CN115060909A

CN115060909A - Detection kit for detecting anti-aconitate hydratase-IgG antibody

Info

Publication number: CN115060909A
Application number: CN202210508484.5A
Authority: CN
Inventors: 叶青; 毛建华; 张俊峰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2022-09-16
Also published as: CN113447657B; CN113447657A

Abstract

The invention provides a detection kit for detecting an anti-aconitate hydratase-IgG antibody, which consists of aconitatehydratase antigen protein, labeled antibody liquid, a solid phase carrier coated with an aconitate hydratase antigen, sample diluent, antibody and antigen diluent, substrate developing solution, washing solution, stop solution, a standard substance and positive and negative quality control products. The invention contacts the detected serum sample with the target antigen aconitatehydratase polypeptide or binding fragment thereof to generate immunoreaction to form an antigen-antibody complex, and the formed antigen-antibody complex is detected by the anti-aconitate hydratase-IgG autoantibody. The invention identifies the anti-aconitate hydratase-IgG antibody for the first time, and provides a better detection method for identifying autoimmune nephrotic syndrome related to the anti-aconitate hydratase-IgG antibody at home and abroad.

Description

Detection kit for detecting anti-aconitate hydratase-IgG antibody

The application is a divisional application of a detection kit for detecting the anti-aconitate hydratase-IgG antibody, which has the application number of 202110742938.0 and the application date of 2021, 7 and 1.

Technical Field

The invention belongs to the technical field of biomedicine, and relates to a detection kit for detecting an anti-aconitate hydratase-IgG antibody.

Background

Primary renal syndrome (PNS) is a clinical syndrome in which there are many factors that cause increased permeability of glomerular basement membrane and increased plasma protein leaching, loss from the urine, and thus a series of pathological changes. PNS is a common glomerular disease of children, and the annual incidence rate is reported to be 1.15-16.9/10 and 0000. The pathological type, which is characterized by minimal change renal syndrome (MCNS), is the most common, and is classified into hormone sensitive type (SSNS), hormone dependent type (SDNS), and hormone resistant type (SRNS) according to the response effect of PNS to hormone therapy. Although most of the children patients are sensitive to the hormone treatment and have good prognosis, 10% -20% of the children patients still have the dependence or drug resistance to the hormone treatment, and if other drug intervention treatment is not carried out, the children patients can progress to glomerulosclerosis and finally end-stage nephropathy, the health of the children patients is seriously affected, and the families and the society cannot bear heavy economic burden. And the pathogenesis of the disease is not yet clear. The clinical symptoms of PNS are usually manifested as edema, profuse proteinuria, hypoproteinemia, hyperlipidemia, etc., but the causes of these clinical manifestations are very complex, and it is difficult to clearly diagnose PNS. Therefore, if the exact cause of PNS can be found, the PNS can be diagnosed as soon as possible in the cause, so as to intervene and treat as soon as possible, which has important clinical significance.

At present, the disease of most of PNS patients clinically treated by hormones and immunosuppressants can be improved, and the fact that the PNS is closely related to autoimmunity of the patients is indirectly suggested. It is well known that B lymphocytes play the most important role in humoral immunity, whereas the parts of the kidneys of PNS children have been devoid of antibody deposition during the past years, thus neglecting the role of B lymphocyte-mediated humoral immunity in PNS diseases. In recent years, it has been found that, in a patient with relapsed hormone-sensitive nephrotic syndrome, the number of B cells in peripheral blood is significantly increased, and that activated B cells are greatly increased in a patient with hormone-dependent nephrotic syndrome, while in a patient with remission-stage hormone-dependent nephrotic syndrome, the number of B cells is significantly decreased, suggesting that B lymphocyte dysfunction plays an important role in PNS. In recent years, much global research has found that a significant therapeutic effect is achieved in the treatment of children's refractory nephrotic syndrome with rituximab specific for CD20+ B cells. However, in the treatment of SDNS with rituximab, it was found that the B cell scavenging efficacy was maintained for about 5 months, but at 6 and 7 months, the disease was recurring due to the rise in B cell numbers. Thus, pathological B cell clones in the PNS patient are suggested to be identified and accurately eliminated, so that the PNS recovery is facilitated, and the risk of humoral immune deficiency caused by indiscriminate B cell elimination by rituximab and other methods is reduced.

