CN114895024A - Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody - Google Patents

Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody Download PDF

Info

Publication number
CN114895024A
CN114895024A CN202210532620.4A CN202210532620A CN114895024A CN 114895024 A CN114895024 A CN 114895024A CN 202210532620 A CN202210532620 A CN 202210532620A CN 114895024 A CN114895024 A CN 114895024A
Authority
CN
China
Prior art keywords
serine
arginine
splicing factor
rich
antibody
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210532620.4A
Other languages
Chinese (zh)
Inventor
叶青
毛建华
周希
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhejiang University ZJU
Original Assignee
Zhejiang University ZJU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Zhejiang University ZJU filed Critical Zhejiang University ZJU
Priority to CN202210532620.4A priority Critical patent/CN114895024A/en
Publication of CN114895024A publication Critical patent/CN114895024A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306Solid-phase reaction mechanisms
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/948Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/34Genitourinary disorders
    • G01N2800/347Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention provides a kit for detecting an anti-serine/arginine-rich splicing factor 9-IgG antibody, which consists of an antigen protein serine/arginine-rich splicing factor 9, 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 substance, a positive quality control product and a negative quality control product. The autoantibodies against serine/arginine-rich splicing factor 9-IgG were detected by a specific kit. The invention identifies the autoantibody which is rich in the serine/arginine splicing factor 9 and aims at the target antigen 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 serine/arginine-rich splicing factor 9 and the serine/arginine-rich splicing factor 9-IgG autoantibody at home and abroad.

