on the inside of blood vessels with high metabolic activity and play key roles in many physiological processes, including regulation of vasomotor tension, blood cell transport between blood and tissues, maintenance of blood fluidity, permeability, vascular Generative and innate and adaptive immunity, involved in the pathophysiological processes of most diseases, are major determinants or victims of pathophysiology. The kidney has the most abundant and diverse population of endothelial cells compared to the rest of the organ. This broad diversity includes the ability of renal endothelial cells to contribute to different transport capabilities across various parts of the kidney and the ability of different endothelial cells to withstand oxygen in the environment. Content and osmotic pressure are different. Therefore, vascular endothelial cell damage can lead to a variety of organ diseases, including nephrotic syndrome, and cause serious harm.

Nephrotic syndrome (NS) is a group of clinical syndromes characterized by massive proteinuria, hypoalbuminemia, high edema, and hyperlipidemia. It can be caused by a variety of causes and is divided into three categories: primary, secondary and hereditary. Primary nephrotic syndrome is a primary glomerular disease and consists of a variety of pathological types. Minimal change disease (MCD) is the main cause of nephrotic syndrome in children and one of autoimmune nephrotic syndrome, accounting for 10-15% of the pathogenic factors of nephrotic syndrome in adults. The glomeruli of patients with minimal change disease appear essentially normal on light microscopy, and the only histopathological abnormality seen on electron microscopy is diffuse fused loss of podocyte foot processes. Therefore, MCD is considered a primary podocyte disease. Corticosteroids are a common drug for the treatment of MCD, and proteinuria can be completely relieved after treatment with corticosteroids, and progressive renal failure caused by MCD is rare. However, MCD can lead to serious complications, and the complications associated with MCD disease observed in adults mainly include venous thrombosis and severe acute kidney injury requiring temporary dialysis. Furthermore, because MCD is characterized by a chronic, relapsing course, prolonged immunosuppressive therapy is often required to maintain remission of proteinuria. However, long-term immunosuppressive therapy increases the risk of serious infections and carries a long-term risk of malignancy.

Currently, the underlying pathogenesis of MCD in existing studies remains poorly understood. Since the pathogenesis of primary focal segmental glomerulosclerosis (FSGS) is very similar to that of MCD, many scholars believe that MCD and FSGS are phenotypes of the same disease at different stages. T cells were first suspected to be the source of circulating permeability factors based on the association between MCD and non-Hodgkin lymphoma, measles infection-induced remission, and prolonged remission after cyclophosphamide therapy. However, the therapeutic efficacy of rituximab and other specific B-cell depleting drugs in recent years has challenged the source of T cells. Notably, the direct effects of corticosteroids and rituximab on podocytes are also thought to be therapeutic.

Although the currently observed podocyte injury is the main classic feature of MCD, the disease mechanism may also involve glomerular endothelial cells. As early as 2000, Futrakul N et al reported that patients with idiopathic nephrotic syndrome (INS) were often accompanied by insufficient renal perfusion. It was speculated that the damage of glomerular endothelial cells may be the cause of insufficient renal perfusion in patients with INS. Purohit S et al. found that syndecan 1, a marker of endothelial cell injury, was elevated in the circulatory system of MCD patients, but it was unclear whether there was concomitant glomerular endothelial cell injury. Trachtman H et al. observed co-deposition of IgM with complement components in the kidney tissue of FSGS and MCD patients and demonstrated that IgM is an antibody against GEC and cardiolipin epitopes. In 2022, BauerC et al. found that endothelial cell markers in the serum of MCD patients increased, and renal histopathology confirmed that the expression of caveolin-1 in glomerular endothelial cells was significantly increased. Co-incubation significantly increased the expression of thrombomodulin, a marker of glomerular endothelial cell damage, thus proving the existence of glomerular endothelial cell damage in MCD patients.

Nonetheless, the current research does not know exactly what the causative factor that causes glomerular endothelial cell damage is. The applicant's team screened and identified a series of glomerular vascular endothelial cell autoantibodies in patients with MCD and FSGS nephrotic syndrome through previous studies. Animal experiments confirmed that these glomerular endothelial cell autoantibodies can cause severe damage to mouse glomerular vascular endothelial cells. In vitro cell culture experiments also showed that these autoantibodies could affect the morphology and function of vascular endothelial cells. Clinical studies have shown that these glomerular endothelial cell autoantibodies are related to the hypercoagulable state and poor prognosis of patients.

Anti-Cytoskeleton-associated protein-IgG antibody is one of the important glomerular endothelial cell autoantibodies, which is closely related to the occurrence and development of MCD and FSGS nephrotic syndrome, and can guide clinical diagnosis and treatment. However, the current research on Cytoskeleton-associated protein 4 and anti-Cytoskeleton-associated protein 4-IgG antibodies in patients with renal disease at home and abroad is limited to the research on the molecular mechanism level, and there is no quantitative detection of their levels in the serum of patients, and the market Lack of corresponding clinical test kits.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of an antibody that specifically binds to Cytoskeleton-associated protein 4 as a biomarker in the preparation of a reagent or a kit for detecting vascular endothelial cell damage, and the antibody that specifically binds to Cytoskeleton-associated protein 4 is Biomarkers can detect diseases related to vascular endothelial cell damage and increase the medical use of Cytoskeleton-associated protein 4.

The invention provides the application of a reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibody in preparing a kit for detecting vascular endothelial injury.

Preferably, the reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies includes Cytoskeleton-associated protein 4 protein, Cytoskeleton-associated protein 4 recombinant protein or polypeptide containing tags.

Preferably, the tags include His tag, thioredoxin, GST tag, maltose binding protein, SA tag of glutathione transferase, c-Myc tag, Flag tag or biotin tag.

Preferably, when the tag is a His tag, the amino acid sequence of the Cytoskeleton-associated protein 4 protein containing the tag includes that shown in SEQ ID NO.1.

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

The present invention also provides a kit for detecting anti-Cytoskeleton-associated protein 4-IgG antibodies, the kit comprising: a method for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies in the application described in any one of the above technical solutions Reagents, solid supports and labeled antibodies.

Preferably, the labeled antibody comprises an enzyme-labeled secondary antibody or a chemiluminescent agent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescently labeled secondary antibody;

Preferably, the secondary antibody includes an anti-human IgG antibody.

Preferably, the enzyme-labeled secondary antibody includes horseradish peroxidase-labeled anti-human IgG antibody; the chemiluminescent agent-labeled secondary antibody includes acridine ester-labeled anti-human IgG antibody or fluorescently labeled anti-human IgG antibody; The biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.

Preferably, the solid phase carrier comprises one or more of nitrocellulose membrane, fluorescently encoded microspheres, magnetic stripe chips, magnetic particles and enzyme-labeled microplates.

Beneficial effects:

The invention provides the application of an antibody specifically binding to Cytoskeleton-associated protein 4 as a biomarker in preparing a reagent or kit for detecting vascular endothelial cell damage, and the antibody specifically binding to Cytoskeleton-associated protein 4 is used as a biomarker The drug can detect diseases related to vascular endothelial cell damage. In addition, the present invention detected an anti-Cytoskeleton-associated protein 4-IgG autoantibody in some patients with nephrotic syndrome for the first time, and determined that the target antigen of the autoantibody was Cytoskeleton-associated protein on glomerular vascular endothelial cells. 4. The present invention finds that the Cytoskeleton-associated protein 4 protein antibody is an important glomerular vascular endothelial cell autoantibody, is closely related to the occurrence and development of MCD and FSGS nephrotic syndrome, and can guide clinical diagnosis and treatment. The detection of anti-Cytoskeleton-associated protein 4-IgG autoantibodies can realize the detection of vascular endothelial damage, which provides a basis for the study of the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment. The kit for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies provided by the present invention can both qualitatively and quantitatively detect anti-Cytoskeleton-associated protein 4-IgG antibodies in the serum of patients with nephrotic syndrome, and the kit of the present invention utilizes human anti-tag The IgG antibody of the peptide is used as a standard and combined with a biotin-avidin amplification system and a magnetic particle chemiluminescence immunoassay to greatly improve the detection accuracy, sensitivity, specificity and detection speed. Specifically, compared with the prior art, the advantages of the kit of the present invention are as follows:

1. The kit of the present invention can realize the efficient detection of vascular endothelial injury, and the detection of anti-Cytoskeleton-associated protein 4-IgG autoantibodies can determine the existence of vascular endothelial injury.

2. At present, Cytoskeleton-associated protein 4 and anti-Cytoskeleton-associated protein 4-IgG antibodies in patients with renal disease at home and abroad are only limited to the molecular mechanism research, and there is no quantitative detection of their serum levels in patients. The invention identifies the IgG autoantibody against Cytoskeleton-associated protein 4 for the first time, and invents a detection kit for the autoantibody, which fills the gap at home and abroad. The anti-Cytoskeleton-associated protein 4-IgG antibody in the serum of 298 patients with nephrotic syndrome was detected by the kit of the invention, and the results showed that 116 patients were positive for the anti-Cytoskeleton-associated protein 4-IgG antibody, that is, the positive detection rate was 38.93%. The detection of the anti-Cytoskeleton-associated protein 4-IgG antibody in the present invention can provide a basis for studying the molecular mechanism of nephrotic syndrome and clinical diagnosis and treatment.

3. The kit of the present invention involves the qualitative analysis of anti-Cytoskeleton-associated protein 4-IgG antibodies in human serum by solid-phase membrane immunoassay. The IgG antibody against human anti-tag peptide is used as a standard, which greatly improves the detection accuracy. The solid-phase membrane immunoqualitative detection is easy to operate, requires less reagents, and saves nearly 10 times compared to traditional ELISA; in addition, the adsorption capacity of the NC membrane is extremely strong, close to 100%, and trace antigens can be completely adsorbed and fixed on the NC membrane; The NC membrane with results can be stored for a long time (-20°C for half a year) without affecting its activity; in addition, the kit of the present invention for the immunoqualitative detection of anti-Cytoskeleton-associated protein 4-IgG antibodies in human serum by the solid phase membrane introduces biotin - Avidin amplification system, which greatly improves the detection sensitivity.