However, to date, the target antigens to which pathological B cells are directed in PNS infants are still not well understood. Pathologically, minimal morbid nephropathy or focal segmental glomerulosclerosis is considered podocytosis resulting in massive proteinuria due to loss or alteration of podocyte function. Podocytes are renal glomerular epithelial cells that attach to the outside of the glomerular basement membrane. It is the last barrier to protein loss, and podocyte injury often causes massive proteinuria.

Aconitase (aconitase) is an essential enzyme located in mitochondria, catalyzing the interconversion of citric acid and isocitric acid in the tricarboxylic acid cycle. Current studies indicate that the expression of aconitate hydratase may be altered in certain types of cancer. Wang et al performed real-time quantitative reverse transcription polymerase chain reaction, Western blot and immunohistochemical staining of gastric cancer specimens to measure aconinate hydratase expression in tumor tissue and matched adjacent non-tumor tissue. The results show that the expression of aconate hydratase, which may play an important role in gastric cancer and may be a prognostic biomarker, is significantly down-regulated in gastric cancer tissue compared to matching adjacent non-tumor tissue and is associated with TNM staging of gastric cancer (Wang, et al. degraded expression of the mitochonic metabolic enzyme) is associated with bone cement in gastric cancer. Aconitate hydratase activity is elevated in prostate cancer and reduced in rectal cancer. A recent study on breast Cancer found that the expression of acrylonitrile hydrate is reduced, possibly by inhibiting cell proliferation by increasing the levels of enzymes in the cell, or that this is associated with increased oxidative metabolism (facial Ciccarone, et al. Aconitase 2 inhibitors the promotion of MCF-7cell promoting pathological metabolism and ROS/FoxO1-mediated autophagic response. Br J Cancer,2020 Jan; 122(2): 182-.

However, there is no report on the relationship between aconitate hydratase and nephrotic syndrome. In addition, in the prior art, the study for identifying autoimmune nephrotic syndrome by detecting serum anti-aconate hydratase-IgG antibody is currently blank.

Disclosure of Invention

The invention aims to provide a detection kit for detecting serum anti-aconite hydratase-IgG autoantibody, which detects the antibody in a detected sample through immunoreaction with aconite hydratase antigen protein (particularly shown by a sequence identification number SEQ ID NO. 1).

The kit comprises: aconate hydratase antigen protein, labeled antibody liquid (enzyme-labeled or chemiluminescent-labeled anti-human IgG solution), solid phase carrier coating aconate hydratase antigen, sample diluent, antibody diluent, antigen diluent, substrate developing solution, washing solution, stop solution, standard substance, positive quality control substance and positive quality control substance.

The sequence of the aconitate hydratase antigen protein is shown as SEQ ID NO: 1, and the following components:

MAPYSLLVTRLQKALGVRQYHVASVLCQRAKVAMSHFEPNEYIHYDLLEKNINIVRKRLNRPLTLSEKIVYGHLDDPASQEIERGKSYLRLRPDRVAMQDATAQMAMLQFISSGLSKVAVPSTIHCDHLIEAQVGGEKDLRRAKDINQEVYNFLATAGAKYGVGFWKPGSGIIHQIILENYAYPGVLLIGTDSHTPNGGGLGGICIGVGGADAVDVMAGIPWELKCPKVIGVKLTGSLSGWSSPKDVILKVAGILTVKGGTGAIVEYHGPGVDSISCTGMATICNMGAEIGATTSVFPYNHRMKKYLSKTGREDIANLADEF。

according to the invention, the aconitate hydratase antigen protein is purified by a molecular sieve, gel filtration chromatography, affinity chromatography, an ion exchange column and a hydrophobic column.