Description

Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody
The application is a divisional application of a kit for detecting the anti-serine/arginine-rich splicing factor 9-IgG antibody, which has the application number of 202110743511.2 and the application date of 2021, 7 and 1.
Technical Field
The invention belongs to the technical field of biomedicine, relates to a kit for detecting an anti-serine/arginine-rich splicing factor 9-IgG antibody, and is a kit for detecting the anti-serine/arginine-rich splicing factor 9-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 Serine/arginine splicing factor 9-rich protein (spring/arginine-rich splicing factor 9) in some patients with primary nephrotic syndrome. Serine/arginine-rich splicing factor 9 is a splice site selection in which splicing factors are involved in alternative splicing (Matsumoto E, Matsumoto Y, Inoue J, Yamamoto Y, Suzuki T.AMP-activated protein kinase regulation beta-protein synthesis by phosphorylation serine/area-rich splicing factor 9.Biochem Biophys Res Commun (2021)534: 347-52).
However, the autoantibodies rich in serine/arginine splicing factor 9 and serine/arginine splicing factor 9 are not reported in urinary system diseases, particularly nephrotic syndrome. In addition, the prior art does not relate to the application of the target-based serine/arginine-rich splicing factor 9 or the autoantibody thereof as a serological marker in autoimmune nephrotic syndrome. The study for identifying autoimmune nephrotic syndrome by detecting serum anti-serine/arginine splicing factor-rich 9-IgG antibodies is blank.
Disclosure of Invention
The invention aims to provide a kit for detecting an anti-serine/arginine-rich splicing factor 9-IgG antibody, which is a detection kit based on a target spot serine/arginine-rich splicing factor 9 and a corresponding autoantibody thereof. The kit can detect autoantibodies from tissues (kidney biopsy) or body fluids (particularly blood, plasma, serum) by reacting with antigen-antibody of the antigenic protein serine/arginine-rich splicing factor 9 (particularly shown according to the sequence identification number SEQ ID NO. 1).
The invention relates to a detection kit for detecting an autoantibody against serine/arginine-rich splicing factor 9, which comprises an antigen protein serine/arginine-rich splicing factor 9, a solid phase carrier, a labeled antibody, an antigen diluent, a sample diluent buffer, 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.
According to the invention, the antigen protein rich in serine/arginine splicing factor 9 can be expressed in bacteria such as Escherichia coli, yeast, and 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, said antigenic protein serine/arginine-rich splicing factor 9 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-serine/arginine splicing factor 9-rich 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 antigen protein rich in serine/arginine splicing factor 9 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 1M Dglucose (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 serine/arginine-rich splicing factor 9 of the 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 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 rich in serine/arginine splicing factor 9, and develops a set of kit suitable for detecting the antibody of the autoimmune nephrotic syndrome patient, which is rich in serine/arginine splicing factor 9-IgG, by taking the recombinant protein as the antigen protein in the kit. Comprises a detection kit for qualitatively or quantitatively analyzing and detecting the anti-serine/arginine-rich splicing factor 9-IgG antibody.
A kit for detecting the anti-serine/arginine-rich splicing factor 9-IgG antibody in serum is based on the principle that an antigen is adsorbed on a solid phase carrier to serve as a coating antigen, then a positive quality control product or a standard product or a serum sample to be detected is added for incubation, a labeled secondary antibody is added for reaction, a ternary complex of the coating antigen-serine/arginine-rich splicing factor 9-IgG antibody-labeled anti-human IgG antibody is formed, and finally a light signal is detected by using a light color development method, a chemiluminescence method and a fluorescence method, so that the aim of qualitatively or quantitatively analyzing the anti-serine/arginine-rich splicing factor 9-IgG antibody in human serum is fulfilled.
The sequence of the antigen protein rich in serine/arginine splicing factor 9 is shown in SEQ ID NO. 1:
MSGWADERGGEGDGRIYVGNLPTDVREKDLEDLFYKYGRIREIELKNRHGLVPFAFVRFEDPRDAEDAIYGRNGYDYGQCRLRVEFPRTYGGRGGWPRGGRNGPPTRRSDFRVLVSGLPPSGSWQDLKDHMREAGDVCYADVQKDGVGMVEYLRKEDMEYALRKLDDTKFRSHEGETSYIRVYPERSTSYGYSRSRSGSRGRDSPYQSRGSPHYFSPFRPY。
specific innovations can be summarized as follows:
(1) the invention identifies the autoantibody rich in the serine/arginine splicing factor 9 for the first time, and invents a detection kit aiming at the autoantibody.
(2) At present, no research related to the serine/arginine-rich splicing factor 9 and the autoantibody against the serine/arginine-rich splicing factor 9 exists in nephrotic syndrome at home and abroad, and no kit for detecting the level of the autoantibody against the serine/arginine-rich splicing factor 9 exists, so that the invention fills the blank of detection kits for the autoantibody against the serine/arginine-rich splicing factor 9 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-serine/arginine splicing factor 9-IgG antibody in human serum introduces a biotin-avidin amplification system, so that the detection sensitivity is greatly improved.
Drawings
FIG. 1: the serine/arginine splicing factor 9-rich protein on podocytes is a target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. 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 serine/arginine splicing factor 9-rich proteins as target antigens.
FIG. 2: SDS-PAGE identification picture of the recombinant protein rich in serine/arginine splicing factor 9.
FIG. 3: the solid-phase membrane immunoassay kit is used for detecting the serine/arginine splicing factor-rich 9-IgG antibody in the serum of the patient with autoimmune nephrotic syndrome.
FIG. 4: the detection principle of the chemiluminescence kit for detecting the antibody rich in the serine/arginine splicing factor 9-IgG is shown in the schematic diagram.
FIG. 5: schematic diagram of coated carboxyl magnetic particles of antigen protein rich in serine/arginine splicing factor 9.
