CN113447656A

CN113447656A - Kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody

Info

Publication number: CN113447656A
Application number: CN202110742935.7A
Authority: CN
Inventors: 叶青; 毛建华; 周希
Original assignee: Childrens Hospital of Zhejiang University School of Medicine
Current assignee: Zhejiang University ZJU
Priority date: 2021-07-01
Filing date: 2021-07-01
Publication date: 2021-09-28
Anticipated expiration: 2041-07-01
Also published as: CN113447656B; CN115060908A

Abstract

The invention provides a kit for detecting an anti-filamentous actin capping protein beta-IgG antibody, which consists of an antigen protein, namely, an F-actin-capping protein subbunit beta, a solid phase carrier, a labeled antibody, an antigen diluent, a sample diluent, an antibody diluent, a substrate color developing agent, a washing solution, a standard product, a positive quality control product and a negative quality control product. The autoantibodies against the cap-forming protein beta-IgG of the filamentous actin are detected by a specific kit. The invention identifies the autoantibody of the target antigen filamentous actin cap-forming protein beta for the first time, and provides a detection kit aiming at the autoantibody. The kit provided by the invention provides a basis for molecular mechanism research and clinical diagnosis and treatment of autoimmune nephrotic syndrome related to the filamentous actin cap-forming protein beta and the filamentous actin cap-forming protein beta-IgG autoantibody at home and abroad.

Description

Kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody

Technical Field

The invention belongs to the technical field of biomedicine, relates to a kit for detecting an anti-filamentous actin cap-forming protein beta-IgG antibody, and is a kit for detecting an anti-filamentous actin cap-forming protein beta-IgG antibody in a patient sample.

Background

Autoimmune diseases are diseases caused by abnormal immune reactions of the human body to substances and tissues normally present in the body. May be limited to certain organs, involve specific tissues in different locations, and may be systemic. The treatment of autoimmune diseases often involves immunosuppression, i.e. drugs that reduce the immune response.

Primary Nephrotic Syndrome (PNS) is characterized by edema, increased protein in the urine, decreased protein in the blood, and increased fat in the blood of a patient (Roth KS, Amaker BH, Chan JC: nephritic syndrome: pathological and management. patient Rev (2002)23(7): 237-48). More than 90% of the pathological types of primary nephrotic syndrome in children are microscopic lesions or focal segmental glomerulosclerosis, which are mainly characterized by podocyte injury. Inflammation of the podocytes increases permeability to proteins, resulting in increased secreted proteins. When the amount of protein excreted in urine exceeds the compensatory capacity of the liver, less protein is detected in the blood, particularly albumin, which accounts for the majority of circulating proteins. As the protein in the blood decreases, the osmotic pressure in the blood decreases. Edema can result because the osmotic pressure in the tissue remains constant. This condition is exacerbated by secretion of aldosterone by the adrenal gland, which is secreted in response to a decrease in circulating blood, resulting in sodium and water retention. Hyperlipidemia is considered to be The result of increased liver activity (Brinkkoetter PT, Ising C, Benzing T: The role of The lipid in albumin filtration. nat Rev Nephrol (2013)9(6): 328-36). Evidence has shown that the majority of children's hormone-sensitive nephrotic syndrome and 2/3 have a role in the development of hormone-resistant nephrotic syndrome in association with autoimmune dysfunction (Li J, Wang L, Wan L, Lin T, ZHao W, Cui H, et al: biological spectra and novel cancer genes in Chinese childhood with systemic steroid syndrome. Peditar Res (2019)85(6): 816-21). We screened autoantibodies against filamentous actin-capping protein subBunit beta (F-actin-capping protein beta) in patients with autoimmune nephrotic syndrome. Filamentous actin capping protein β is a member of the F-actin capping protein family. This gene encodes the β subunit of the barb-terminal actin-binding protein. The protein regulates actin filament growth by covering the barbed ends of the growing actin filament. In muscle, the interaction of the cap-forming protein β of filamentous actin with actin is crucial in myofibrillating (Pyre WG, Hart MC, Cooper JA, Sumandea MP, de Tombe PP, Solaro RJ. active trapping protein: an infectious element in protein kinase signaling to the myofibers. circulation Research. (2002)90(12): 1299-306).