The aconate hydratase antigenic protein according to the invention may be a fusion protein using a tag with a biological or physical function, in particular an N-terminal or C-terminal tag, preferably a C-terminal tag. These tags facilitate purification, immobilization, and precipitation of antigenic proteins. In a preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, and the tag peptide is selected from the group consisting of: his tag, GST tag, maltose binding protein, thioredoxin, and fluorescent tag or biotin tag.

According to the invention, the antigenic protein aconinate hydratase can be expressed in bacteria such as escherichia coli, fungal yeast and mammalian cells.

In a preferred embodiment, the aconate hydratase antigenic protein of the present invention is presented in an immobilized form, the term "immobilized" referring to binding to an insoluble solid support in aqueous solution. Preferably, the binding is by electrostatic interaction, hydrophobic interaction, covalent bond. The solid phase carrier is preferably polystyrene, a material microporous plate, a nitrocellulose membrane and magnetic beads.

The immobilization mode of the acoonite hydratase antigen protein comprises a reversible immobilization mode or an irreversible immobilization mode. For example, the molecules are bound by a cleavable covalent bond (e.g., a disulfide bond that can be cleaved by addition of a thiol-containing reagent), and this immobilization is reversible. In addition, if the molecule is immobilized to the support through a covalent bond that does not cleave in aqueous solution (a bond formed by the reaction of an epoxide group with the amine group coupling the lysine side chain to the affinity column), the immobilization is irreversible. The immobilization may also be an indirect way of immobilizing proteins: such as immobilizing an antibody having a specific affinity for the molecule, and then forming a complex to achieve the effect of immobilizing the molecule-antibody complex.

The method for fixing the aconate hydratase antigen protein disclosed by the invention is a direct coating method: (1) the antigen is combined on the nitrocellulose membrane or the polystyrene microporous plate in a physical adsorption mode or by non-covalent bonds; (2) the magnetic particle belt with carboxyl functional group is combined with protein amino, and the antigen is combined on the magnetic particle by means of chemical coupling.

The substrate color developing solution is TMB, luminol, hydrogen peroxide and acridine ester; the antigen diluent is: 1xPBS, pH7.40; the antibody diluent is as follows: 0.15% BSA +0.01M PBS (pH7.40); the sample diluent is: 6% fetal bovine serum +0.01M PBS (pH7.40); the washing solution is as follows: 1xPBS (pH7.40) +0.1 Tween-20; the stop solution is: 2M sulfuric acid.

In one embodiment of the present invention, the standard and the positive quality control substance are preferably prepared from human homologous antigen, and are recombinant human anti-tag peptide immunoglobulin G or fragments thereof, or anti-aconate hydratase-IgG antibody extracted from patient serum as the positive quality control substance and the standard, and the negative quality control substance is serum of a healthy physical examiner.

The labeled antibody liquid can be Horse Radish Peroxidase (HRP) labeled anti-human IgG, biotin labeled anti-human IgG and acridinium ester labeled anti-human IgG.

According to the invention, the sample to be tested is a liquid sample containing antibodies, preferably pleural fluid, ascites, urine, whole blood, plasma, most preferably serum. The serum is mammalian serum, preferably human serum. The sample to be tested may be further processed prior to testing, including fractionation, centrifugation, or enrichment.

The invention relates to a detection kit which takes recombinant protein aconate hydratase expressed and purified by a gene recombination method as antigen protein in the kit, then coats the antigen protein on a solid phase carrier, adds a positive quality control product or a standard product or serum to be detected for incubation, then adds a labeled secondary antibody for reaction, combines the labeled secondary antibody with an anti-aconate hydratase-IgG antibody in the serum to form a coated antigen aconate hydratase-anti-aconate hydratase-IgG antibody-labeled anti-human IgG antibody compound of the serum to be detected, detects a light signal by an optical method such as a light color development method, a chemiluminescence method and a fluorescence method, and qualitatively or quantitatively analyzes the concentration of the anti-aconate hydratase-IgG antibody in the serum.

By applying the kit, an anti-aconate hydratase-IgG autoantibody is detected in a part of autoimmune nephrotic syndrome patients for the first time, and the target antigen aimed by the autoantibody is determined to be mesangial podocyte aconitase (aconate hydratase). Therefore, the kit can be used for detecting the anti-aconitate hydratase-IgG autoantibody, and provides a basis for researching the autoimmune nephrotic syndrome.