4. The magnetic particle chemiluminescence immunoassay kit for quantitative detection of anti-Cytoskeleton-associated protein 4-IgG antibodies in human serum involved in the present invention uses magnetic particles as a solid-phase carrier, and its diameter is only 1.0 μm, which greatly increases the package size. The surface area increases the adsorption capacity of the antigen, improves the reaction speed, and also makes the cleaning and separation easier, thereby reducing pollution and reducing the probability of cross infection. On the other hand, using acridinium ester luminescent agent to directly label anti-human IgG, the chemical reaction is simple, fast, and does not require a catalyst; acridinium ester chemiluminescence is a flash type, which is activated by the luminescent agent (H ₂ O ₂ , NaOH) for 0.4 s After the emission intensity can reach the maximum, the half-life is 0.9s, and it basically ends within 2s, which is convenient for rapid detection.

Description of drawings

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

Figure 1-1 shows that the Cytoskeleton-associated protein 4 protein on glomerular endothelial cells is the main target antigen for autoantibodies in patients with nephrotic syndrome. A shows that the primary antibody is a two-dimensional electrophoresis protein spot in healthy human serum, B Two-dimensional electrophoresis protein spots showing that the primary antibody is the serum of patients with nephrotic syndrome;

Figure 1-2 shows the mass spectrometry identification of Cytoskeleton-associated protein 4 protein on glomerular endothelial cells;

Figure 2 shows the SDS-PAGE identification of the expressed recombinant protein Cytoskeleton-associated protein 4. Lane A is the supernatant of cell lysates induced at 15°C for 16 hours, and lane B is the supernatant of cell lysates. Induction at 37°C for 4 hours;

Figure 3 shows the detection of anti-Cytoskeleton-associated protein 4 1-IgG antibody in serum of patients with nephrotic syndrome by solid-phase membrane immunoassay kit;

Figure 4 is a schematic diagram of the principle of the magnetic particle chemiluminescence immunoassay kit for detecting anti-Cytoskeleton-associated protein 4-IgG antibodies;

Figure 5 is a schematic diagram of the antigenic protein Cytoskeleton-associated protein 4 coated with carboxyl magnetic particles;

Figure 6 shows the detection of anti-Cytoskeleton-associated protein 4-IgG antibodies in patients with various types of nephropathy, in which NS: nephrotic syndrome, HSP: allergic purpura, HSPN: purpura nephritis, KD: Kawasaki disease, NC: healthy children;

Figure 7 is a linear correlation diagram of anti-Cytoskeleton-associated protein 4-IgG antibody and vascular endothelial injury markers.

Detailed ways

The invention provides the application of a reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibody in preparing a kit for detecting vascular endothelial injury. The reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibody according to the present invention preferably includes Cytoskeleton-associated protein 4 protein, Cytoskeleton-associated protein 4 recombinant protein or polypeptide containing tags. The accession number of Cytoskeleton-associated protein 4 in the present invention in NCBI is BC082972. The vascular endothelial cell injury in the present invention preferably includes nephrotic syndrome, more preferably includes autoimmune nephrotic syndrome. Blood, blood vessels and heart make up the blood circulatory system of the human body. Vascular endothelial cells are attached to the innermost layer of blood vessels, and vascular endothelial cell autoantibodies can cause damage to vascular endothelial cells, induce dysfunction of the blood circulatory system, and then lead to damage to organs such as the heart, lungs, and liver, and cause nephrotic syndrome. Various organ-related diseases, and the vascular endothelial cells of different organs are the same. Therefore, based on the detection of vascular endothelial cell autoantibodies in the blood circulation system, it can be used for clinical vascular endothelial cell injury. The invention can realize the detection of vascular endothelial cell damage by using the antibody that specifically binds to Cytoskeleton-associated protein 4 as a biomarker.

The reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies of the present invention uses Cytoskeleton-associated protein 4 protein as a target to detect Cytoskeleton-associated protein 4 autoantibodies (that is, anti-Cytoskeleton-associated protein 4-IgG autoantibodies are used to detect blood vessels). Endothelial cell damage biomarkers), the reagent can achieve efficient detection of vascular endothelial damage. In the present invention, the agent is capable of immunoreacting with Cytoskeleton-associated protein 4 protein autoantibodies from tissue (kidney biopsy) or body fluids (especially blood, plasma, serum). In the present invention, the reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies preferably includes Cytoskeleton-associated protein 4 protein or Cytoskeleton-associated protein 4 recombinant protein or polypeptide containing a tag; the Cytoskeleton-associated protein 4 protein The NCBI protein accession number is BC082972. In the present invention, the tags are preferably tags with certain biological or physical functions, especially N-terminal or C-terminal; the presence of these tags is beneficial to the purification, immobilization and precipitation of antigenic proteins; the tags are more preferably is a sequence or domain capable of specifically binding 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.在本发明中，当所述标签为His标签时，所述含标签的 Cytoskeleton-associated protein 4重组蛋白的氨基酸序列优选如SEQ ID NO.1所示： MIFTEVQKRSQKEINDMKAKVASLEESEGNKQDLKALKEAVKEIQTSAKSRE WDMEALRSTLQTMESDIYTEVRELVSLKQEQQAFKEAADTERLALQALTEKL LRSEESVSRLPEEIRRLEEELRQLKSDSHGPKEDGGFRHSEAFEALQQKSQGL DSRLQHVEDGVLSMQVASARQTESLESLLSKSQEHEQRLAALQGRLEGLGSS EADQDGLASTVRSLGETQLVLYGDVEELKRSVGELPSTVESLQKVQEQVHTL LSQDQAQAARLPPQDFLDRLSSLDNLKASVSQVEADLKMLRTAVDSLVAYSV KIETNENNLESAKGLLDDLRNDLDRLFVKVEKIHEKVHHHHHH。 The vascular endothelial cell injury in the present invention preferably includes nephrotic syndrome, more preferably includes autoimmune nephrotic syndrome. The present invention uses the Cytoskeleton-associated protein 4 as a detection target, which can detect vascular endothelial cell damage, especially nephrotic syndrome, etc., and then use it for the preparation of a vascular endothelial cell damage related detection kit, which increases the cytoskeleton. Medicinal uses of related protein 4.

The present invention also provides a kit for detecting anti-Cytoskeleton-associated protein 4-IgG antibodies, the kit includes the reagents for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies in the application described in the above technical solution, a solid phase carrier and labeled antibody.

In the present invention, the reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies (Cytoskeleton-associated protein 4 protein or Cytoskeleton-associated protein 4 recombinant protein containing tags) is preferably immobilized on a solid phase carrier. The term "fixation" in the present invention refers to binding to a water-insoluble solid-phase carrier of Cytoskeleton-associated protein 4 antigen protein, and the solid-phase carrier or support is water-insoluble, more preferably by covalent bonding, electrostatic interaction, Hydrophobic interactions, or interactions via disulfide bonds, most preferably via one or more covalent bonds. Immobilization can be by direct immobilization, for example by filtration, centrifugation or chromatography, to separate the immobilized molecules from the aqueous solution together with the insoluble support. Also included are reversible or irreversible immobilization of Cytoskeleton-associated protein 4 antigen proteins. For example, the antigenic protein is immobilized to the support via cleavable covalent bonds (eg, disulfide bonds that can be cleaved by the addition of thiol-containing reagents), and this immobilization is reversible. Alternatively, if the antigenic protein is immobilized to the support via a covalent bond that does not cleave in aqueous solution (a bond formed by the reaction of an epoxide group with an amine group that couples the lysine side chain to the affinity column), The fixation is irreversible. The immobilization can also be an indirect way: such as immobilizing an antibody with specific affinity to the antigenic protein, and then forming an antigenic protein-antibody complex to achieve the effect of immobilization.

The antigen protein Cytoskeleton-associated protein 4 immobilization method of the present invention is preferably a direct coating method: (1) The antigen protein Cytoskeleton-associated protein 4 is bound to a nitrocellulose membrane or polystyrene by physical adsorption or non-covalent bond (2) Magnetic particles with carboxyl functional groups bind to the amino group of the antigenic protein Cytoskeleton-associated protein 4, and the antigenic protein Cytoskeleton-associated protein 4 is bound to the magnetic particles by chemical coupling. In the present invention, the solid phase carrier includes one or more of nitrocellulose membrane, fluorescently encoded microspheres, magnetic strip chips, magnetic particles and enzyme-labeled microplates.

The present invention preferably adopts the gene recombinant prokaryotic expression method to successfully express and purify the recombinant protein Cytoskeleton-associated protein 4, which is used as the antigen protein in the kit to develop a set of autoantibodies suitable for detecting glomerular vascular endothelial cells in patients with nephrotic syndrome. The kit for anti-Cytoskeleton-associated protein 4-IgG antibody includes a detection kit for qualitative or quantitative analysis and detection of anti-Cytoskeleton-associated protein 4-IgG antibody in human serum.

In the present invention, the Cytoskeleton-associated protein 4 protein is preferably expressed in bacteria (eg E. coli), yeast, insect or mammalian cells. After the Cytoskeleton-associated protein 4 protein is obtained by expression, the present invention preferably uses Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography, hydrophobic column purification and other methods to purify the Cytoskeleton-associated protein 4 protein.

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

In the present invention, the enzyme-labeled secondary antibody preferably includes horseradish peroxidase-labeled anti-human IgG antibody; the chemiluminescent agent-labeled secondary antibody includes acridinium ester-labeled anti-human IgG antibody or fluorescent-labeled anti-human IgG antibody 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 particle chemiluminescence immunoassay kit; when the kit is a solid-phase membrane immunoassay kit, the kit preferably further includes Antigen dilution solution, sample dilution buffer, antibody dilution solution, substrate chromogenic solution, washing solution, enzyme working solution, standard substance, positive quality control substance and negative quality control substance; when the kit is magnetic particle chemiluminescence immunoassay When analyzing the kit, the kit preferably further includes a chemiluminescence pre-excitation solution A, a chemiluminescence excitation solution B, a standard and a cleaning solution. In the present invention, the standard product and the positive quality control product are preferably recombinant human anti-tag peptide immunoglobulin G or a fragment thereof, or anti-Talin-1-IgG autoantibody extracted from patient serum; the negative quality control product is preferably Serum for healthy people.

Specifically, when the kit is a solid-phase membrane immunization kit, in the kit, the reagent for detecting anti-Cytoskeleton-associated protein 4-IgG autoantibodies, that is, the antigen, is preferably a recombinant protein Cytoskeleton-associated protein 4 (The amino acid sequence includes SEQ ID NO.1); the solid phase carrier is preferably Satourius CN140 nitrocellulose membrane; the positive quality control substance (standard product) is preferably human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang); The negative quality control substance is preferably the serum of healthy people; the labeled antibody is preferably a biotin-labeled anti-human IgG antibody; the enzyme working solution is preferably alkaline phosphatase-streptavidin; the substrate color reagent is preferably TMB, Hydrogen peroxide, AMPPD, 4-MUP or BCIP; the antigen dilution is preferably 1×PBS pH7.4 containing 163mM NaCl, 1% TritonX-100; the sample dilution buffer is preferably 0.01M PBS containing 10% BSA pH7.4; the antibody diluent is preferably 0.01M PBS pH7.4 containing 1M D-glucose, 2% glycerol, 0.35% Tween20; the washing solution is preferably: 163mM NaCl, 10% glycerol, 1% TritonX- 100 in 1x PBS pH7.4.

When the kit is a magnetic particle chemiluminescence immunoassay kit, in the kit, the antigen is preferably a recombinant protein Cytoskeleton-associated protein 4 (the amino acid sequence includes SEQ ID NO. 1); the solid phase carrier is preferably a Carboxyl magnetic beads; the labeled antibody is preferably acridinium ester-labeled anti-human IgG antibody; the chemiluminescence pre-excitation solution A and the chemiluminescence excitation solution B are preferably conventional commercially available products, and the standard is preferably anti-Cytoskeleton-associated protein 4- IgG autoantibody; the washing solution is preferably a pH 7.2, 25 mmol/L Tris-HCL solution containing 0.15 mol/L NaCl and 0.05% Tween-20.

In the present invention, the samples to be tested in the kit are preferably from whole blood, serum, plasma, urine, lymph, pleural and ascites; more preferably, mammalian (human) serum.

In the present invention, the principle of the kit for detecting anti-Cytoskeleton-associated protein 4-IgG antibodies in serum is preferably as follows: using the indirect method reaction principle, firstly, the Cytoskeleton-associated protein 4 antigen is adsorbed on a solid phase carrier as a coating antigen, and then added The positive quality control substance or standard substance or the serum sample to be tested is incubated, and then the labeled antibody (labeled secondary antibody) is added to react. If the serum to be tested contains anti-Cytoskeleton-associated protein 4-IgG antibody, the coated antigen Cytoskeleton-associated protein is formed. protein 4-anti-Cytoskeleton-associated protein 4-IgG antibody of the serum to be tested-labeled anti-human IgG antibody ternary complex, and finally use light color method, chemiluminescence method, fluorescence luminescence method to detect the light signal to achieve qualitative or quantitative The purpose of analyzing anti-Cytoskeleton-associated protein 4-IgG antibodies in human serum.

The application of the reagent for detecting anti-Cytoskeleton-associated protein4-IgG autoantibodies of the present invention in the preparation of a kit for detecting vascular endothelial injury will be further introduced in detail below with reference to specific examples. The technical solutions of the present invention include but are not limited to the following Example.

Example 1

Cytoskeleton-associated protein 4 on vascular endothelial cells is the main target antigen for autoantibodies in patients with nephrotic syndrome:

Through a large number of clinical and molecular mechanism studies in the early stage, the present invention finds for the first time that the serum IgG level of patients with nephrotic syndrome is higher, and confirms that Cytoskeleton-associated protein 4 on vascular endothelial cells is the main target of autoantibodies in patients with autoimmune nephrotic syndrome. antigen. Therefore, detecting the presence and quantitative level of anti-Cytoskeleton-associated protein 4-IgG antibody in serum is helpful to clarify vascular endothelial cell injury.

The specific implementation is as follows:

(1) Extraction of total protein from vascular endothelial cells: culture vascular endothelial cell line (ECV 304), wash 2-3 times with PBS, and then use a focused ultrasound instrument (Covaris S220, Gene) in a solution containing 30mm Tris-HCl, 8m urea, Fully lysed on ice in a lysis buffer of 4% CHAPS and protease inhibitors (#ab65621; Abcam, diluted 1:200), the samples were then centrifuged at 12000g, 4°C for 30min. The supernatant was collected, which was the total protein of vascular endothelial cells collected. The total protein concentration of the collected vascular endothelial cells was determined using the BCA protein concentration assay kit.

(2) Two-dimensional electrophoresis: The total protein of vascular endothelial cells was extracted for two-dimensional electrophoresis, and then transferred to nitrocellulose membrane, incubated with serum from healthy people and patients with autoimmune nephrotic syndrome as primary antibodies, and then added with two Antibody was developed, see panels A and B in Figure 1.

(3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis: After developing step (2), carry out differential analysis of positive spots, and select proteins with strong positive nephrotic syndrome on two-dimensional electrophoresis gel, while negative or weakly positive proteins in healthy people are selected. The selected protein spots were removed from the gel, the dried gel was digested with trypsin (0.1 μg/μL), and 10 μL of 25 mM ammonium bicarbonate was added to the reaction mixture, incubated at 37°C overnight, and then Peptides were 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 peptide mass spectra, which were identified as Cytoskeleton-associated protein 4 protein, as shown in panel C in Figure 1.

Example 2

Expression and purification of recombinant antigen protein Cytoskeleton-associated protein 4

The gene encoding the Cytoskeleton-associated protein 4 protein was used as a template for PCR amplification by genetic engineering, and then an expression vector was constructed for protein expression. The antigenic protein expressed in the present invention contains a His-tagged tag peptide. The expressed recombinant protein was purified by nickel column affinity chromatography. Finally, the molecular weight of the recombinant protein Cytoskeleton-associated protein 4 was identified by SDS-PAGE as 40KDa, as shown in Figure 2 (the SDS-PAGE identification of the expressed recombinant protein Talin-1) , where lane A: supernatant of cell lysate, induced at 15°C for 16 hours; lane B: supernatant of cell lysate, induced at 37°C for 4 hours.

Example 3

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

According to the coating concentration of antigen Cytoskeleton-associated protein 4 (50μg, 80μg, 100μg, 150μg four coating concentrations), each reaction time (15min, 30min, 45min) and temperature (25℃, 37℃), the enzyme-labeled secondary antibody is the most suitable. Four factors including the optimal dilution (1:100, 1:500, 1:1000, 1:1500) and other four factors were selected in an orthogonal table, and each factor was repeated at 2 levels to determine the standard positive serum and standard negative serum. The ratio (P/N) of the highest light signal value (P) of positive serum to the lowest light signal value (N) of negative serum. The best antigen-Cytoskeleton-associated protein 4 coating concentration of this kit is 250ug/mL, and the optimal antigen-antibody reaction temperature of the solid-phase membrane immunodetection anti-Cytoskeleton-associated protein 4-IgG antibody kit is obtained by orthogonal design. 25℃, the best antigen-antibody reaction time is 30min, and the best working dilution of biotin-labeled anti-human IgG antibody is 1:500; the best anti-Cytoskeleton-associated protein 4-IgG antibody kit is detected by magnetic particle chemiluminescence immunoassay The antigen-antibody reaction temperature was 37℃, the optimal antigen-antibody reaction time was 15min, and the optimal working dilution of acridinium ester-labeled anti-human IgG antibody was 1:500.

Example 4

Preparation of solid-phase membrane immunoassay kit for detection of anti-Cytoskeleton-associated protein 4-IgG antibodies:

Antigen: recombinant protein Cytoskeleton-associated protein 4

Solid support: Satourius CN140 nitrocellulose membrane

Positive quality control substance (standard substance): human anti-His tag immunoglobulin G (purchased from Huzhou Yingchuang)

Negative control substance: serum of healthy people

Labeled antibody: Biotin-labeled anti-human IgG antibody

Antigen Diluent

Sample Dilution Buffer

Antibody Diluent

detergent

Enzyme working solution: alkaline phosphatase-streptavidin

Substrate chromogenic solution: BCIP chromogenic solution.

4.2 The detection steps of the solid-phase membrane immunoassay kit for the detection of anti-Cytoskeleton-associated protein 4-IgG antibodies are as follows:

4.2.1 Coating and blocking: Spot 8 μL of Cytoskeleton-associated protein 4 antigen with a concentration of 250 μg/mL directly on the nitrocellulose membrane and place it in a 37°C incubator to dry for 30 minutes, and place the nitrocellulose membrane on the detection plate. Add 200 μL of 5% BSA to block in a 37°C incubator for 30 min, discard the blocking solution and wash twice with washing solution;

4.2.2 Antigen incubation: Add 10 μL of antibody standard or serum to be tested diluted with diluent to the detection plate, and at the same time as negative control and positive control, incubate at 25°C for 30 minutes, and set 3 parallel wells for each sample;

4.2.3 Secondary antibody incubation: discard the liquid in the detection plate, wash 5 times with washing solution for 1 min, add 20 μL of 1:500 biotin-labeled anti-human IgG antibody, and incubate at 25°C for 30 min;

4.2.4 Color development: discard the liquid in the detection plate, wash 5 times with washing solution for 1 min, add 500 μL of alkaline phosphatase-streptavidin, incubate at room temperature for 20 min, discard the liquid in the detection plate, wash 5 times with washing solution ×1min, then add BCIP chromogenic solution, react at room temperature for 20min, rinse the detection plate with running water, and stop the enzyme reaction. Take out the test nitrocellulose membrane strip and dry the membrane strip with a hair dryer, and qualitatively judge with the naked eye with a colorimetric card. Those with obvious brown spots are positive as shown in Figure 3 (solid-phase membrane immunoassay kit to detect anti-Cytoskeleton-associated serum in patients with nephrotic syndrome). protein 4-IgG antibody result picture), or place the membrane strip on the developing instrument to scan, the analysis software that comes with the developing instrument takes the reference standard concentration as the ordinate and the gray value read by the instrument as the abscissa to draw the standard curve Semi-quantitative analysis of anti-Cytoskeleton-associated protein 4-IgG antibody levels in serum.

Example 5

Preparation of magnetic particle chemiluminescence immunoassay kit for detection of anti-Cytoskeleton-associated protein 4-IgG antibody

5.1 Anti-Cytoskeleton-associated protein 4-IgG antibody chemiluminescence detection kit, including the following components:

(1) acridinium ester-labeled anti-human IgG;

(2) Carboxyl magnetic beads coupled with Talin-1 antigen;

(3) chemiluminescence pre-excitation solution A (H ₂ O ₂ ) and chemiluminescence excitation solution B (NaOH);

(4) Anti-Talin-1-IgG antibody series standard solution, standard concentration: 0μg/mL, 2μg/mL, 4μg/mL, 8μg/mL, 16μg/mL, 20.0μg/mL, the buffer is 0.5mol/L Tris-HCl 5.0% BSA and 0.1~0.5% PC300;

(5) Cleaning solutions, especially pH 7.2, 25 mmol/LTris-HCl solutions containing 0.15 mol/L NaCl and 0.05% Tween-20.

5.2 Preparation of magnetic bead-coupled antigen

(1) Take 1 mg of carboxyl magnetic particles into a 0.5 mL centrifuge tube, add 200 μL of 0.1 mol/L MES buffer, vortex and mix, place on a magnetic stand, and let stand for 5 minutes to remove the magnetic particles from the liquid and discard the clear liquid. Wash 3 times, then add 200 μL of MES (pH 5.0) buffer and vortex;

(2) Add 18μL (18μg) Cytoskeleton-associated protein 4 antigen, vortex, rotate the reaction tube, and incubate at room temperature for 30min;

(3) Add 10 μL of 10 mg/mL coupling reagent EDC, vortex, rotate the reaction tube, and incubate at room temperature for 2 h;

(4) Remove the supernatant, add 200 μL of washing buffer (TBS+0.05% Tween-20) and wash for 3 times;

(5) Blocking with 1% BSA-containing buffer was repeated 4 times for 10 min each time. Magnetic particle suspensions were stored at 2-8°C.

5.3 Preparation of acridinium ester-labeled antibodies

(1) Put 100 μL of anti-human IgG antibody into the dialysis bag, put the dialysis bag into not less than 1L of labeled buffer for dialysis, change the buffer at least 3 times during the period, the last dialysis overnight, the labeled buffer is Na ₂ CO ₃ -NaHCO ₃ buffer, pH 10.1, concentration 0.1 mol/L;

(2) Weigh 1.7 mg of acridine ester NSP-DMAE-NHS and dissolve it in 447 μL of anhydrous dimethylformamide DMF to form a 6.5 mmol/L NSP-DMAE-NHS DMF solution;

(3) Put the dialyzed antibody solution in a 500 μL centrifuge tube, add 100 μL of 6.5 mmol/L NSP-DMAE-NHS DMF solution, the molar ratio of acridine ester to antibody is 7.4:1, add 200 μL of labeling buffer, React at room temperature for 45 minutes, add 10 μL of lysine and 10 μL, and continue to react for 15 minutes to terminate the labeling reaction;

(4) The marker NSP-DMAE-NHS-Ab was separated from the free NSP-DMAE-NHS by a Sephadex G-50 column (1×25cm), and the pH of the purification buffer was 6.3 and the concentration was 0.1mol/L;

(5) During the separation process, detect the protein peak with a chromatograph, and measure the chemiluminescence intensity of the effluent and the absorbance at 430 nm respectively;

(6) Collect the eluate with high luminosity and high absorbance, add 1% BSA (volume), and store on ice.

5.4 Sample preparation: Dilute the sample at a ratio of 1:10

5.5 The detection steps of the chemiluminescence kit for the detection of anti-Talin-1-IgG antibodies are as follows:

(1) 100 μL of the sample to be tested, 150 μL of coupled magnetic powder suspension, and 150 μL of acridine ester-labeled secondary antibody were added to the reaction tube in turn, shaken and mixed, and incubated at 37°C for 15 minutes;

(2) 5 times of isolation washing;

(3) Shake the washed reaction vessel sufficiently to disperse the magnetic particles uniformly;

(4) 100 μL of chemiluminescence pre-excitation solution A was added, followed by 100 μL of chemiluminescence excitation solution B, and the relative luminescence intensity was measured. The content of anti-Cytoskeleton-associated protein 4-IgG antibody in the sample is proportional to its luminescence intensity.

Example 6

Clinical application of the kit for detecting serum anti-Cytoskeleton-associated protein 4-IgG antibody

6.1 Subjects included: Patients diagnosed with various types of nephropathy from June 2018 to June 2020, including 298 cases of nephrotic syndrome (NS), 100 cases of allergic purpura (HSP), 100 cases of purpura nephritis (HSPN) ), 100 cases of Kawasaki disease (KD), and 100 healthy children (NC) during the same period. Serum samples were obtained from various renal disease patients and healthy controls. All subjects had their first serum sample collection before immunosuppressive therapy.

6.2 Detection of anti-Cytoskeleton-associated protein 4-IgG antibodies in patients with various types of nephropathy The kit of the present invention was used to detect anti-Cytoskeleton-associated protein 4 in the serum of patients diagnosed with various types of nephropathy from June 2018 to June 2020 -IgG antibody levels, including 298 cases of nephrotic syndrome, 100 cases of allergic purpura, 100 cases of purpura nephritis, 100 cases of Kawasaki disease and 100 healthy children in the same period, the results showed that some patients with nephrotic syndrome in anti-Cytoskeleton-associated protein 4 -IgG antibody positive (116 patients were anti-Cytoskeleton-associated protein 4-IgG antibody positive, that is, anti-Talin-1-IgG antibody positive detection rate was 38.93%), while purpura nephritis, allergic purpura, Kawasaki disease and healthy Anti-Talin-1-IgG antibody in children was negative, see Figure 6 (the detection situation of anti-Cytoskeleton-associated protein 4-IgG antibody in patients with various types of nephropathy, in which NS: nephrotic syndrome, HP: allergic purpura, HPN: Purpuric nephritis, KD: Kawasaki disease, NC: healthy children). Detection of the presence of anti-Cytoskeleton-associated protein 4-IgG antibodies in serum is helpful for the determination of vascular endothelial damage in nephrotic syndrome. .

6.3 Serum anti-Cytoskeleton-associated protein 4-IgG antibody in patients with nephrotic syndrome was linearly correlated with the expression of vascular endothelial injury markers

The kit of the present invention was used to detect the expression of anti-Cytoskeleton-associated protein 4-IgG antibody in the serum of patients diagnosed with nephrotic syndrome from June 2018 to June 2020, and to detect the expression of the vascular endothelial injury marker Plvap in the serum of the patients The results showed that the expression of anti-Cytoskeleton-associated protein 4-IgG antibody in patients with nephrotic syndrome was linearly correlated with the expression of vascular endothelial injury markers, and nephrotic syndrome was related to vascular endothelial injury. The detection can be used to determine vascular endothelial injury, that is, if anti-Cytoskeleton-associated protein 4-IgG antibody is detected, it is determined that there is vascular endothelial injury, as shown in Figure 7 (anti-Cytoskeleton-associated protein 4-IgG antibody is linearly correlated with vascular endothelial injury markers). result graph).

It can be concluded from the above examples that the anti-Cytoskeleton-associated protein 4-IgG of the present invention can be used as the detection target of vascular endothelial injury; and the kit prepared by using it as the detection target can be used to detect the anti-cytoskeleton-associated protein. 4-IgG antibody, with high sensitivity and accuracy, safe and fast.

Although the above-mentioned embodiment has made a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments according to the present embodiment without creativity. These embodiments All belong to the protection scope of the present invention.

sequence listing

<110> Zhejiang University

<120> Application of reagents for detecting anti-cytoskeleton-related protein 4-IgG autoantibodies in the preparation of kits for detecting vascular endothelial injury

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 356

<212> PRT

<213> Artificial Sequence

<400> 1

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

His His His His

355

Claims (10)

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

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

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

4. The use according to claim 3, wherein the amino acid sequence of the tagged Cytoskeleton-associated protein4 protein comprises SEQ ID No.1 when the tag is a His-tag.

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

6. A kit for detecting an anti-Cytoskeleton-associated protein4-IgG antibody, comprising: a reagent for detecting autoantibodies against Cytoskeleton-associated protein4-IgG, a solid phase carrier and a labeled antibody for use according to any one of claims 1 to 5.

7. The kit of claim 6, wherein the labeled antibody comprises an enzyme-labeled secondary antibody or a chemiluminescent-labeled secondary antibody or a biotin-labeled secondary antibody or a fluorescent-labeled secondary antibody.

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

9. The kit of claim 7 or 8, wherein the enzyme-labeled secondary antibody comprises a horseradish peroxidase-labeled anti-human IgG antibody; the secondary antibody marked by the chemiluminescence agent comprises an acridinium ester marked anti-human IgG antibody or a fluorescence marked anti-human IgG antibody; the biotin-labeled secondary antibody includes a biotin-labeled anti-human IgG antibody.

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

Images