FIG. 6: the detection condition of the serine/arginine splicing factor-rich 9-IgG antibody in various nephropathy patients is shown in the specification, wherein the content of PNS: autoimmune nephrotic syndrome, HSP: allergic purpura, HSPN: purpuric nephritis, IgAN: IgA nephropathy, NC: a healthy child.
FIG. 7: the ROC curve evaluates the application value of the autoantibody for resisting the serine/arginine-rich splicing factor 9 as a serological marker for differential diagnosis of autoimmune nephrotic syndrome.
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 serine/arginine-rich splicing factor 9 on podocytes is a target antigen for autoantibodies in patients with autoimmune nephrotic syndrome. According to the invention, through a large number of clinical and molecular mechanism researches at the early stage, the serum IgG level of a nephrotic syndrome patient is found to be high for the first time, and the serine/arginine splicing factor 9 rich in podocytes is proved to be one of target antigens aiming at an autoantibody of an autoimmune nephrotic syndrome patient. It would therefore be advantageous to detect the presence and quantitative levels of antibodies against serine/arginine splicing factor-rich 9-IgG 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 serine/arginine splicing factor 9-rich protein, as shown in FIG. 1C.
Example 2 serine/arginine-rich splicing factor 9 antigen expression and purification
The gene rich in serine/arginine splicing factor 9 is coded by a gene engineering method as a template 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 and the like, and finally the molecular weight of the recombinant protein rich in serine/arginine splicing factor 9 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 antigen-rich serine/arginine splicing factor 9 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 ℃), enzyme-labeled secondary antibody optimal dilution (four dilutions of 1:100, 1:500, 1:1000, 1: 1500), and each factor was determined at 2 levels in duplicate for standard positive and negative sera. 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 solid-phase Membrane immunoassay kit for 9-IgG antibody rich in serine/arginine splicing factor
4.1 composition of solid-phase membrane immunoassay kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody:
TABLE 1
Component name Description of the invention
Antigens Recombinant protein rich in serine/arginineSplicing factor 9
Solid phase carrier Sataurus CN140 nitrocellulose membrane
Positive quality control (Standard) Human anti-His tag IgG
Negative quality control product Serum 10 of healthy physical examiner
Labeled secondary antibody Biotin-labeled anti-human IgG antibody
Substrate Alkaline phosphatase-streptavidin
Color developing liquid BCIP color developing solution
4.2 detection procedure of the solid-phase membrane immunoassay kit for detecting anti-serine/arginine-rich splicing factor 9-IgG is as follows:
(1) coating and sealing: placing 8 μ l of serine/arginine splicing factor-rich 9 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 serine/arginine-rich splicing factor 9-IgG in the serum by placing the membrane strip on a developing instrument for scanning, wherein the developing instrument is provided with analysis software, the concentration of a reference standard is used as a vertical coordinate, and a gray value read by the instrument is used as a horizontal coordinate.
EXAMPLE 5 chemiluminescence assay kit for anti-serine/arginine-rich splicing factor 9-IgG antibody
5.1 anti serine/arginine splicing factor-rich 9-IgG antibody chemiluminescence detection kit, including the following components:
(1) acridinium ester labeled anti-human IgG;
(2) a carboxyl magnetic bead coupled to a serine/arginine-rich splicing factor 9 antigen;
(3) a chemiluminescent pre-excitation liquid A and a chemiluminescent excitation liquid B;
(4) anti serine/arginine splicing factor-rich 9-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 solution, especially Tris-HCl solution (25 mmol/L) with pH7.2 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 serine/arginine-rich splicing factor 9 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 Na 2 CO 3 -NaHCO 3 A 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-serine/arginine-rich splicing factor 9-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-serine/arginine splicing factor 9-IgG antibody in the sample was 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 antibodies against serine/arginine-rich splicing factor 9 in various renal disease patients. The kit is used for detecting the level of the serine/arginine splicing factor-rich 9-IgG antibody in the serum of various nephrotic patients, 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 results showed that autoantibodies rich in serine/arginine splicing factor 9 were present in some patients with autoimmune nephrotic syndrome (FIG. 6). The application value of the antibody for resisting the serine/arginine-rich splicing factor 9-IgG as a serological marker for differential diagnosis of autoimmune nephrotic syndrome is evaluated by the ROC curve. When autoimmune nephrotic syndrome was diagnosed with an autoantibody against serine/arginine-rich splicing factor 9 greater than 21.9 as a diagnostic cutoff, the sensitivity was 69.3% and the specificity was 60.9% (fig. 7). The result shows that the anti-serine/arginine splicing factor-rich 9-IgG antibody is a good serological marker for identifying and diagnosing patients with autoimmune nephrotic syndrome.
SEQUENCE LISTING
<110> Zhejiang university
<120> kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody
<130> 2022.5.10
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 221
<212> PRT
<213> Artificial sequence (Unknow)
<400> 1
Met Ser Gly Trp Ala Asp Glu Arg Gly Gly Glu Gly Asp Gly Arg Ile
1 5 10 15
Tyr Val Gly Asn Leu Pro Thr Asp Val Arg Glu Lys Asp Leu Glu Asp
20 25 30
Leu Phe Tyr Lys Tyr Gly Arg Ile Arg Glu Ile Glu Leu Lys Asn Arg
35 40 45
His Gly Leu Val Pro Phe Ala Phe Val Arg Phe Glu Asp Pro Arg Asp
50 55 60
Ala Glu Asp Ala Ile Tyr Gly Arg Asn Gly Tyr Asp Tyr Gly Gln Cys
65 70 75 80
Arg Leu Arg Val Glu Phe Pro Arg Thr Tyr Gly Gly Arg Gly Gly Trp
85 90 95
Pro Arg Gly Gly Arg Asn Gly Pro Pro Thr Arg Arg Ser Asp Phe Arg
100 105 110
Val Leu Val Ser Gly Leu Pro Pro Ser Gly Ser Trp Gln Asp Leu Lys
115 120 125
Asp His Met Arg Glu Ala Gly Asp Val Cys Tyr Ala Asp Val Gln Lys
130 135 140
Asp Gly Val Gly Met Val Glu Tyr Leu Arg Lys Glu Asp Met Glu Tyr
145 150 155 160
Ala Leu Arg Lys Leu Asp Asp Thr Lys Phe Arg Ser His Glu Gly Glu
165 170 175
Thr Ser Tyr Ile Arg Val Tyr Pro Glu Arg Ser Thr Ser Tyr Gly Tyr
180 185 190
Ser Arg Ser Arg Ser Gly Ser Arg Gly Arg Asp Ser Pro Tyr Gln Ser
195 200 205
Arg Gly Ser Pro His Tyr Phe Ser Pro Phe Arg Pro Tyr
210 215 220