Compared with the prior art, the kit has the advantages that:

(1) the invention firstly identifies the existence of the anti-aconitate hydratase-IgG autoantibody in the body of the patient with autoimmune nephrotic syndrome, and invents a detection kit aiming at the antibody. At present, research related to the aconitate hydratase-IgG antibody is not seen at home and abroad, and the method fills the blank of detection of the aconitate hydratase-IgG antibody at home and abroad.

(2) The kit relates to qualitative detection of an anti-aconate hydratase-IgG antibody in serum, wherein solid-phase membrane immune qualitative detection is simple and convenient to operate, the dosage of a reagent is small, a trace amount of antigen can be adsorbed by the strong adsorption capacity of an NC membrane, and the adsorbed NC membrane can be stored for a long time (the temperature is minus 20 ℃ and can be stored for half a year) without influencing the activity of the NC membrane. Is suitable for large-scale screening.

(3) The invention relates to a magnetic particle chemiluminescence immune analysis quantitative detection kit, which utilizes magnetic particles as solid phase carriers, the diameter of the magnetic particles is only 1.0 mu m, thus greatly increasing the surface area of the coating, increasing the adsorption quantity of antigens, improving the reaction speed, and ensuring that the cleaning and the separation are simpler and more convenient, thereby reducing pollution and reducing the probability of cross infection. On the other hand, in the case of a liquid,the acridine ester luminescent agent is adopted to directly mark the anti-human IgG, the chemical reaction is simple and quick, and no catalyst is needed; the acridinium ester chemiluminescence is of the scintillation type by initiating the luminescent reagent (H) 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.

(4) At present, the relation between the aconate hydratase and the nephrotic syndrome is not reported, and the invention discovers that an aconate hydratase-IgG antibody exists in the nephrotic syndrome for the first time, thereby defining the cause of autoimmunity. In the prior art, the research for identifying the autoimmune nephrotic syndrome by detecting the serum anti-aconitate hydratase-IgG antibody is blank, the invention provides a non-invasive detection method for the disease, and compared with the existing kidney biopsy detection method, the application of the kit can reduce the pain and the economic burden of a patient.

Drawings

FIG. 1: the aconitate hydratase protein on podocytes is a target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. FIG. 1A: the primary antibody is a two-dimensional electrophoresis protein spot of human serum of healthy people; FIG. 1B: the first antibody is a two-dimensional electrophoresis protein spot of serum of an autoimmune nephrotic syndrome patient; FIG. 1C: mass spectrometric identification of the target antigen, aconitate hydratase protein.

FIG. 2 is a schematic diagram: SDS-PAGE identification picture of the expressed recombinant aconitate hydratase antigen protein.

FIG. 3: a solid-phase membrane immunoassay kit is used for detecting antibody membrane maps of aconintate hydratase-IgG in serum of patients with autoimmune nephrotic syndrome.

FIG. 4: a schematic diagram of a magnetic particle chemiluminescence kit for detecting an anti-aconitate hydratase-IgG antibody.

FIG. 5 is a schematic view of: schematic diagram of coated carboxyl magnetic particles of antigenic protein aconintate hydratase.

FIG. 6: detection condition of anti-aconitate hydratase-IgG antibody of different nephropathy patients, wherein NC: healthy people; HP: allergic purpura; HPN: purpuric nephritis; IgAN: IgA nephropathy; and NS: autoimmune nephrotic syndrome.

FIG. 7: the application value of the anti-aconitate hydratase-IgG antibody for detecting the autoimmune nephrotic syndrome is evaluated by the ROC curve.

Detailed Description

The invention is further described below with reference to the following figures and specific examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.

Example 1 Aconitate hydratase on podocytes is a target antigen for autoantibodies in autoimmune nephrotic syndrome patients

(1) Extraction of glomerular podocyte total protein: the podocyte strain (MPC5) was cultured, washed 2-3 times with PBS, then sufficiently lysed on ice using a focused ultrasound machine (Covaris S220, Gene) in lysis buffer containing 30mm Tris-HCl, 8m urea, 4% CHAPS and protease inhibitor (# ab 65621; Abcam, 1: 200 dilution), and then the samples were centrifuged at 12000g for 30min at 4 ℃. Collecting the supernatant, namely the total protein of the glomerular podocyte. The total protein concentration of the collected glomerular podocytes was determined using the BCA protein concentration assay kit. (2) Two-dimensional electrophoresis: extracting total protein of glomerular podocyte, performing two-dimensional electrophoresis, transferring to nitrocellulose membrane, incubating with serum of autoimmune nephrotic syndrome patient and healthy person as primary antibody, and developing with secondary antibody as shown in fig. 1A and 1B. (3) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: differential analysis of positive spots was performed after visualization in step (2), and protein spots strongly positive for nephrotic syndrome patients on two-dimensional electrophoresis gel and negative or weakly positive for healthy persons were selected, the selected protein spots were cut out from the gel, the dried gel was digested with trypsin (0.1. mu.g/. mu.L), 10. mu.L of 25mM ammonium bicarbonate was added to the reaction mixture, incubated overnight at 37 ℃, and then peptides were extracted from the gel with trifluoroacetic acid (0.1%). The extracted peptides were analyzed by matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) mass spectrometer to obtain a peptide mass spectrum, which was identified as aconitate hydratase protein, see FIG. 1C.

EXAMPLE 2 expression and purification of recombinant aconitate hydratase antigenic protein

The gene of coding aconitate hydratase protein is used as a template by utilizing a genetic engineering method to carry out PCR amplification, and then an expression vector is constructed to carry out protein expression. The expressed antigen protein of the invention contains tag peptide of His tag. 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 aconate hydratase is identified by SDS-PAGE, and the result is shown in figure 2.

Example 3 optimization of the reaction conditions of the kit according to the invention by orthogonal assay design

Orthogonal tables were selected based on 4 factors, such as the concentration of antigen aconate hydratase coating (four coating concentrations of 50. mu.g/mL, 100. mu.g/mL, 150. mu.g/mL, and 200. mu.g/mL), each reaction time (30min, 45min), and temperature (25 ℃, 35 ℃), the optimal dilution of enzyme-labeled secondary antibody (four dilutions of 1: 100, 1: 500, 1: 1000, and 1: 1500), and each factor was measured in 2-level replicates for standard positive serum and standard negative serum. 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 measurement is statistically processed to determine the optimal coating condition and the optimal dilution of the secondary antibody to carry out orthogonal optimization, thereby obviously improving the positive detection rate of the standard positive serum. Through orthogonal design, the optimal antigen coating concentration of the kit is 100 mu g/mL, the optimal antigen-antibody reaction temperature is 25 ℃, the optimal antigen-antibody reaction time is 45 minutes, and the optimal working dilution of the optimal labeled anti-human IgG antibody is 1: 500.

EXAMPLE 4 preparation of solid-phase Membrane Immunity kit for detecting anti-aconitate hydratase-IgG antibody

4.1 composition of solid phase membrane immunoassay kit for detecting anti-aconitate hydratase-IgG:

1. a nitrocellulose membrane coated with an aconitate hydratase antigen protein;

2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro lake),

3. the dilution liquid of the antibody is used as the antibody,

4. an antigen diluent is added to the antigen-containing solution,

5. an anti-human IgG antibody marked by horseradish peroxidase,

6. the washing liquid is used for washing the glass fiber,

7. a TMB color-developing agent,

8. and (4) stopping the solution.

4.2 the detection steps are as follows:

4.2.1 coating, sealing: the antigen was diluted with 0.01M PBS (pH7.4) and 10. mu.l of the diluted antigen was spotted on a nitrocellulose membrane, which was placed in a 37 ℃ incubator for 30 minutes, the nitrocellulose membrane was placed in a plate tank, 150. mu.l of 3% BSA was added thereto, the resulting mixture was sealed in a 37 ℃ incubator for 15 minutes, and after the blocking solution was aspirated off, the membrane was washed 2 times with a washing solution.

4.2.2 serum incubation (first incubation): 100 microliter of the standard substance diluted by the antigen releasing solution and the detected serum sample are loaded in the reaction tank, a negative control and a positive control are simultaneously carried out, the nozzle of the sample loader does not touch the surface of the membrane, and one nozzle is replaced when each serum is loaded. The reaction vessel with the added sample was placed on a shaker and incubated at room temperature (20-25 ℃) for 45 minutes.

4.2.3 Wash (first wash): pouring out the liquid in the reaction tank, and washing for 10 seconds by using diluted washing liquid; in washing, the washing solution is allowed to sufficiently flow through the reaction vessel. The cleaning is repeated for 5 times, and the liquid is poured and washed while the liquid flows down along the reaction tank, so that the cross contamination is avoided. And (5) drying the reaction tank after cleaning.

4.2.4 incubation with secondary antibody working solution (second incubation): the horseradish peroxidase-labeled anti-human IgG antibody was diluted with the antibody diluent, and then 6 drops (300. mu.l) of the secondary antibody working solution were added to the reaction tank, and incubated on a shaker at room temperature (20-25 ℃) for 45 minutes.

4.2.5 Wash (second wash): the process is the same as step 3.

4.2.6. Chromogenic incubation (third incubation): 6 drops (300. mu.l) of the chromogenic solution were added to the strips and incubated on a shaker at room temperature (20-25 ℃) for 20 minutes.

4.2.7. And (3) terminating the reaction: the reaction vessel was flushed with running water to terminate the reaction.

4.2.8. Interpretation results: the test strips were taken out and dried by air blow (about 5 minutes) or placed in an oven at 37-50 ℃ for over 20 minutes. And (3) carrying out naked eye qualitative judgment, wherein the person with obvious brown spots is positive (see figure 3) or placing the membrane strip on a developing instrument for scanning, and drawing a standard curve by analysis software carried by the developing instrument by taking the concentration of a reference standard substance as a vertical coordinate and the gray value read by the instrument as a horizontal coordinate to carry out semi-quantitative analysis on the level of the anti-aconate hydratase-IgG in the serum.

EXAMPLE 5 preparation of chemiluminescent immunoassay kit for detection of anti-aconitate hydratase-IgG antibody

5.1 composition of chemiluminescence immunoassay kit for detecting anti-aconitate hydratase-IgG:

1. coating the magnetic particle solution of aconitate hydratase antigen protein,

2. and (3) standard substance: human anti-His tag immunoglobulin G (purchased from invitro hu),

3. the dilution liquid of the sample is used for diluting the sample,

4. the acridinium ester is marked on the anti-human IgG solution,

5. the quality control material is prepared by the following steps of,

6. the pre-excitation liquid is mixed with the water,

7. the excitation liquid is used for exciting the liquid,

8. and (4) washing liquid.

5.2 detection principle: the kit adopts an indirect method to detect the antibody of the anti-aconintate hydratase-IgG in human serum, and the whole process comprises two steps of reactions: firstly, mixing the magnetic bead solution with the diluted sample, binding a specific anti-aconite hydratase-IgG antibody on the magnetic bead, and washing to remove residual solution. Secondly, adding acridinium ester labeled anti-human IgG antibody to form a magnetic bead-antigen-anti-aconitate hydratase-IgG antibody-acridinium ester antibody compound, washing to remove residual liquid, and adding pre-excitation liquid (H) ₂ O ₂ ) And (3) carrying out luminescence reaction with an excitation solution (NaOH), recording a luminescence value, wherein the concentration of the antibody is in direct proportion to the luminescence value, and calculating a concentration value through a calibration curve (see figure 4).

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 are covalently combined with antigen (amino residue) to form magnetic particle solution. The coating step is as follows:

a) adding 40 microliters of magnetic bead stock solution into 400 microliters of 0.05M phosphate buffer solution, uniformly mixing for 8 minutes, then carrying out magnet separation, and removing the supernatant;

b) adding 200 microliters of 10mg/mL sodium periodate into 200 microliters of 2% dextran solution to react;

c) after the reaction is finished, adding 10mg/mL EDC solution prepared by phosphate buffer solution and 10mg/mLSulfo-NHS solution, and uniformly mixing for 60 minutes;

d) magnet separation, abandoning the supernatant, taking 400 microliter 0.05M phosphate buffer solution to wash the magnetic beads, adding 400 microliter of sealing solution to fix the 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 aconintate hydratase into the activated magnetic beads, fully and uniformly mixing, and standing at room temperature for reaction for 16 hours; after the reaction is finished, adding pH7.4PBS buffer solution 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 a pH7.4PBS buffer containing 0.2% Tween20 and 0.2% BSA, and the crosslinking of the activated magnetic beads with the antigenic protein aconintate hydratase was completed (see FIG. 5).

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;

c) uniformly mixing and stirring acridinium ester and an anti-human IgG antibody in a molar ratio of 4:1, and reacting for 40 minutes;

d) the reaction was stopped by adding 20. mu.l of carbonate buffer containing 5% lysine for 30 minutes;

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 according to a certain proportion;

5.6.2 taking the diluted sample, adding the magnetic particle liquid and the sample diluent, and reacting for 12min at 37 ℃;

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 12 min;

5.6.5 washing solution for 3 times;

5.6.6 adding pre-exciting liquid (H) ₂ O ₂ ) Reacting with an excitation solution (NaOH), and collecting a luminescence measured value;

5.6.7 the concentration measurement is calculated from the calibration curve.

EXAMPLE 6 detection of anti-acnonite hydratase-IgG antibodies in patients with renal diseases of all types

6.1 Subjects were enrolled in patients diagnosed with nephrotic syndrome from 6 months 2018 to 6 months 2020, and healthy controls were selected from healthy examiners who had a contemporaneous visit. Serum samples were taken from nephrotic syndrome patients and healthy controls. All subjects had a first serum sample collection prior to no immunosuppressive treatment.

6.2 detection of serum anti-acntate hydratase-IgG antibodies in patients with various nephropathies who were diagnosed from 6.2018 to 6.2020 by using the kit of the present invention, the serum anti-acntate hydratase-IgG antibodies levels included 466 nephrotic syndrome, 168 anaphylactoid purpura, 137 purpura nephritis, 133 IgA nephropathy and 195 healthy children at the same time, and the results showed that anti-acntate hydratase-IgG antibodies were positive in some patients with autoimmune nephrotic syndrome, while anti-acntate hydratase-IgG antibodies were negative in patients with purpura nephritis, allergic purpura, IgA nephropathy and healthy children (see FIG. 6).

Example 7 evaluation of value of ROC curve for detection of autoimmune nephrotic syndrome by anti-aconitate hydratase-IgG antibody as a serological marker the results of detection of anti-aconitate hydratase-IgG antibody in autoimmune nephrotic syndrome patients 6.2 of example 6 were analyzed using the ROC curve to determine the value of the antibody for detection of autoimmune nephrotic syndrome. The results show that the anti-aconitate hydratase-IgG antibody as a serum marker has good value for the diagnosis of patients with autoimmune nephrotic syndrome, and when the diagnosis is defined as more than 53.4, the diagnosis sensitivity is 56.8%, the diagnosis specificity is 87.2%, and the area under the curve is 0.773 (see figure 7).

SEQUENCE LISTING

<110> Zhejiang university

<120> detection kit for detecting anti-aconitate hydratase-IgG antibody

<130> 2022.5.10

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 322

<212> PRT

<213> Artificial sequence (Unknow)

<400> 1

Met Ala Pro Tyr Ser Leu Leu Val Thr Arg Leu Gln Lys Ala Leu Gly

1 5 10 15

Val Arg Gln Tyr His Val Ala Ser Val Leu Cys Gln Arg Ala Lys Val

20 25 30

Ala Met Ser His Phe Glu Pro Asn Glu Tyr Ile His Tyr Asp Leu Leu

35 40 45

Glu Lys Asn Ile Asn Ile Val Arg Lys Arg Leu Asn Arg Pro Leu Thr

50 55 60

Leu Ser Glu Lys Ile Val Tyr Gly His Leu Asp Asp Pro Ala Ser Gln

65 70 75 80

Glu Ile Glu Arg Gly Lys Ser Tyr Leu Arg Leu Arg Pro Asp Arg Val

85 90 95

Ala Met Gln Asp Ala Thr Ala Gln Met Ala Met Leu Gln Phe Ile Ser

100 105 110

Ser Gly Leu Ser Lys Val Ala Val Pro Ser Thr Ile His Cys Asp His

115 120 125

Leu Ile Glu Ala Gln Val Gly Gly Glu Lys Asp Leu Arg Arg Ala Lys

130 135 140

Asp Ile Asn Gln Glu Val Tyr Asn Phe Leu Ala Thr Ala Gly Ala Lys

145 150 155 160

Tyr Gly Val Gly Phe Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile

165 170 175

Ile Leu Glu Asn Tyr Ala Tyr Pro Gly Val Leu Leu Ile Gly Thr Asp

180 185 190

Ser His Thr Pro Asn Gly Gly Gly Leu Gly Gly Ile Cys Ile Gly Val

195 200 205

Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Ile Pro Trp Glu Leu

210 215 220

Lys Cys Pro Lys Val Ile Gly Val Lys Leu Thr Gly Ser Leu Ser Gly

225 230 235 240

Trp Ser Ser Pro Lys Asp Val Ile Leu Lys Val Ala Gly Ile Leu Thr

245 250 255

Val Lys Gly Gly Thr Gly Ala Ile Val Glu Tyr His Gly Pro Gly Val

260 265 270

Asp Ser Ile Ser Cys Thr Gly Met Ala Thr Ile Cys Asn Met Gly Ala

275 280 285

Glu Ile Gly Ala Thr Thr Ser Val Phe Pro Tyr Asn His Arg Met Lys

290 295 300

Lys Tyr Leu Ser Lys Thr Gly Arg Glu Asp Ile Ala Asn Leu Ala Asp

305 310 315 320

Glu Phe

Claims

1. Use of an aconitate hydratase (aconate hydratase) polypeptide or antibody binding fragment thereof capable of forming any antigen antibody complex in contact with a biological sample obtained from the patient for the preparation of a nephrotic syndrome detection reagent or kit; wherein the antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody (anti-aconate hydratase-IgG antibody) complex.

2. The use of claim 1, wherein the nephrotic syndrome is autoimmune nephrotic syndrome.

3. The use of claim 2, wherein the nephrotic syndrome is childhood autoimmune nephrotic syndrome.

4. The use of claim 1 wherein the aconitate hydratase (aconate hydratase) antibody binding fragment sequence is set forth in SEQ ID No. 1.

5. The use of claim 1, wherein the biological sample is serum.

6. The use of claim 1, wherein the biological sample is of a patient prior to undergoing immunotherapy.

7. Use of an aconitate hydratase (aconitite hydratase) polypeptide or antibody binding fragment capable of forming any antigen-antibody complex in contact with a biological sample obtained from the patient for the preparation of a reagent or kit for the specific detection of nephrotic syndrome with respect to purpuric nephritis, henoch-schonlein purpura, IgA nephropathy; wherein the antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody (anti-aconate hydratase-IgG antibody) complex.

8. A reagent for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome with respect to purpuric nephritis, henoch-schonlein purpura, IgA nephropathy, comprising: an aconitate hydratase (aconate hydratase) polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex in contact with a biological sample obtained from the patient; wherein the antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody (anti-aconate hydratase-IgG antibody) complex.

9. A kit for diagnosing nephrotic syndrome or specifically detecting nephrotic syndrome relative to purpuric nephritis, henoch-schonlein purpura, IgA nephropathy, comprising: an aconitate hydratase (aconate hydratase) polypeptide or antibody binding fragment thereof capable of forming any antigen-antibody complex in contact with a biological sample obtained from the patient; wherein the antigen-antibody complex comprises an anti-aconitate hydratase-IgG antibody (anti-aconate hydratase-IgG antibody) complex.