However, the filamentous actin capping protein beta and autoantibodies against the filamentous actin capping protein beta are not reported in urinary system diseases, particularly in nephrotic syndrome. In addition, the prior art does not relate to the application of target-based filamentous actin-capping protein beta or an autoantibody thereof as a serological marker in autoimmune nephrotic syndrome. The study for identifying autoimmune nephrotic syndrome by detecting serum anti-filamentous actin cap-forming protein beta-IgG antibodies is blank.

Disclosure of Invention

The invention aims to provide a kit for detecting an anti-filamentous actin capping protein beta-IgG antibody, which is a detection kit based on target filamentous actin capping protein beta and a corresponding autoantibody thereof. The kit can detect autoantibodies from tissues (kidney biopsy) or body fluids (in particular blood, plasma, serum) by reacting with an antigen antibody of the antigenic protein filamentous actin cap-forming protein beta (in particular according to the sequence identification number SEQ ID NO. 1).

The invention relates to a detection kit for detecting an autoantibody against filamentous actin capping protein beta, which comprises an antigen protein filamentous actin capping protein beta, a solid phase carrier, a labeled antibody, an antigen diluent, a sample dilution buffer, an antibody diluent, a substrate color developing agent, a washing solution, a stop solution, a standard substance, a positive quality control product and a negative quality control product.

According to the invention, the antigenic protein filamentous actin cap-forming protein beta can be expressed in bacteria such as escherichia coli, yeast, mammalian cells.

According to the invention, the antigenic protein may be a fusion protein, using tags having certain biological or physical functions, the presence of which facilitates purification, immobilization, precipitation or identification of the antigenic protein or of other sequence motifs or polypeptides of the polypeptide of the invention. In a more preferred embodiment, the tag is a sequence or domain capable of specifically binding to a ligand, e.g., selected from the group consisting of: a GST tag, a c-Myc tag, a His tag, a Flag tag, or a biotin tag.

According to the invention, the antigenic protein filamentous actin cap-forming protein β is immobilized on a solid support, preferably a solid support comprising: nitrocellulose membrane, magnetic beads and enzyme-labeled microporous plate.

In one embodiment of the invention, the standard substance and the positive quality control substance are recombinant human anti-tag peptide IgG or fragments thereof, or anti-filamentous actin cap-forming protein beta antibody extracted from patient serum is used as the positive quality control substance and the standard substance, and the serum of a healthy examiner is the negative quality control substance.

According to the invention, the antigenic protein filamentous actin cap-forming protein beta is purified by Ni column affinity chromatography, molecular sieve chromatography, ion exchange chromatography and hydrophobic column.

The sample herein is a sample of a body fluid or tissue to be tested. Preferred test samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusion. Furthermore, certain test samples will be more easily analyzed after separation or purification procedures, such as separation of whole blood into serum or plasma components. Thus, in a preferred embodiment of the invention, the sample is selected from the group consisting of a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, a saliva sample and a urine sample or an extract of any of the above mentioned samples. Preferably, the sample is a blood sample, more preferably a serum sample or a plasma sample.

The detection kit further comprises a substrate color developing agent, an antigen diluent, a sample dilution buffer solution, an antibody diluent, a washing solution and a stop solution. The substrate color developing agent is TMB, BCIP, AMPPD and 4-MUP; the antigen diluent is pH7.41x PBS, 163mM NaCL and 1% TritonX-100; the sample dilution buffer was 0.01M PBS buffer containing 10% BSA, ph 7.4; the antibody diluent is prepared by adding 1MD glucose (19.82g) and 2% glycerol (2ml) into 0.01M PBS containing 0.2% Tween20 to 100 ml; the washing solution is as follows: 1 XPBS pH7.4, NaCL 163mM, 1% TritonX-100, glycerol 10%; the stop solution is: 2M sulfuric acid.

The method for fixing the cap-forming protein beta of the antigen protein filamentous actin 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 non-covalent bond; 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 labeled antibody selected by the invention can be Horseradish Peroxidase (HRP) labeled anti-human IgG, acridinium ester labeled anti-human IgG and biotin labeled anti-human IgG.

The invention adopts a gene recombination prokaryotic expression method to successfully express and purify recombinant protein filamentous actin cap-forming protein beta, takes the recombinant protein filamentous actin cap-forming protein beta as antigen protein in a kit, and develops a kit suitable for detecting anti-filamentous actin cap-forming protein beta-IgG antibody of patients with autoimmune nephrotic syndrome. Comprises a detection kit for qualitatively or quantitatively analyzing and detecting the anti-filamentous actin capping protein beta-IgG antibody in human serum.

The principle of the kit for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody in serum adopts the principle that an antigen is adsorbed on a solid phase carrier to serve as an envelope antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled secondary antibody is added for reaction, a ternary complex of the envelope antigen-coated filamentous actin cap-forming protein beta-IgG antibody of the serum to be detected and a labeled anti-human IgG antibody is formed, and finally, an optical signal is detected by using a chromogenic method, a chemiluminescence method and a fluorescence method, so that the aim of qualitatively or quantitatively analyzing the anti-filamentous actin cap-forming protein beta-IgG antibody in human serum is fulfilled.

The sequence of the antigen protein filamentous actin cap-forming protein beta is shown in SEQ ID NO. 1:

MSDQQLDCALDLMRRLPPQQIEKNLSDLIDLVPSLCEDLLSSVDQPLKIARDKVVGKDYLLCDYNRDGDSYRSPWSNKYDPPLEDGAMPSARLRKLEVEANNAFDQYRDLYFEGGVSSVYLWDLDHGFAGVILIKKAGDGSKKIKGCWDSIHVVEVQEKSSGRTAHYKLTSTVMLWLQTNKSGSGTMNLGGSLTRQMEKDETVSDCSPHIANIGRLVEDENKIRSTLNEIYFGKTKDIVNGLRSIDAIPDNQKFKQLQRELSQVLTQRQIYIQPDN。

specific innovations can be summarized as follows:

(1) the invention identifies the autoantibody of the anti-filamentous actin cap-forming protein beta for the first time, and invents a detection kit aiming at the autoantibody.

(2) At present, no kit for detecting the level of the anti-filamentous actin capping protein beta autoantibody exists at home and abroad, and the invention fills the blank of the detection kit for the anti-filamentous actin capping protein beta autoantibody at home and abroad.

(3) The advantage of the chemiluminescence method is the simplicity of the assay. The magnetic separation system is adopted, and the acridinium ester labeling chemiluminescence technology has the advantages that the acridinium ester can be used as a luminescent agent in the absence of a catalyst and can also emit light in a dilute alkali solution containing hydrogen peroxide; the molecular weight is small, the antibody binding site is prevented from being shielded, the overall sensitivity of the system is improved, and the reaction is rapid; the background is low, the signal-to-noise ratio is high, and the chemiluminescent label is effective.

(4) The solid-phase membrane immunity method greatly shortens the detection time and can achieve the effect of rapid detection. The kit for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody introduces a biotin-avidin amplification system, so that the detection sensitivity is greatly improved.

Drawings

FIG. 1: the filamentous actin cap-forming protein beta protein on podocytes is one of the target antigens for autoantibodies in autoimmune nephrotic syndrome patients. FIG. 1A shows two-dimensional electrophoretic protein spots of human healthy serum; FIG. 1B two-dimensional electrophoresis protein spots of serum from patients with autoimmune nephrotic syndrome; FIG. 1C Mass Spectrometry identification of the target antigen, the filamentous actin cap-forming protein beta protein.

FIG. 2: SDS-PAGE identification of recombinant protein filamentous actin cap-forming protein beta.

FIG. 3: the solid-phase membrane immunoassay kit is used for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody in the serum of the patient with autoimmune nephrotic syndrome.

FIG. 4 is a drawing: the detection principle of the chemiluminescence kit for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody is shown in the schematic diagram.

FIG. 5: schematic diagram of coated carboxyl magnetic particles of antigenic protein filamentous actin capping protein beta.

FIG. 6: detection of anti-filamentous actin cap-forming protein beta-IgG antibody in various nephrotic patients, wherein PNS: autoimmune nephrotic syndrome, HSP: allergic purpura, HSPN: purpuric nephritis, IgAN: IgA nephropathy, NC: a healthy child.

FIG. 7: the application value of the anti-filamentous actin cap-forming protein beta antibody serving as a serological marker for differential diagnosis of autoimmune nephrotic syndrome is evaluated by an 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 filamentous actin capping protein β on podocytes is one of the target antigens to which autoantibodies are directed in patients with autoimmune nephrotic syndrome. According to the invention, a large number of clinical and molecular mechanism researches at the early stage are carried out, the serum IgG level of a patient with nephrotic syndrome is found to be high for the first time, and the filamentous actin cap forming protein beta on podocytes is proved to be a target antigen aiming at an autoantibody of the patient with autoimmune nephrotic syndrome. It would therefore be advantageous to detect the presence and quantitative levels of anti-filamentous actin cap-forming protein β -IgG antibodies in serum to aid in the early identification of autoimmune nephrotic syndrome, particularly in screening patients for symptoms of interest.

Specifically, the following (1) extraction of total protein of glomerular podocyte is carried out: 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 healthy people and autoimmune nephrotic syndrome patients 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), protein spots which were 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 excised 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, which was identified as the filamentous actin cap-forming protein β protein, see fig. 1C.

Example 2 expression and purification of Cap protein beta antigen from filamentous actin

The gene of the cap-forming protein beta protein of the filamentous actin 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 antigen protein expressed by the invention contains a tag peptide of His tag. The expressed recombinant protein is purified by nickel column affinity chromatography, ion affinity chromatography, hydrophobic column, molecular sieve, etc., and finally the molecular weight of the recombinant protein filamentous actin cap-forming protein beta is identified by SDS-PAGE, and the result is shown in FIG. 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 antigen anti-filamentous actin capping protein β -coating concentration (four coating concentrations of 50. mu.g/ml, 90. mu.g/ml, 120. mu.g/ml, 150. mu.g/ml), each reaction time (30min, 45min) and temperature (25 ℃, 35 ℃), the enzyme-labeled secondary antibody optimal dilution (four dilutions of 1:100, 1:500, 1:1000, 1: 1500), and each factor was determined repeatedly at 2 levels 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 90 mu g/ml, the optimal antigen-antibody reaction time is 30min, and the optimal acridinium ester labeled anti-human IgG working dilution is 1: 1000.

example 4 anti-filamentous actin capping protein beta-IgG antibody solid phase membrane immunoassay kit

4.1 composition of solid-phase membrane immunoassay kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody:

TABLE 1

4.2 detection procedure of solid-phase membrane immunoassay kit for detecting anti-filamentous actin capping protein beta-IgG is as follows:

(1) coating and sealing: placing 8 μ l of filamentous actin cap-forming protein β antigen direct contact with concentration of 90ug/ml on nitrocellulose membrane, drying in 37 deg.C incubator for 30min, placing NC membrane in detection plate, adding 200 μ l of 5% BSA, sealing in 37 deg.C incubator for 30min, discarding the sealing solution, and washing with washing solution for 2 times;

(2) antigen incubation: adding 10 μ l of antibody standard or serum to be detected diluted with diluent into the detection plate, performing negative control and positive control, incubating at room temperature for 30min, and arranging 3 parallel holes in each sample;

(3) and (3) secondary antibody incubation: discarding the liquid in the detection plate, washing with the washing solution for 5 times × 1min, adding 20 μ l of 1:500 biotin-labeled anti-human IgG antibody, and incubating at room temperature for 30 min;

(4) color development: discarding the liquid in the detection plate, washing with washing solution for 5 times multiplied by 1min, adding 500 μ l of alkaline phosphatase-streptavidin, incubating at room temperature for 20min, discarding the liquid in the detection plate, washing with washing solution for 5 times multiplied by 1min, then adding BCIP color development solution, reacting at room temperature for 20min, washing the detection plate with running water, and terminating the enzyme reaction;

(5) and (3) taking out the tested NC membrane strip, drying the membrane strip by using a blower, qualitatively judging by using a colorimetric card by naked eyes, and drawing a standard curve to perform semi-quantitative analysis on the level of the anti-filamentous actin capping protein beta-IgG in the serum by placing the membrane strip on a developing instrument to scan, wherein the developing instrument is provided with analysis software which takes the concentration of a reference standard as a vertical coordinate and takes a gray value read by the instrument as a horizontal coordinate.

Example 5 detection kit for anti-filamentous actin cap-forming protein beta-IgG antibody by chemiluminescence method

5.1 anti-filamentous actin cap-forming protein beta-IgG antibody chemiluminescence detection kit, including the following components:

(1) acridinium ester labeled anti-human IgG;

(2) carboxyl magnetic beads coupled with filamentous actin cap-forming protein beta antigen;

(3) a chemiluminescent pre-excitation liquid A and a chemiluminescent excitation liquid B;

(4) anti-filamentous actin capping protein beta-IgG antibody series standard solution, standard concentration: 0. mu.g/ml, 2. mu.g/ml, 4. mu.g/ml, 8. mu.g/ml, 16. mu.g/ml, 20.0. mu.g/ml, buffer of 0.5-containing Tris-HCl 5.0% BSA and 0.1-0.5% PC 300;

(5) cleaning solutions, especially Tris-HCl solution at pH 7.2, 25mmol/L containing 0.15mol/L NaCL and 0.05% Tween-20.

5.2 preparation of magnetic bead-coupled antigen (FIG. 4)

(1) Taking 1mg carboxyl magnetic particles into a 0.5mL centrifuge tube, adding a certain amount of 0.1mol/L MES buffer solution, uniformly mixing by vortex, placing on a magnetic frame, standing for 5min to ensure that the magnetic particles are separated from liquid, and discarding the supernatant. Washing 3 times, then adding a certain amount of MES (pH 5.0) buffer and vortexing;

(2) adding 18 μ L (18 μ g) of filamentous actin cap-forming protein β antigen, vortexing, rotating the reaction tube, and incubating at room temperature for 30 min;

(3) adding 10 mu L of 10mg/mL coupling reagent EDC, vortexing, rotating the reaction tube, and incubating for 2h at room temperature;

(4) the supernatant was removed and washed 3 times with 200. mu.L of washing buffer (TBS + 0.05% Tween-20);

(5) blocking with 1% BSA in buffer was repeated 4 times for 10min each. The magnetic particle suspension was stored at 2-8 ℃.

5.3 preparation of acridinium ester-labeled antibody

(1) Putting a certain amount of antihuman IgG antibody into a dialysis bag, putting the dialysis bag into not less than 1L of labeled buffer solution for dialysis, at least replacing buffer solution for 3 times, and finally dialyzing overnight, wherein the labeled buffer solution is Na₂CO₃-NaHCO₃A buffer solution with the pH of 10.1 and the concentration of 0.1 mol/L;

(2) weighing 1.7mg of acridinium ester NSP-DMAE-NHS, and dissolving in 447 mu L of anhydrous dimethylformamide DMF to form 6.5mmol/L of NSP-DMAE-NHS DMF solution;

(3) placing the dialyzed antibody solution into a 500-mu-L centrifuge tube, adding a certain amount of 6.5mmol/L NSP-DMAE-NHS DMF solution, wherein the molar ratio of the acridinium ester to the antibody is 7.4:1, adding 200-mu-L labeling buffer solution, reacting at room temperature for 45min, adding 10-mu-L lysine 10-mu-L, and continuing to react for 15min to terminate the labeling reaction;

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

(5) during the separation process, detecting protein peaks by using a chromatograph, and respectively measuring the chemiluminescence intensity of effluent and the absorbance at 430 nm;

(6) the high-light, high-absorbance eluate was collected, 1% BSA (by volume) was added, and stored on ice.

5.4 sample preparation, diluting the sample according to a certain proportion

5.5 detection procedure of the chemiluminescence assay kit for detecting the anti-filamentous actin capping protein beta-IgG antibody is as follows:

(1) sequentially adding 100 mu L of sample to be detected, 150 mu L of coupled magnetic powder suspension and 150 mu L of acridinium ester labeled secondary antibody into a reaction tube, shaking up and mixing, and keeping the temperature at 37 ℃ for 15 min;

(2) washing for 5 times in an isolation way;

(3) fully shaking the washed reaction container to uniformly disperse the magnetic particles;

(4) 100. mu.L of the chemiluminescent pre-excitation liquid A was added, followed by 100. mu.L of the chemiluminescent excitation liquid B, and the relative luminescence intensity was measured. The content of anti-filamentous actin capping protein β -IgG antibody in the sample was directly proportional to its luminescence intensity (fig. 5).

Example 6 application of the detection kit

6.1 subjects included: patients with various types of renal diseases were diagnosed from 6 months in 2018 to 6 months in 2020, and include nephrotic syndrome patients (n ═ 466), anaphylactoid purpura patients (n ═ 168), purpuric nephritis patients (n ═ 137), IgA nephropathy patients (n ═ 133), and healthy persons (n ═ 195). Serum samples were taken from various renal patients and healthy controls. All subjects received a first serum sample collection prior to no immunosuppressive treatment.

6.2 detection of anti-filamentous actin cap-forming protein beta antibody in various nephrotic patients. The kit is used for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody level in the serum of patients with various nephropathies, and the detection result is analyzed by adopting an ROC curve so as to judge the value of the antibody in diagnosing nephrotic syndrome. The prevalence of Ab against filamentous actin capping protein β was significantly higher in the patient cohort compared to the control group (fig. 6). The ROC curve assesses the value of the application of the anti-filamentous actin cap-forming protein β -IgG antibody as a serological marker for PNS patient diagnosis. When the diagnostic cutoff was greater than 57.3, the sensitivity of the anti-filamentous actin capping protein β autoantibody diagnostic autoantibody for nephrotic syndrome was 67.0% and the specificity was 73.5% (fig. 7). The result shows that the anti-filamentous actin cap-forming protein beta-IgG antibody is a good serological marker for differential diagnosis of autoimmune nephrotic syndrome.

Sequence listing

<110> Zhejiang university college of medicine subsidiary children hospital

<120> kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 276

<212> PRT

<213> Cap protein of filamentous actin beta protein (Artificial sequence Unknow)

<400> 2

Met Ser Asp Gln Gln Leu Asp Cys Ala Leu Asp Leu Met Arg Arg Leu

1 5 10 15

Pro Pro Gln Gln Ile Glu Lys Asn Leu Ser Asp Leu Ile Asp Leu Val

20 25 30

Pro Ser Leu Cys Glu Asp Leu Leu Ser Ser Val Asp Gln Pro Leu Lys

35 40 45

Ile Ala Arg Asp Lys Val Val Gly Lys Asp Tyr Leu Leu Cys Asp Tyr

50 55 60

Asn Arg Asp Gly Asp Ser Tyr Arg Ser Pro Trp Ser Asn Lys Tyr Asp

65 70 75 80

Pro Pro Leu Glu Asp Gly Ala Met Pro Ser Ala Arg Leu Arg Lys Leu

85 90 95

Glu Val Glu Ala Asn Asn Ala Phe Asp Gln Tyr Arg Asp Leu Tyr Phe

100 105 110

Glu Gly Gly Val Ser Ser Val Tyr Leu Trp Asp Leu Asp His Gly Phe

115 120 125

Ala Gly Val Ile Leu Ile Lys Lys Ala Gly Asp Gly Ser Lys Lys Ile

130 135 140

Lys Gly Cys Trp Asp Ser Ile His Val Val Glu Val Gln Glu Lys Ser

145 150 155 160

Ser Gly Arg Thr Ala His Tyr Lys Leu Thr Ser Thr Val Met Leu Trp

165 170 175

Leu Gln Thr Asn Lys Ser Gly Ser Gly Thr Met Asn Leu Gly Gly Ser

180 185 190

Leu Thr Arg Gln Met Glu Lys Asp Glu Thr Val Ser Asp Cys Ser Pro

195 200 205

His Ile Ala Asn Ile Gly Arg Leu Val Glu Asp Glu Asn Lys Ile Arg

210 215 220

Ser Thr Leu Asn Glu Ile Tyr Phe Gly Lys Thr Lys Asp Ile Val Asn

225 230 235 240

Gly Leu Arg Ser Ile Asp Ala Ile Pro Asp Asn Gln Lys Phe Lys Gln

245 250 255

Leu Gln Arg Glu Leu Ser Gln Val Leu Thr Gln Arg Gln Ile Tyr Ile

260 265 270

Gln Pro Asp Asn

275

Claims

1. The kit for detecting the anti-filamentous actin cap-forming protein beta-IgG antibody is characterized by consisting of an antigen protein anti-filamentous actin cap-forming protein beta, a solid phase carrier, a labeled antibody, an antigen diluent, a sample diluent, an antibody diluent, a substrate color developing agent, a washing solution, a stop solution, a standard substance, a positive quality control substance and a negative quality control substance.

2. The kit according to claim 1, wherein the sequence of the anti-filamentous actin cap-forming protein beta antigen protein is shown in SEQ ID: 1.

3. The kit of claim 1, wherein the labeled antibody is an enzyme-labeled, chemiluminescent, or biotin-labeled secondary antibody.

4. The kit according to claim 1, wherein said antigenic protein anti-filamentous actin cap-forming protein β carries a tag peptide comprising: a GST tag, a c-Myc tag, a His tag, a Flag tag, or a biotin tag.

5. The kit according to claim 1, wherein the anti-filamentous actin cap-forming protein beta antigen protein is purified by nickel column affinity chromatography, molecular sieve chromatography, gel filtration chromatography, ion exchange column chromatography, and hydrophobic column chromatography.

6. The kit of claim 1, wherein the standard substance and the positive quality control substance are recombinant human anti-tag peptide IgG or fragments thereof, or anti-filamentous actin cap-forming protein beta antibody extracted from serum is used as the positive quality control substance and the standard substance, and the negative quality control substance is serum of a healthy physical examiner.

7. The kit of claim 1, wherein the antigenic protein anti-filamentous actin cap-forming protein β is immobilized on a solid support, and the solid support is selected from nitrocellulose membranes, nylon membranes, ion exchange resins, polystyrene microwell plates, and magnetic beads.

8. The kit according to claim 7, wherein the anti-filamentous actin cap-forming protein β antigen is directly immobilized on the solid support by physical adsorption or covalent or chemical bonding.

9. The kit according to claim 1, characterized in that the chromogenic solution is TMB, hydrogen peroxide, luminol, acridinium ester; the antigen diluent is 1x PBS pH7.4 containing 163mM NaCl and 1% TritonX-100; the sample dilution buffer was 0.01M PBS ph7.4 containing 10% BSA; the antibody diluent is 0.01M PBS containing 1M D-glucose, 2% glycerol and 0.35% Tween 20; the washing solution is as follows: 1 XPBS pH7.4 containing 163mM NaCl, 10% glycerol, 1% TritonX-100; the stop solution is: 2M sulfuric acid.