CN116199763A - ATRX antigen epitope peptide and application thereof in detecting brain glioma - Google Patents

ATRX antigen epitope peptide and application thereof in detecting brain glioma Download PDF

Info

Publication number
CN116199763A
CN116199763A CN202310402167.XA CN202310402167A CN116199763A CN 116199763 A CN116199763 A CN 116199763A CN 202310402167 A CN202310402167 A CN 202310402167A CN 116199763 A CN116199763 A CN 116199763A
Authority
CN
China
Prior art keywords
atrx
epitope peptide
protein
antigen
detecting
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
CN202310402167.XA
Other languages
Chinese (zh)
Inventor
卜令斌
张瑞
曹金良
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.)
Koran Biomedical Technology Shanghai Co ltd
Original Assignee
Koran Biomedical Technology Shanghai Co ltd
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 Koran Biomedical Technology Shanghai Co ltd filed Critical Koran Biomedical Technology Shanghai Co ltd
Priority to CN202310402167.XA priority Critical patent/CN116199763A/en
Publication of CN116199763A publication Critical patent/CN116199763A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Organic Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses an ATRX epitope peptide and application thereof in detecting glioma. The amino acid sequence of the ATRX epitope peptide is shown in a sequence table SEQ ID NO:1 or SEQ ID NO: 2. The ATRX epitope peptide has good antigenicity, and the antigen prepared by the ATRX epitope peptide can be used for immunizing animals to generate monoclonal antibodies and polyclonal antibodies with high specificity. The human ATRX in-vitro diagnostic kit can effectively detect the ATRX expression level in brain tissue samples, and can be used for evaluating the risk of cerebral glioma.

Description

ATRX antigen epitope peptide and application thereof in detecting brain glioma
Technical Field
The invention belongs to the technical field of immunology, and particularly relates to an ATRX epitope peptide and application thereof in detecting glioma.
Background
Gliomas are a relatively common primary tumor of the central nervous system, and the incidence rate in China is about seven ten thousandths. Different histological subtypes of gliomas are as follows: astrocytomas account for 70% of all cases, with 9% of oligodendrogliomas, including classical oligodendrogliomas as well as mixed oligooptic neurocytomas, and 6% of ependymomas.
ATRX gene is a gene located at xq21.1 that regulates chromosomal remodeling, nucleosome assembly, and maintenance of telomere length, encoding a280 kDa nuclear protein, mutations of which were found early in X-linked thalassemia a and mental retardation, and ATRX mutations have been found in recent studies in at least 15 types of cancer, including neuroblastoma, osteosarcoma, and pancreatic neuroendocrine tumor. ATRX protein plays an important epigenetic role, it deposits histones on heterochromatin and telomere DNA, which also maintains a close relationship with telomere length.
ATRX protein binds strongly to promyelocytic nucleosomes and when ATRX interacts with DAXX, the complex deposits histone variant H3.3 around the center of heterochromatin repeats including telomere and ribosomal DNA repeats as a chaperone complex. Functional mutations in this gene, which constitute defects with their sister chromatids, correlate with aberrant DNA methylation and telomerase maintenance of independent telomeres, can lead to replacement of prolonged telomeres. ATRX mutations and reduced ATRX nuclear expression were observed in IDH mutant gliomas, IDH mutant GBM and grade II and grade III astrocytomas of pediatric advanced gliomas. 31% of primary GBM patients have ATRX mutations, accompanied by TP53 mutations and point mutations of the gene encoding histone H3.3 variant H3F 3A.
The ATRX gene plays a critical role in nervous system development. Berube NG et al found that ATRX gene mutations are closely related to the aggressiveness of central nervous system tumors. NB as a tumor of peripheral neuroectodermal origin, it is also necessary to determine whether NB patients have ATRX mutations and whether NB with ATRX gene mutations has longer telomeres. A more intensive study is required for the mechanism of action of ATRX protein expression deficiency in the progression and invasion progression of neuroblastoma. The Wadat et al medical records report that ATRX gene mutations affect two important regions of functionality: ADD domain and helicase domain.
ATRX has repeatedly proven potential as a biomarker, and the most ideal method for detecting ATRX levels in serum is immunodetection, which can be checked by routine blood drawing using ATRX antibodies and advanced enzyme immunoassay, chemiluminescence techniques. However, because ATRX protein has a large molecular weight, it is difficult to prepare an antibody by using its whole antigen, and the prepared antibody has poor specificity, so searching for a suitable ATRX epitope peptide with immunogenicity and preparing a specific ATRX antibody become an important point of research.
Disclosure of Invention
The invention aims to provide an ATRX epitope peptide and application thereof in detecting glioma.
An ATRX antigen epitope peptide has an amino acid sequence shown in a sequence table SEQ ID NO:1 or SEQ ID NO: 2.
An antigen, wherein the antigen is formed by coupling the ATRX epitope peptide and carrier protein.
The invention also provides an ATRX monoclonal antibody or polyclonal antibody prepared from the antigen.
Application of the ATRX antibody in preparation of a reagent for detecting brain glioma.
The ATRX antibody is used for detecting the expression level of ATRX protein in vivo; the expression level of ATRX protein is reduced in glioma patients relative to normal humans.
A kit for detecting brain glioma, comprising the ATRX monoclonal antibody or polyclonal antibody.
Preferably, it is used for quantitative detection of human ATRX protein in serum.
The invention has the beneficial effects that: the ATRX epitope peptide has good antigenicity, and the antigen prepared by the ATRX epitope peptide can be used for immunizing animals to generate monoclonal antibodies and polyclonal antibodies with high specificity. The human ATRX in-vitro diagnostic kit can effectively detect the ATRX expression level in brain tissue samples, and can be used for evaluating the risk of cerebral glioma.
Drawings
FIG. 1 is a western blot detection diagram of monoclonal and polyclonal antibodies;
in the figure, 1 is a monoclonal antibody, and 2 is a polyclonal antibody.
FIG. 2 shows the concentration of ATRX protein in brain glioma tissue samples and normal brain tissue samples.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
The specific techniques or conditions not identified in the examples are all conducted as described in the literature or as conventional in the art.
Example 1 epitope peptide screening
And (3) carrying out sequence analysis according to the human ATRX protein sequence published by NCBI by adopting antheprot software, wherein the analysis contents comprise transmembrane differentiation, signal peptide analysis, modification site analysis and full sequence analysis, finding out possible antigen epitopes from the obtained antigen epitopes, and adding cysteine to the sequence end without cysteine in the antigen epitopes. And finally screening to obtain the ATRX epitope peptide with the amino acid sequence shown in a sequence table SEQ ID NO:1 or SEQ ID NO: 2.
The epitope peptide is synthesized by adopting an artificial synthesis method and is synthesized by the Kirschner Biotech Co., ltd.
Coupling of epitope peptide to carrier protein:
1) 20mg of SMCC was dissolved in 2ml DMF,0.8ml KLH and added to a 25ml round bottom flask, supplemented with 1 XPBS to give a final protein concentration of 15mg/ml;
2) Slowly dripping the dissolved SMCC solution into 120mg KLH protein system, and stirring at room temperature to react for 1h;
3) Dialysis was performed with 1L of 1 XPBS solution at 4℃for 6h to remove free SMCC;
4) Pouring the dialyzed KLH protein into a 50ml centrifuge tube, determining the volume of the dialyzed KLH protein by the scale of the centrifuge tube, calculating the concentration of the dialyzed KLH protein according to the amount of the KLH protein added before the reaction, and transferring 2.5mg KLH-SMCC solution into a 5ml centrifuge tube according to the concentration of the dialyzed KLH protein; the amount of KLH protein added is 120mg, and the volume of KLH protein after dialysis is 20ml;
5) 3.0mg of epitope peptide was dissolved in 0.6ml of 1 XPBS solution;
6) Detection of thiol groups in polypeptides with Ellman reagent: 100 mu l of Ellman reagent stock solution and 10 mu l of antigen epitope peptide solution are added into a 96-well plate, ultraviolet absorption value is measured at lambda=412 nm by a Nano spectrophotometer, and if OD value is more than 0.15, the next step is performed; the OD value is less than 0.05, and the epitope peptide is added until the requirement is met; OD value <0.03 indicates that the crosslinking rate of the epitope peptide and KLH protein is over 80 percent; and if OD value is more than 0.03, supplementing the KLH protein activated by SMCC to continue crosslinking; if the Ellman reagent shows yellow color, the antigen epitope peptide is incompletely coupled with the KLH protein; if the Ellman reagent does not appear yellow it indicates that the epitope peptide has been coupled to the KLH protein in its entirety.
Example 2 polyclonal antibody preparation
Animals were immunized with the antigen prepared in example 1 by selecting healthy male New Zealand white rabbits at an age of 3 months and a weight of 2.5.+ -. 0.1 kg.
Blood was taken through the ear vein as a negative control prior to the first immunization. Antigen was treated with PBS (137mM NaCl,2.7mM KCl,10mM Na) 2 HPO 4 ，2mM KH 2 PO 4 pH 7.4) was diluted to 1mg/mL, and stored at-20℃after packaging. Taking 500 μl of 1mg/mL antigen (i.e. 0.5mg, adding 300 μl PBS for dilution again, then adding equal volume of Freund's complete adjuvant (first immunization) or Freund's incomplete adjuvant (2-4 th immunization), performing multi-point injection subcutaneously on four limbs, armpit and back of rabbit, performing second immunization after the first immunization for three weeks, and boosting every two weeks (4 total immunizations) at intervals, measuring antibody titer by taking blood from ear vein after 7d of 3 rd immunization, and obtaining the final product after 11d of immunizationBleeding and collecting a blood sample. Standing blood sample at 4deg.C overnight or 37 deg.C for 3 hr, centrifuging at 4deg.C at 6000rpm for 10min, collecting serum, packaging, and preserving at-80deg.C.
Concentrating protein samples by an ammonium sulfate precipitation method: 20mL of serum was diluted to 100mL with TBS buffer, and then saturated ammonium sulfate solution was added thereto while stirring until the concentration of the ammonium sulfate solution reached 55%, and stirred overnight at 4℃using a magnetic stirrer. The next day the solution containing a large amount of sediment was centrifuged and the supernatant was discarded and the sediment was retained. 10mL of PBS buffer solution containing sodium azide is added to dissolve the precipitate, and dialysis treatment is performed by using a dialysis bag to remove ammonium sulfate. After dialysis, the solution in the dialysis bag was centrifuged and the supernatant was collected.
Purifying the anti-polypeptide antibody by affinity chromatography: the protein solution after dialysis and centrifugation is added into a chromatographic column (protein A FF) at a speed of 0.5mL/min, and the column is required to be continuously loaded twice in order to ensure the sufficient combination of the antibody protein and the filler. Washing the chromatographic column with TBS until A280nm of the eluent is less than 0.008, eluting with an elution buffer (50 mM glycine, pH 2.7) at the same speed, collecting the eluent with a centrifuge tube which is added with a neutralization buffer (1M Tris-HCl, pH8.0,1.5M NaCl,1mM EDTA) in advance, checking the pH of the solution with a pH test paper after mixing uniformly, adjusting the pH to about pH7.4 with the neutralization buffer if the pH is less than 7.4 so as to prevent antibody denaturation, detecting the concentration of the antibody, adding an equal volume of glycerol, and preserving at-20 ℃ to obtain the purified polyclonal antibody.
EXAMPLE 3 monoclonal antibody preparation
10 healthy female BALB/c mice of 6-8 weeks old are selected, the antigen prepared in example 1 is first immunized and dissolved in normal saline and mixed with equal volume of Freund's complete adjuvant, after full emulsification, the back part of the mice is injected subcutaneously at 4 points, and each mouse is injected with 40ug of antigen. 14d: boosting, namely dissolving an antigen in normal saline, mixing with an equal volume of Freund's incomplete adjuvant, fully emulsifying, and injecting subcutaneously at 4 points on the back of a mouse, wherein each mouse is injected with 40ug of antigen; 21d: the inner canthus vein of the mice is used for blood collection, serum is separated, and the indirect ELISA method is used for detecting the titer of the antiserum, and the 28d: boosting, namely dissolving an antigen in normal saline, mixing with an equal volume of Freund's incomplete adjuvant, fully emulsifying, and injecting subcutaneously at 4 points on the back of a mouse, wherein each mouse is injected with 40ug of antigen; collecting blood from inner canthus vein of mice at 35d, separating serum, detecting the titer of the antiserum by indirect ELISA method, and injecting 100 mug antigen into abdominal cavity of the mice with highest titer of the antiserum at 42 d; day 45: spleen cells were harvested on a super clean bench and prepared for fusion.
Selecting a mouse with high anti-serum antibody titer, performing boosting immunization for the last 1 time, taking spleen cells after 3 days, and fusing the spleen cells with SP2/0 myeloma cells to prepare a hybridoma cell strain. Screening out positive hybridoma cell strains by adopting an indirect ELISA method, identifying the positive hybridoma cell strains by using a western blot method, subcloning the positive hybridoma cell strains by using a limiting dilution method, detecting the titer of antisera by using the indirect ELISA method, and performing expanded culture on the cells with positive detection to finally obtain the hybridoma cell strains capable of stably secreting monoclonal antibodies.
Selecting 3 healthy BALB/c mice, injecting 0.5ml pristane into each mouse abdominal cavity, injecting hybridoma cells into the abdominal cavity after 14d, changing the liquid of the hybridoma cells in the logarithmic phase by using fresh culture medium 1d before injection, mixing the hybridoma cells to prepare cell suspension on the injection day, adding the cell suspension into a 15ml centrifuge tube after counting, centrifuging at room temperature of 200 Xg for 5min, suspending the cells with PBS after removing the supernatant to make the cell concentration be 5X 106/ml, and injecting 1ml into each mouse; the health condition and ascites production condition of the mice are observed, and after 12d, the ascites is collected and the titer is determined. The collected ascites was centrifuged at 1500 Xg for 10 minutes, and the supernatant was purified by protein A affinity chromatography.
Example 4 determination of polyclonal and monoclonal antibody detection Capacity
In the three atmosphere (CO) 2 /N 2 /O 2 ) In a cell incubator, human endothelial HUVEC cells grown in log phase are inoculated into a 6-well plate and subjected to 5% CO at 37 DEG C 2 And culturing under saturated humidity condition; after the cells are attached, the cells are transferred to a three-gas incubator at 37 ℃ and 5 ℃ CO 2 ，2％O 2 And culturing at 37deg.C under saturated humidity for 24 hr, and controlling at 5% CO 2 And saturation ofCulturing for 24h under the humidity condition, and lysing the cells.
Cell lysis and protein electrophoresis: the cultured cells were washed with ice-chilled PBS, collected and then lysed in RIPA buffer containing a mixture of protease inhibitors, the lysis process being performed on ice for 30min. The supernatant was collected after centrifugation at 12,000r/min for 10min at 4℃and then quantified using the conventional BCA method. The quantitated samples were mixed with 5 Xloading buffer and denatured in a water bath at 100℃for 5min. Protein electrophoresis was performed in SDS-polyacrylamide gel and buffer system, and samples were loaded at 50. Mu.g/well and subjected to constant pressure electrophoresis at 80/120V.
After 60ug of human endothelial HUVEC cell total protein was separated by 6% SDS-PAGE and transferred onto nitrocellulose membrane, after the nitrocellulose membrane was blocked by a TBST solution containing 10% skimmed milk powder for 2h at room temperature, nitrocellulose membrane was immersed in a TBST solution containing 1. Mu.g/ml polyclonal and monoclonal antibodies overnight at 4℃and was removed the next day, washed 3 times by shaking the TBST solution, nitrocellulose membrane was immersed in a TBST solution containing HRP-labeled goat anti-mouse IgG (dilution: 1:5000), washed 3 times by shaking the TBST solution (240 rpm, 15min each time) after a water bath of 1h at 27℃for 1 min, washed 1 time by shaking the TBS solution (240 rpm,5 min) and protein bands were observed in a chemiluminescent imaging system (Syngene, cambridge, UK) using an enhanced chemiluminescent detection kit (Thermo Fisher Scientific, walsh, USA).
As a result of the measurement, as shown in FIG. 1, a single band appeared around 280kDa, which is consistent with the theoretical molecular weight of ATRX protein, and both monoclonal and polyclonal antibodies recognized ATRX protein.
Example 5
38 samples of brain glioma tissue are adopted, 35 samples of normal brain tissue are taken from the medical treatment of people in Beijing university at 1 month-2022 1 month in 2015, and are proved to be pathological specimens of brain glioma through surgical pathology. The world health organization classifies the central nervous system tumor into I to IV grades according to histology and molecular characteristics, wherein the grade I of 38 brain glioma tissue samples selected by the invention has 10, the grade II has 8, the grade III has 10 and the grade IV has 10; patients participating in the trial of the present invention, all signed informed consent and approved by the ethics committee of the hospital.
And extracting total protein of the sample by adopting a conventional protein extraction method, and detecting the concentration of ATRX protein by using a self-prepared monoclonal antibody and polyclonal antibody detection kit.
The preparation and operation of the kit are as follows:
coating buffer solution preparation: CB (carbonate buffer), na, at 0.050M, pH9.6 2 CO 3 16.0g、NaHCO 3 29.0g, distilled water was dissolved to 1000ml.
Sample/wash buffer formulation: 10 XPBS-Tween 20, na at pH7.2 2 HPO 4 ·12H 2 O 58g，KH 2 PO 4 4g,NaCl 100g,KCl 4g,Tween 20 20ml, distilled water was dissolved to 1000ml.
Preparing enzyme marker diluent: 10 XPBS-Tween 20 10ml, FCS (calf serum) 20ml, enzyme stabilizer 1g, biological preservative 1ml, distilled water to 1000ml.
And (3) preparing a color developing agent A: 35.5g of citric acid, 10g,Tween 20 10ml g of carbamide peroxide and distilled water are dissolved to 1000ml.
And (3) preparing a color-developing agent B: 120g of citric acid, 1g of EDTA-2Na, 2g of TMB.2HCl and distilled water to 1000ml.
Preparing a stop solution: 2M H 2 SO 4 22.2ml of concentrated sulfuric acid and 177.3ml of distilled water, and the concentrated sulfuric acid is slowly dripped into the distilled water during preparation, and is uniformly shaken while being added.
Preparation of pre-coated plates: the ATRX monoclonal antibody is dissolved in 0.05M carbonate buffer solution with pH=9.6 to prepare a pre-coating liquid, 100 mu l of the pre-coating liquid is added to each hole of an ELISA plate according to 0.1 mu g/hole, the mixture is placed at 4 ℃ for 24 hours, the mixture is taken out, the coating liquid is thrown off, washed by a sample/washing buffer solution, blocked by 1 (w/v)% BSA-0.05M ethanolamine for 16 hours, and the mixture is placed in an aluminum platinum bag for vacuum sealing after being dried overnight, and is stored at 4 ℃.
The dilution ratio of the binding antibody (ATRX polyclonal antibody) and the enzyme conjugate (horseradish peroxidase-labeled goat anti-rabbit IgG antibody) was determined by a square titration experiment, and the horseradish peroxidase-labeled goat anti-rabbit IgG antibody was diluted with an enzyme-labeled diluent.
Composition of the kit:
pre-coating plate: 48/96 well
ATRX calibrator: 6: 6X 0.5ml (concentration 0.0ng/ml, 8.0ng/ml, 15.0ng/ml, 50.0ng/ml, 100.0ng/ml, 200.0ng/ml, respectively)
ATRX binding antibody: 1X 2.5 ml/1X 5ml (diluted 1:5000)
Enzyme-linked product: 2X 5 ml/2X 5ml (diluted 1:5000)
ATRX quality control: 1X 0.5mL (serum with ATRX concentration of 60.0 ng/mL)
Concentrated wash (25 XPBS-Tween 20): 1X 20ml
Color developer A: 1X 5.0ml
Color developer B: 1X 5.0ml
Stop solution: 1X 5.0ml.
The operation steps of the kit are as follows:
50 μl/well of total protein sample to be detected, calibrator and quality control material are respectively added into each well of the pre-coated plate, and the wells are double-well, incubated at 37deg.C for 30min, washed 5 times with 300ul of 1 Xwashing buffer, and patted dry. 100. Mu.l/well of ATRX-conjugated antibody was added to each well, incubated at 37℃for 30min, washed 5 times with 300ul of 1 Xwashing buffer per well, and patted dry. Then, 100. Mu.l/well of the ELISA was added to each well, incubated at 37℃for 30min, and each well was washed 5 times with 200-300ul of 1 Xwashing buffer, and the wells were dried by pipetting. Color-developing agent A, B solution was added, 50. Mu.l each well was mixed and incubated at 37℃for 15 minutes. The reaction was stopped by adding 50. Mu.l of the stop solution per well, and the absorbance was measured by an ELISA (model RT-6000, lei Du) using a dual wavelength (450 nm, 620 nm).
And drawing a standard curve according to the logarithmic value of the concentration of the calibrator and the corresponding absorbance, and calculating the ATRX concentration result in the detected specimen according to the standard curve, wherein the measurement result is shown in figure 2, and the ATRX concentration in the glioma patient is obviously lower than that of normal human brain tissue.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (7)

1. An ATRX antigen epitope peptide is characterized in that the amino acid sequence is shown in a sequence table SEQ ID NO:1 or SEQ ID NO: 2.
2. An antigen, wherein the antigen is formed by coupling the ATRX epitope peptide of claim 1 with a carrier protein.
3. An ATRX monoclonal or polyclonal antibody prepared from the antigen of claim 2.
4. Use of the ATRX antibody of claim 3 for the preparation of a reagent for detecting glioma.
5. The use of ATRX antibody according to claim 4 for the preparation of a reagent for detecting glioma, wherein the ATRX antibody is used for detecting the expression level of ATRX protein in vivo; the expression level of ATRX protein is reduced in glioma patients relative to normal humans.
6. A kit for detecting brain glioma, comprising the ATRX monoclonal or polyclonal antibody of claim 2.
7. The kit for detecting glioma according to claim 6, which is used for quantitatively detecting human ATRX protein in serum.
CN202310402167.XA 2023-04-14 2023-04-14 ATRX antigen epitope peptide and application thereof in detecting brain glioma Pending CN116199763A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310402167.XA CN116199763A (en) 2023-04-14 2023-04-14 ATRX antigen epitope peptide and application thereof in detecting brain glioma

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310402167.XA CN116199763A (en) 2023-04-14 2023-04-14 ATRX antigen epitope peptide and application thereof in detecting brain glioma

Publications (1)

Publication Number Publication Date
CN116199763A true CN116199763A (en) 2023-06-02

Family

ID=86507920

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310402167.XA Pending CN116199763A (en) 2023-04-14 2023-04-14 ATRX antigen epitope peptide and application thereof in detecting brain glioma

Country Status (1)

Country Link
CN (1) CN116199763A (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040141977A1 (en) * 2002-10-31 2004-07-22 Paz Einat ATRX and uses thereof
CN103765212A (en) * 2011-06-27 2014-04-30 杰克逊实验室 Methods and compositions for treatment of cancer and autoimmune disease
CN112940133A (en) * 2021-04-09 2021-06-11 福州迈新生物技术开发有限公司 Monoclonal antibody of anti-ATRX protein, cell strain, preparation method and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040141977A1 (en) * 2002-10-31 2004-07-22 Paz Einat ATRX and uses thereof
CN103765212A (en) * 2011-06-27 2014-04-30 杰克逊实验室 Methods and compositions for treatment of cancer and autoimmune disease
CN112940133A (en) * 2021-04-09 2021-06-11 福州迈新生物技术开发有限公司 Monoclonal antibody of anti-ATRX protein, cell strain, preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SATOSHI OGASAWARA: "Establishment of Anti-Human ATRX Monoclonal Antibody AMab-6", MONOCLONAL ANTIBODIES IN IMMUNODIAGNOSIS AND IMMUNOTHERAPY, vol. 35, no. 5, 31 December 2016 (2016-12-31), pages 254 - 258 *
唐双阳;刘志敏;李冉辉;陈艳;赵兰华;申海艳;万艳平;: "抗ATRX-C_(2193-2492)多克隆抗体的制备和鉴定", 细胞与分子免疫学杂志, no. 04, 18 April 2017 (2017-04-18), pages 536 - 539 *

Similar Documents

Publication Publication Date Title
EP3152231B1 (en) Peptides, and methods and apparatus utilising same
US7196169B2 (en) Isolated post-translationally modified mammalian proteins for monitoring and diagnosing muscle damage
KR20120057562A (en) Detection of fibrin and fibrinogen degradation products and associated methods of production and use for the detection and monitoring of cancer
CN114989313B (en) P-tau181 epitope peptide and application thereof in Alzheimer disease detection
WO2008054724A9 (en) Monoclonal antibodies against osteopontin
KR0174528B1 (en) Cancer related haptoglobin (hpr)
CN108997500B (en) Anti-human PD-L1 antibody and application thereof
Poulik et al. Post-gamma globulin. II. Radioimmunoassay determination of levels of post-gamma globulin and β2-microglobulin
CN116199763A (en) ATRX antigen epitope peptide and application thereof in detecting brain glioma
JP2000512123A (en) Nephropathy-related immunoglobulin G and antibodies therefor
JP3857468B2 (en) Immunological analysis method and analysis kit for D-dimer and DD / E complex
JPS63258898A (en) Monoclonal antibody against human pancreas phospholipase a2, its production, hybridoma producing said monoclonal antibody and determination of human pancreas phospholipase a2 using said monoclonal antibody
JPWO2006004207A1 (en) Anti-Synoviolin antibody
JP6630116B2 (en) Monoclonal anti-AGEs antibody and method for producing the same
JPH07304800A (en) Monoclonal antibody against human pepsinogen
CN118005798B (en) Anti-vitamin K2Antibodies and uses thereof
CN116731165B (en) Anti-serotonin antibody and application thereof
JP2848705B2 (en) Monoclonal antibody against human lung surfactant apoprotein D and its use
DE60216482T2 (en) COMPOSITIONS AND METHODS FOR DETERMINING AA4RP
JPH0249595A (en) Monoclonal antibody and use thereof
EP2731968A1 (en) Monoclonal antibodies specific for beta-amyloid x-37 and uses thereof
JP2008058194A (en) Discrimination method of westnile virus infection
CN112679596B (en) Adducin antigen peptide, antibody and application thereof
KR102202082B1 (en) Monoclonal antibody with specificity for the envelope protein domain Ⅱ of chikungunya virus, hybridoma cell line producing the same and use thereof
JP2000034300A (en) Phosphopylation-resistant tau protein antibody and detection of alzheimer&#39;s disease by using the same

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