Claims (9)

1. Use of a Serine/arginine-rich splicing factor 9(Serine/arginine-rich splicing factor 9) polypeptide or an antibody-binding fragment thereof, capable of forming any antigen-antibody complex in contact with a biological sample obtained from said patient, for the preparation of a nephrotic syndrome detection reagent or kit; wherein the antigen-antibody complex comprises an anti-Serine/arginine-rich splicing factor 9-IgG antibody (anti-Serine/arginine-rich splicing factor 9-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 according to claim 1, wherein the Serine/arginine-rich splicing factor 9 (spring/arginine-rich splicing factor 9) antibody binding fragment has the sequence shown 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 a Serine/arginine-rich splicing factor 9 (Serine/allergic-rich splicing factor 9) polypeptide or antibody binding fragment capable of forming any antigen-antibody complex in contact with a biological sample obtained from said 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-Serine/arginine-rich splicing factor 9-IgG antibody (anti-Serine/arginine-rich splicing factor 9-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: a Serine/arginine-rich splicing factor 9(Serine/arginine-rich splicing factor 9) polypeptide or an antibody-binding fragment thereof, which is capable of forming any antigen-antibody complex in contact with a biological sample obtained from said patient; wherein the antigen-antibody complex comprises an anti-Serine/arginine-rich splicing factor 9-IgG antibody (anti-Serine/arginine-rich splicing factor 9-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: a Serine/arginine-rich splicing factor 9(Serine/arginine-rich splicing factor 9) polypeptide or an antibody-binding fragment thereof, which is capable of forming any antigen-antibody complex in contact with a biological sample obtained from said patient; wherein the antigen-antibody complex comprises an anti-Serine/arginine-rich splicing factor 9-IgG antibody (anti-Serine/arginine-rich splicing factor 9-IgG antibody) complex.
CN202210532620.4A 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody Pending CN114895024A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210532620.4A CN114895024A (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110743511.2A CN113447648B (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody
CN202210532620.4A CN114895024A (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202110743511.2A Division CN113447648B (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody

Publications (1)

Publication Number Publication Date
CN114895024A true CN114895024A (en) 2022-08-12

Family

ID=77814652

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202110743511.2A Active CN113447648B (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody
CN202210532620.4A Pending CN114895024A (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202110743511.2A Active CN113447648B (en) 2021-07-01 2021-07-01 Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody

Country Status (1)

Country Link
CN (2) CN113447648B (en)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002081683A2 (en) * 2001-04-05 2002-10-17 Nextgen Sciences Ltd. Protein analysis by means of immobilized arrays of antigens or antibodies
WO2012074904A2 (en) * 2010-11-29 2012-06-07 Precision Therapeutics, Inc. Methods and systems for evaluating the sensitivity or resistance of tumor specimens to chemotherapeutic agents
EP3825416A3 (en) * 2014-05-22 2021-09-15 The Scripps Research Institute Gene expression profiles associated with sub-clinical kidney transplant rejection
CA2981455A1 (en) * 2015-03-30 2016-10-06 Hycor Biomedical Llc Automated immunoanalyzer system for performing diagnostic assays for autoimmune and infectious diseases
CN107561288B (en) * 2017-08-30 2020-06-30 上海市肺科医院 Lung cancer diagnostic kit for detecting blood autoantibody and application thereof
BR112021016121A2 (en) * 2019-02-15 2022-01-04 Atreca Inc Isolated antibody that binds to tumor tissue, antibody that binds to tumor tissue, method of inducing an immune response, method of treating a cancer patient with a tumor, method of identifying a patient that has a tumor, expression vector, cell host, method of producing an antibody, method of identifying an antibody with antitumor activity, use of an antibody and polypeptide

Also Published As

Publication number Publication date
CN113447648A (en) 2021-09-28
CN113447648B (en) 2022-04-19

Similar Documents

Publication Publication Date Title
CN113447659B (en) Kit for detecting anti-proteasome subunit alpha 1-IgG antibody
CN113447658B (en) Kit for detecting anti-peroxiredoxin-1-IgG antibody
CN110850100A (en) Detection of anti-Annexin A in serum2Kit for IgG antibodies
US20230333097A1 (en) KIT FOR DETECTING ANTI-VINCULIN-IMMUNOGLOBULIN G (IgG) ANTIBODY
JP2023017986A (en) Direct immunoassay measurement of autoantibodies
CN113588942B (en) Kit for detecting antigen myosin1-IgG antibody
CN113447656B (en) Kit for detecting anti-filamentous actin cap-forming protein beta-IgG antibody
WO2024001044A1 (en) Biomarker combination related to lung cancer, kit containing same, and use thereof
CN113447648B (en) Kit for detecting anti-serine/arginine-rich splicing factor 9-IgG antibody
CN113466451B (en) Kit for detecting anti-Prelamin A/C-IgG antibody
CN114966045A (en) Application of reagent for detecting anti-myosin light chain1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury
CN114895023A (en) Application of reagent for detecting anti-Talin-1-IgG autoantibody in preparation of kit for detecting vascular endothelial injury
CN114994308A (en) Kit for detecting Desmoglein1-IgG antibody
CN114924081A (en) Application of neuroblast differentiation related protein-IgG in preparation of vascular endothelial injury kit
CN114720700A (en) Application of reagent for detecting anti-cytoskeleton-associated protein4-IgG autoantibody in preparation of kit for detecting vascular endothelial injury
CN114910647A (en) Application of filamin-A-IgG antibody in preparation of kit for detecting vascular endothelial injury
CN114994330A (en) Kit for detecting anti-HSP 90-beta-IgG autoantibody and application thereof
CN114910650A (en) Application of reagent for detecting anti-moesin-IgG antibody in preparation of kit for detecting vascular endothelial injury
CN114910649A (en) Application of reagent for detecting anti-alpha-enolase-IgG antibody in preparation of kit for detecting vascular endothelial injury
CN113447657B (en) Detection kit for detecting anti-aconitate hydratase-IgG antibody
CN113447650B (en) Detection kit for anti-peptidyl prolyl cis-trans isomerase D-IgG antibody
CN108948173B (en) Citrulline modified peptide and application thereof
CN113563479B (en) Echinococcosis diagnostic kit
CN114910643B (en) Method and reagent for identifying antibody combined with mutant antigen
CN118005733A (en) Peptide aptamer for detecting inflammation, test strip, kit and application of peptide aptamer

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination