CN116769040B - Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application - Google Patents
Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application Download PDFInfo
- Publication number
- CN116769040B CN116769040B CN202311030863.9A CN202311030863A CN116769040B CN 116769040 B CN116769040 B CN 116769040B CN 202311030863 A CN202311030863 A CN 202311030863A CN 116769040 B CN116769040 B CN 116769040B
- Authority
- CN
- China
- Prior art keywords
- antibody
- seq
- variable region
- gdh
- chain variable
- 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.)
- Active
Links
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 27
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 11
- 238000002965 ELISA Methods 0.000 claims abstract description 8
- 238000003745 diagnosis Methods 0.000 claims abstract description 6
- 238000000338 in vitro Methods 0.000 claims abstract description 6
- 101000950981 Bacillus subtilis (strain 168) Catabolic NAD-specific glutamate dehydrogenase RocG Proteins 0.000 claims description 28
- 102000016901 Glutamate dehydrogenase Human genes 0.000 claims description 28
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 22
- 238000004321 preservation Methods 0.000 claims description 22
- 239000000427 antigen Substances 0.000 claims description 18
- 102000036639 antigens Human genes 0.000 claims description 18
- 108091007433 antigens Proteins 0.000 claims description 18
- 239000002773 nucleotide Substances 0.000 claims description 13
- 125000003729 nucleotide group Chemical group 0.000 claims description 13
- 241001529936 Murinae Species 0.000 claims description 12
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000003018 immunoassay Methods 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 5
- 238000003127 radioimmunoassay Methods 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 102000040430 polynucleotide Human genes 0.000 claims 1
- 108091033319 polynucleotide Proteins 0.000 claims 1
- 239000002157 polynucleotide Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 17
- 101710088194 Dehydrogenase Proteins 0.000 abstract description 7
- 238000000746 purification Methods 0.000 abstract description 4
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 abstract description 2
- 238000003757 reverse transcription PCR Methods 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 230000003248 secreting effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 25
- 239000000243 solution Substances 0.000 description 22
- 238000001514 detection method Methods 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 17
- 239000000523 sample Substances 0.000 description 15
- 241000699670 Mus sp. Species 0.000 description 13
- 241000193163 Clostridioides difficile Species 0.000 description 10
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000005406 washing Methods 0.000 description 10
- 239000011248 coating agent Substances 0.000 description 9
- 238000000576 coating method Methods 0.000 description 9
- 238000001035 drying Methods 0.000 description 9
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 8
- 150000001413 amino acids Chemical group 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 206010003445 Ascites Diseases 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000283707 Capra Species 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 229910002091 carbon monoxide Inorganic materials 0.000 description 4
- 208000028235 central diabetes insipidus Diseases 0.000 description 4
- 238000007865 diluting Methods 0.000 description 4
- 238000009629 microbiological culture Methods 0.000 description 4
- 229910052759 nickel Inorganic materials 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 210000000952 spleen Anatomy 0.000 description 4
- 210000004989 spleen cell Anatomy 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 208000037384 Clostridium Infections Diseases 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000007928 intraperitoneal injection Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000004806 packaging method and process Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010009657 Clostridium difficile colitis Diseases 0.000 description 2
- 206010054236 Clostridium difficile infection Diseases 0.000 description 2
- 235000009161 Espostoa lanata Nutrition 0.000 description 2
- 240000001624 Espostoa lanata Species 0.000 description 2
- 101710116034 Immunity protein Proteins 0.000 description 2
- 101710084578 Short neurotoxin 1 Proteins 0.000 description 2
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 2
- 101710182223 Toxin B Proteins 0.000 description 2
- 101710182532 Toxin a Proteins 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000011091 antibody purification Methods 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 210000005252 bulbus oculi Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 238000003892 spreading Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- ZMPYMKAWMBVPQE-UHFFFAOYSA-N 2-[(6-chloropyridin-3-yl)methyl-ethylamino]-2-methyliminoacetic acid Chemical compound CCN(CC1=CN=C(C=C1)Cl)C(=NC)C(=O)O ZMPYMKAWMBVPQE-UHFFFAOYSA-N 0.000 description 1
- 101710145634 Antigen 1 Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 238000009007 Diagnostic Kit Methods 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 241000581650 Ivesia Species 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 208000003100 Pseudomembranous Enterocolitis Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 231100000599 cytotoxic agent Toxicity 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 231100000676 disease causative agent Toxicity 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000013595 supernatant sample Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000033 toxigenic Toxicity 0.000 description 1
- 230000001551 toxigenic effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- 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
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a mouse anti-GDH hybridoma cell strain, a monoclonal antibody and application, wherein Ig variable region genes are cloned through mouse hybridoma monoclonal antibody screening and RT-PCR method, hybridoma cell strain for stably secreting mouse anti-GDH antibody and variable region sequences thereof are obtained, antibody binding specificity is identified in ELISA mode, antibody subtype of antibodies 1HF9 and 1GE10 is IgG 2a, and the antibody is diluted to 1 mg/ml after purification, and the titer is more than 1:1280000; the anti-glutamate dehydrogenase monoclonal antibody has better performance in all aspects and is suitable for being used as an immunodiagnosis reagent for preparing an in-vitro diagnosis kit.
Description
Technical Field
The invention belongs to the field of antibody preparation, and particularly relates to a mouse anti-GDH hybridoma cell strain, a monoclonal antibody and application.
Background
Clostridium difficile @Clostridioides difficile) Is an obligate anaerobic gram-positive bacillus, which was first isolated from neonatal faeces in 1935 Hall and O' Toole. In 1978, bartlett et al reported that clostridium difficile secreted cytotoxins were the main causative agent of antibiotic-associated diarrhea, pseudomembranous enteritis. For the last 20 years, the incidence, recurrence and severity of clostridium difficile infection (Clostridioides difficile infection, CDI) has been on the rise due to the increasing proportion of the general use of antibiotics to the elderly population. Particularly, in recent years, with the emergence of clostridium difficile virulent strains, the incidence of CDI has increased significantly, and public health safety has been seriously threatened.
At present, CDI has become one of important nosocomial infections, and CDI is diagnosed early and correctly according to relevant symptoms and test results, so that clinical diagnosis and disease prevention are facilitated, reasonable use of antibiotics can be guided, and unnecessary medical expenses can be reduced. In 2021, a rapid diagnosis method for clinical samples is proposed in the standard of clostridium difficile infection diagnosis (T/CPMA 008-2020) formulated by the China medical society for prevention, firstly, glutamate Dehydrogenase (GDH) and toxin A/B in the samples are detected simultaneously, and if the results are inconsistent, PCR detection is combinedtcdBGene, judging whether toxigenic clostridium difficile exists in sampleBacteria. Glutamate dehydrogenase (glutamate dehydrogenase, GDH) is a membrane protein of Clostridium difficile, has good stability and high conservation in each type of Clostridium difficile, but the protein is not unique to virulent Clostridium difficile, and non-virulent strains and other bacteria of the genus Clostridium can produce the enzyme, and therefore cannot be used alone as a diagnostic method, often as a primary screening diagnostic antigen. At present, no clostridium difficile GDH detection kit for obtaining medical instrument registration lot parts is available in China, and imported registration products are expensive, and some of the clostridium difficile GDH detection kits are required to be matched with corresponding instruments and equipment for detection, so that development of similar domestic kits for replacing imported products is needed to enable conventional clinical detection to be carried out in China and disease progression to be monitored early.
The clostridium difficile glutamate dehydrogenase antigen and toxin detection kit adopting a colloidal gold method is developed, firstly, glutamate dehydrogenase antigen (GDH) and toxin A and toxin B antigens thereof are required to be respectively obtained, after high-purity antigens are obtained, good immune response is stimulated in a mouse body, and then, a hybridoma technology is adopted to screen antibodies with high affinity and specificity for developing related in-vitro diagnostic reagents.
Disclosure of Invention
In order to solve the technical problems, the invention provides a mouse anti-GDH hybridoma cell strain, a monoclonal antibody and application.
The technical scheme adopted by the invention is as follows: the mouse anti-GDH hybridoma cell strain is named as 1HF9, the preservation number is CGMCC No.45517, or is named as 1GE10, and the preservation number is CGMCC No.45516.
The mouse anti-GDH monoclonal antibody, the antibody 1HF9 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 shown as SEQ ID NO. 1, CDRH2 shown as SEQ ID NO. 2 and CDRH3 shown as SEQ ID NO. 3, and the light chain variable region comprises CDRL1 shown as SEQ ID NO.4, CDRL2 shown as SEQ ID NO. 5 and CDRL3 shown as SEQ ID NO. 6;
SEQ ID NO:1 CDRH1: GFTFNTNAMS
SEQ ID NO:2 CDRH2: RIRSKSDNHGTFYADSVKD
SEQ ID NO:3 CDRH3: SWFGF
SEQ ID NO:4 CDRL1: SASSSVSSIYLY
SEQ ID NO:5 CDRL2: RTSHLAS
SEQ ID NO:6 CDRL3: LQWSSYPYT
or,
antibody 1GE10 comprises a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO. 11, CDRH2 as shown in SEQ ID NO. 12 and CDRH3 as shown in SEQ ID NO. 13, and a light chain variable region comprising CDRL1 as shown in SEQ ID NO. 14, CDRL2 as shown in SEQ ID NO. 15 and CDRL3 as shown in SEQ ID NO. 16;
SEQ ID NO:11 CDRH1: GFTFNTNSMS
SEQ ID NO:12 CDRH2: RIRSRNNNFETYYADSVKD
SEQ ID NO:13 CDRH3：SWFGY
SEQ ID NO:14 CDRL1: SASSSVSSSYLY
SEQ ID NO:15 CDRL2: RTSNLAS
SEQ ID NO:16 CDRL3: LQWNSYPFT
preferably, the amino acid sequence of the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9;
SEQ ID NO:7
QVQLQQSGGGLVQPKGSLTLSCEASGFTFNTNAMSWVRQAPGKGLEWIARIRSKSDNHGTFYADSVKDRLTISRDDLQNILYLQMNNLKTEDTAMYYCVDSWFGFWGQGTLVTVSA
SEQ ID NO:9
DIVLTQSPEIMSASPGEKVTLTCSASSSVSSIYLYWYQQKPGSSPKLWIYRTSHLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCLQWSSYPYTFGGGTKLEIK
or,
the amino acid sequence of the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19;
SEQ ID NO:17
EVQLQESGGGLVQPKGSLKLSCAASGFTFNTNSMSWVRQAPGKGLEWVARIRSRNNNFETYYADSVKDRFTISRDDSQSMLYLQMNNLKSEDTAMYYCVDSWFGYWGQGTLVTVSA
SEQID NO:19
DIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCLQWNSYPFTFGGGTKLEIK
preferably, the antibody 1HF9 is produced by a mouse anti-GDH hybridoma cell strain with the preservation number of CGMCC No.45517;
antibody 1GE10 was produced by a murine anti-GDH hybridoma cell line with a accession number of CGMCC No.45516.
A nucleic acid molecule comprising nucleotides encoding a murine anti-GDH monoclonal antibody.
Preferably, the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 8, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 10;
SEQ ID NO:8
CAGGTGCAGCTTCAGCAGTCTGGTGGAGGATTGGTACAGCCTAAAGGGTCATTGACACTCTCATGTGAAGCCTCTGGATTCACCTTCAATACCAATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTTTGGAATGGATTGCTCGCATAAGAAGTAAAAGTGATAATCATGGAACATTTTATGCCGATTCAGTGAAAGACAGGTTAACCATCTCCAGAGATGATTTACAAAACATACTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGGACTCCTGGTTTGGTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCCGCA
SEQ ID NO:10
GACATTGTGCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCTGGGGAGAAGGTCACTTTGACCTGCAGTGCCAGTTCAAGTGTAAGTTCCATCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGGACATCCCACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCTTCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
or,
the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 18, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 20;
SEQ ID NO:18
GAGGTGCAGCTGCAGGAATCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCAATTCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAGAAATAATAATTTTGAAACATATTATGCCGATTCAGTGAAAGACAGATTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAATAATTTGAAAAGTGAGGACACAGCCATGTATTACTGTGTGGACTCCTGGTTTGGTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCTGCA
SEQ ID NO:20
GACATTGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAGTGCCAGCTCAAGTGTAAGTTCCAGCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGGACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCTTCAGTGGAATAGTTACCCGTTCACGTTCGGGGGGGGGACCAAGCTGGAAATAAAA
application of mouse anti-GDH monoclonal antibody in preparing reagent for detecting glutamate dehydrogenase antigen.
Preferably, the murine anti-GDH monoclonal antibody is used in an in vitro diagnostic kit, in particular in a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunoassay kit, a fluorescent immunoassay kit or a microfluidic chip.
A kit for detecting glutamate dehydrogenase antigen, comprising a murine anti-GDH monoclonal antibody.
Preferably, the method comprises a double-antibody sandwich immune colloidal gold test strip, wherein the antibody 1HF9 is a coated antibody, and the antibody 1GE10 is a gold-labeled antibody;
or antibody 1GE10 is a coated antibody, and antibody 1HF9 is a gold-labeled antibody.
The invention has the advantages and positive effects that: the mouse anti-GDH hybridoma cell strain and the monoclonal antibody are provided, and the anti-glutamate dehydrogenase monoclonal antibody has better performance in all aspects, and the titer reaches more than 1:1280000, so that the anti-glutamate dehydrogenase monoclonal antibody is suitable for being used as an immunodiagnosis reagent for preparing an in vitro diagnosis kit.
Drawings
FIG. 1 SDS-PAGE electrophoretic identification of GDH antigen; 1 is a purified protein; m is a protein relative molecular mass marker;
FIG. 2 is an antibody purification electrophoretogram; 1 is 1HF9 mab; 2 is 1GE10 mab, M is a protein relative molecular mass marker;
FIG. 3 colloidal Jin Fashu anti-GDH assay card assay results; blank on the left side and antigen detection on the right side;
FIG. 4 colloidal Jin Fashu anti-GDH assay card assay results; the left side is a positive sample, and the right side is a negative sample;
biological material: 1HF9 belongs to hybridoma cells, the preservation number is CGMCC No.45517, the preservation place is China general microbiological culture Collection center (CGMCC), the North Chen Xili No. 1 and 3 of the Korean area of Beijing, the preservation date is 2023, 3 months and 23 days, and the preservation state is survival;
biological material: 1GE10 belongs to hybridoma cells, and the preservation number is CGMCC No.45516; the preservation land is China general microbiological culture Collection center (CGMCC), north Xidelu No. 1, no. 3 in the Korean area of Beijing, the preservation date is 2023, 3 and 23, and the preservation state is survival.
Detailed Description
Embodiments of the present invention are described below with reference to the accompanying drawings.
The invention relates to a mouse anti-GDH hybridoma cell strain, wherein one strain of biological material is named as 1HF9, belongs to hybridoma cells, and has a preservation number of CGMCC No.45517; the preservation land is China general microbiological culture collection center (CGMCC), north Xidelu No. 1, no. 3 in the Korean area of Beijing, the preservation date is 2023, 3 and 23, and the preservation state is survival; the other biological material is named as 1GE10, belongs to hybridoma cells, and has the preservation number of CGMCC No.45516; the preservation land is China general microbiological culture Collection center (CGMCC), north Xidelu No. 1, no. 3 in the Korean area of Beijing, the preservation date is 2023, 3 and 23, and the preservation state is survival.
Monoclonal antibody 1HF9 produced by mouse anti-GDH hybridoma cell line 1HF9, antibody 1HF9 comprising a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO. 1, CDRH2 as shown in SEQ ID NO. 2 and CDRH3 as shown in SEQ ID NO. 3, and a light chain variable region comprising CDRL1 as shown in SEQ ID NO.4, CDRL2 as shown in SEQ ID NO. 5 and CDRL3 as shown in SEQ ID NO. 6;
SEQ ID NO:1 CDRH1: GFTFNTNAMS
SEQ ID NO:2 CDRH2: RIRSKSDNHGTFYADSVKD
SEQ ID NO:3 CDRH3: SWFGF
SEQ ID NO:4 CDRL1: SASSSVSSIYLY
SEQ ID NO:5 CDRL2: RTSHLAS
SEQ ID NO:6 CDRL3: LQWSSYPYT
the amino acid sequence of the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9;
SEQ ID NO:7
QVQLQQSGGGLVQPKGSLTLSCEASGFTFNTNAMSWVRQAPGKGLEWIARIRSKSDNHGTFYADSVKDRLTISRDDLQNILYLQMNNLKTEDTAMYYCVDSWFGFWGQGTLVTVSA
SEQ ID NO:9
DIVLTQSPEIMSASPGEKVTLTCSASSSVSSIYLYWYQQKPGSSPKLWIYRTSHLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCLQWSSYPYTFGGGTKLEIK
the nucleotide sequence of the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 8, and the nucleotide sequence of the light chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 10;
SEQ ID NO:8
CAGGTGCAGCTTCAGCAGTCTGGTGGAGGATTGGTACAGCCTAAAGGGTCATTGACACTCTCATGTGAAGCCTCTGGATTCACCTTCAATACCAATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGTTTGGAATGGATTGCTCGCATAAGAAGTAAAAGTGATAATCATGGAACATTTTATGCCGATTCAGTGAAAGACAGGTTAACCATCTCCAGAGATGATTTACAAAACATACTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGGACTCCTGGTTTGGTTTCTGGGGCCAAGGGACTCTGGTCACTGTCTCCGCA
SEQ ID NO:10
GACATTGTGCTCACCCAGTCTCCAGAAATCATGTCTGCATCTCCTGGGGAGAAGGTCACTTTGACCTGCAGTGCCAGTTCAAGTGTAAGTTCCATCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGGACATCCCACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCTTCAGTGGAGTAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA
the invention discloses a monoclonal antibody 1GE10 generated by another mouse anti-GDH hybridoma cell strain 1GE10, wherein the antibody 1GE10 comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDRH1 shown as SEQ ID NO. 11, CDRH2 shown as SEQ ID NO. 12 and CDRH3 shown as SEQ ID NO. 13, and the light chain variable region comprises CDRL1 shown as SEQ ID NO. 14, CDRL2 shown as SEQ ID NO. 15 and CDRL3 shown as SEQ ID NO. 16;
SEQ ID NO:11 CDRH1: GFTFNTNSMS
SEQ ID NO:12 CDRH2: RIRSRNNNFETYYADSVKD
SEQ ID NO:13 CDRH3：SWFGY
SEQ ID NO:14 CDRL1: SASSSVSSSYLY
SEQ ID NO:15 CDRL2: RTSNLAS
SEQ ID NO:16 CDRL3: LQWNSYPFT
the amino acid sequence of the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19;
SEQ ID NO:17
EVQLQESGGGLVQPKGSLKLSCAASGFTFNTNSMSWVRQAPGKGLEWVARIRSRNNNFETYYADSVKDRFTISRDDSQSMLYLQMNNLKSEDTAMYYCVDSWFGYWGQGTLVTVSA
SEQID NO:19
DIVLTQSPAIMSASPGEKVTLTCSASSSVSSSYLYWYQQKPGSSPKLWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCLQWNSYPFTFGGGTKLEIK
the nucleotide sequence of the nucleic acid molecule encoding the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 18, and the nucleotide sequence of the nucleic acid molecule encoding the light chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 20;
SEQ ID NO:18
GAGGTGCAGCTGCAGGAATCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATACCAATTCCATGAGCTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAGAAATAATAATTTTGAAACATATTATGCCGATTCAGTGAAAGACAGATTCACCATCTCCAGAGATGATTCACAAAGCATGCTCTATCTGCAAATGAATAATTTGAAAAGTGAGGACACAGCCATGTATTACTGTGTGGACTCCTGGTTTGGTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCTGCA
SEQ ID NO:20
GACATTGTGCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAGTGCCAGCTCAAGTGTAAGTTCCAGCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGGACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCTTCAGTGGAATAGTTACCCGTTCACGTTCGGGGGGGGGACCAAGCTGGAAATAAAA
the anti-glutamate dehydrogenase monoclonal antibody has better performance in all aspects through systematic evaluation, including evaluation of antibody subtype and potency, kit sensitivity, specificity and stability, so that the anti-glutamate dehydrogenase monoclonal antibody is suitable for being used as an immunodiagnosis reagent for preparing an in vitro diagnosis kit. Can be made into colloidal gold immunoassay kit, chemiluminescence kit, radioimmunoassay kit, enzyme-linked immunoassay kit or fluorescence immunoassay kit, or made into microfluidic chip; the prepared kit can detect glutamate dehydrogenase antigen.
The kit for detecting the glutamate dehydrogenase antigen contains a mouse anti-GDH monoclonal antibody, the mouse anti-GDH monoclonal antibody is prepared into a double-antibody sandwich immune colloidal gold test strip, the antibody 1HF9 is a coated antibody, and the antibody 1GE10 is a gold-labeled antibody, so that the detection effect is good; in certain embodiments of the invention, antibody 1GE10 may also be coated and antibody 1HF9 may be gold-labeled.
The following description of the present invention is made with reference to the accompanying drawings, wherein the experimental methods without specific description of the operation steps are performed according to the corresponding commodity specifications, and the instruments, reagents and consumables used in the embodiments can be purchased from commercial companies without specific description.
Example 1: GDH antigen preparation
The GDH full-length gene sequence is searched and downloaded from NCBI, the gene is chemically synthesized and then is connected to a recombinant plasmid constructed in pet-28a, and the recombinant plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) and cultured overnight at 37 ℃. Picking single colony and culturing in LB culture medium to OD 600 0.6-0.8, IPTG was added at a final concentration of 1 mM and induced overnight at 18 ℃. Centrifugally collecting induced thalli, ultrasonically crushing, centrifugally collecting supernatant, purifying by a nickel column, balancing the nickel column by using a balancing buffer solution, adding a supernatant sample into the nickel column, and respectively eluting and collecting target proteins by using washing solutions containing 20, 50, 100 and 200 mmol/L imidazole after the sample completely enters the nickel column. SDS-PAGE electrophoresis result shows that the protein with better purity is obtained, as shown in figure 1, the size is 46.3KD, and the concentration is determined by BCA protein quantitative kit2.3 mg/mL, packaging, and storing at-80deg.C.
Example 2: antibody preparation
2.1 Immunization of mice
Antibody preparation was performed by immunizing female Balb/c mice of about 6 weeks of age with purified glutamate dehydrogenase antigen. The immunity protein content is 0.1 mg/mL, and the immunity protein is divided into 2 groups according to the immunity dose, and 5 mice in each group; the first group of immunization doses was 25 μg/dose and the second group of immunization doses was 50 μg/dose, calculated as antigen content. Firstly, diluting a proper amount of glutamate dehydrogenase antigen to 300 mu L by distilled water, adding 300 mu L of equivalent Freund's complete adjuvant, emulsifying uniformly, and subcutaneously injecting a plurality of points to immunize a mouse; two weeks later, the same dose was taken for a second immunization, which was to immunize the mice by intraperitoneal injection, and two weeks later, the mice were immunized by intraperitoneal injection, and 7 days later, the mice were collected from the tail and the serum titers of the mice were measured by ELISA. The method comprises the following specific steps: glutamate dehydrogenase antigen 1. Mu.g/mL, 100. Mu.L/well, ELISA plates were coated overnight at 4 ℃, spin-dried and PBST washed 3 times. 5% skim milk powder, 200. Mu.L/well, was blocked at 37℃for 2h. The mouse tail is sampled at 3000 rpm/min, serum is collected after centrifugation, and PBS is used for dilution to 1:512000 from 1:1000 for later use. Spin-dried, PBST washed 3 times, 1:1000 times and PBS diluted primary antibody was added at 100. Mu.L/well, 37℃for 1h. Spin-drying, PBST washing for 3 times, adding goat anti-mouse secondary antibody diluted 1:6000 times in PBS, 100 μl/hole, 37 ℃ for 45 min. Spin-drying, PBST washing for 5 times, adding 100 mu L TMB/hole, developing at 37 ℃ for 10 min, stopping and reading.
2.2 Cell fusion
Mice were boosted three days prior to fusion and vaccinated at the same dose as the previous immunization without adjuvant and intraperitoneally. Feeder cells were prepared the day before fusion, balb/c 1 mice were taken from 6-8 weeks old, and after the eyeballs were exsanguinated, cervical vertebrae were dislocated and killed, and sterilized in 75% alcohol for 5 min, fixed on a dish, and abdomen skin was aseptically cut off in an ultra clean bench. The HAT selection medium 10 mL was aspirated with a sterile syringe and injected into the abdominal cavity of the mice, which was gently rubbed with an alcohol cotton ball and the medium was withdrawn. Adding into 40 mLHAT culture solution, spreading into 4 96-well cell culture plates, 100 μl/well, 37deg.C, 5% CO 2 Culturing in a cell culture incubator. Melting and meltingOne week prior to confluence myeloma cells (Sp 2/0 cells) were resuscitated and cultured in PRMI-1640 medium containing 10% fetal bovine serum at 37℃with 5% CO 2 Subculturing in an incubator. Collecting cells in logarithmic phase into centrifuge tube, counting, diluting to 10 7 The sample was kept at one/mL. Balb/c mice with the immunity enhanced for 3 days are taken, eyeballs are taken out for bloodletting to prepare positive serum, cervical vertebra is removed for sacrifice, 75% alcohol is used for sterilization for 5 min, spleens are aseptically taken out on an ultra-clean workbench, washed for a plurality of times in an aseptic plate, and connective tissues are stripped. Placing spleen on a microporous copper mesh, adding fresh RPMI-1640 culture solution, sucking the culture solution by a syringe, injecting the culture solution from the spleen for one time, blowing spleen cells, repeating for several times, and lightly grinding the rest spleen by using an inner plug of the syringe until no obvious red tissue block exists. The spleen cell suspension in the plate is gently blown and transferred into a 50 mL centrifuge tube, centrifuged at 1000 r/min for 5 min, and the spleen cells are collected and counted for later use. Mixing the immunized mouse spleen cells with Sp2/0 cells according to the cell number of 10:1, adding into a centrifuge tube of 50 mL, centrifuging at 1000 r/min for 5 min, discarding the supernatant, lightly rubbing the two cells at the palm to fully mix the two cells, placing the centrifuge tube into a 100 mL blue cap bottle, placing hot water at 37 ℃ in the blue cap bottle, dropwise adding preheated 1 mL DMSO/PEG into a fusion tube within 1min, slowly and quickly, and lightly rotating the centrifuge tube while adding. Then, the reaction was terminated by immediately adding the antibiotic-free blood-free RPMI-1640 medium, 1 mL in the first minute, 2 mL in the second minute Zhong Jia, 3 mL in the third minute and 4 mL in the fourth minute. Centrifuging in a water bath at 37deg.C for 5 min at 800 r/min for 5 min, removing supernatant, suspending the precipitate with HAT, mixing to 40 mL HAT selective culture solution containing pre-heated 20% calf serum at 37deg.C, spreading into 96-well cell plate with feeder cells at 100 μl/well, placing the culture plate into 37 deg.C and 5% CO 2 Culturing in an incubator. After 7d, the cell plates were half-plated with fresh HAT medium and 10 days later with HT medium. Cells positive for detection in 96-well plates were subcloned by limiting dilution: firstly, preparing feeder cells according to the above-mentioned method, taking hybridoma cells to be cloned, cell-counting, diluting the cells to 5-8 cells/mL with HT culture medium, adding 100 mul/hole into 96-well cell plate with feeder cells spread, every hybridomaCell cloning a 96-well cell plate, 37 ℃,5% CO 2 Culturing in a cell culture incubator. After about 5 days, the number of clones in the cell well is counted, marked, 7 days later and a new culture medium is changed, and the cell is detected when 1/3-1/2 of the whole bottom of the well is paved. After 2-3 times of cloning, when all cell holes of the 96-well plate are positive, the amplification culture, strain fixing and freezing storage can be carried out. And (5) performing amplification culture and freezing storage on the hybridoma cells of which the positive detection determination strains. The specific process is as follows: the well-grown hybridoma cells were gently blown down from the flask with the use of the nonreactive bloodless DMEM, centrifuged at 1000 r/min for 5 min, and the supernatant was discarded. Adding the frozen stock solution (containing 40% RPMI-1640 culture solution, 50% fetal calf serum and 10% DMSO), blowing off the cells, and packaging into cell frozen stock tubes. And (3) placing the freezing and storing tube into a freezing and storing box, placing the freezing and storing box into a refrigerator at the temperature of-70 ℃, transferring the freezing and storing tube into liquid nitrogen after one day, and recording.
2.3 Ascites preparation
Taking 10-12 week old female Balb/c mice, injecting sterile liquid paraffin into the abdominal cavity, and culturing into logarithmic phase hybridoma cells by intraperitoneal injection after 7d of 0.5: 0.5 mL/mouse, 5×10 6 Individual cells/individual. After the abdomen of the mice is obviously raised, the skin of the lower abdomen is disinfected by a 75% alcohol cotton ball, and the abdominal cavity is pierced by a 16-gauge needle to collect ascites after the observation is carried out for about 7-10 days. And after the ascites is regenerated and accumulated, collecting again. Centrifuging the collected ascites at 3000 r/min for 10 min, collecting the middle clear part, filtering with filter paper, packaging, and preserving at-70deg.C.
2.4 Antibody purification
Ascites purification was performed using Protein-G column as follows: taking ascites 2 mL (n), centrifuging 10000 g, taking a clarified part, adding 2 mL (1:1) washing buffer solution, uniformly mixing, balancing the column by using 8 mL washing solution after 20% ethanol is completely flowed out, allowing a sample to pass through the column, performing washing precipitation by using 15 mL washing buffer solution at a flow rate of 8S/drip deposition for 3 times, eluting by using 10 mL eluting buffer solution after washing, adjusting pH to 7.4 by using 1M Tris PH=9 after eluting, concentrating by using concentrated injection, dialyzing by using 50 kd dialysis bags, PBS and dialyzing at 4 ℃ overnight.
Example 3: antibody identification
3.1 Antibody subtype identification
The monoclonal antibody subclass identification is carried out by a capture ELISA method according to the specification of a SIGMA kit, and specifically comprises the following steps: diluting the monoclonal antibody subclass identification reagent 1:1000, adding the monoclonal antibody subclass identification reagent into an enzyme-labeled hole, and incubating at 37 ℃ for 1h at 100 mu L/hole; washing with PBST for three times, and drying; the antibody is diluted by 1:1000 times and then is added with 100 mu L/hole, and incubated for 1h at 37 ℃; washing with PBST for three times, and drying; the HRP enzyme-labeled goat anti-mouse IgG secondary antibody is diluted by 1:6000 and then is added with 100 mu L/hole and incubated for 30 min at room temperature; color development is carried out for 10-20 min. At OD 450 The read value is obviously higher than that of the subclass reagent added in other holes, and the subclass reagent is of the subclass type to which the monoclonal antibody belongs. The results are shown in Table 1, and the antibody subtypes of antibodies 1HF9 and 1GE10 are IgG 2a.
TABLE 1
3.2 Antibody titer determination
The method for measuring the antibody titer after purification by adopting an indirect ELISA method comprises the following steps: glutamate dehydrogenase is respectively diluted to 0.2 mug/mL and 100 mug/hole, meanwhile, an uncoated control is established, coating is carried out at 4 ℃ overnight, spin-drying is carried out, and PBST is washed for 3 times; 5% skim milk powder, 200 μl/well, block 2h at 37deg.C; spin-dried, PBST washed 3 times, and antibody (concentration 1 mg/mL) was added in a multiple dilution starting from 1:1000, for a total of 12 gradients, while no coating control 100. Mu.L/well was established, 37 ℃,1h. Spin-drying, PBST washing for 3 times, adding goat anti-mouse secondary antibody diluted 1:6000 times in PBS, 100 μl/hole, 37 ℃ for 45 min. Spin-drying, PBST washing 5 times, adding 100 μl TMB/well, developing at 37deg.C for 10 min, stopping, and reading, and the results are shown in Table 2. After purification, the antibody is diluted to 1 mg/ml, and the titer reaches more than 1:1280000.
TABLE 2
3.3 Identification of antibody purity and molecular weight
Performing molecular weight and purity identification of the antibody by adopting an SDS-PAGE method; preparing gel, wherein the separating gel is 12% and the concentrating gel is 5%; preparing a sample, namely, mixing 20 mu L of the sample with 20 mu Lbuffer, and boiling for 3 min; loading 20 mu L of the sample into each hole, and simultaneously setting up a protein pre-dyeing Marker control; 80 volts for 30 min,120 volts for 2 h; after electrophoresis, adding coomassie brilliant blue solution for dyeing; decolorizing, boiling deionized water for 5 min each time for 3 times; the purified monoclonal antibody was identified by SDS-PAGE, and the bands were clear, and there were no bands, as shown in FIG. 2, at 50 kDa and 25 kDa, respectively.
Cloning Ig variable region genes by RT-PCR method
1) Total RNA extraction, single-stranded cDNA synthesis:
total RNA from 1HF9,1GE10 hybridoma cell lines was extracted by the Trizol method (kit from Invitrogen) and inverted into a cDNA library using M-MLV reverse transcriptase (from Invitrogen).
Heavy chain framework region upstream primer
P1：5’SAGGTGMAGCTKCASSARTCWGG3’
Heavy chain variable region downstream primer
P2：5’TGGGGSTGTYGTTTTGGCTGMRGAGACRGTGA3’
Light chain leader peptide upstream primer
P3：5’GACATTGTGCTCACCCAGTCTCCA3’
Light chain variable region downstream primer
P4：5’GGATACAGTTGGTGCAGCATCAGCCCGTTT3’
The PCR reaction system (50. Mu.L) was prepared as follows:
cDNA:2 μl; upstream primer (10. Mu.M): 2 μl; downstream primer (10. Mu.M): 2. mu L; dNTP mix: 2. mu L; pfu DNA polymerase (5U/. Mu.L): 1. mu L;10 X pfu Buffer II: 5. mu L; ddH 2 O: make up to 50 μl.
Reaction conditions: pre-denaturation at 95 ℃ for 5 min; the following cycle was repeated 35 times: 95 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 1min; finally, the extension is carried out at 72℃for 10 min.
The VL and VH fragments were separated and recovered by agarose gel electrophoresis. The recovered VL and VH fragments were ligated with pMD19-T (sKPCle) vector (Takara Co.) in the following manner:
VL PCR product/VH PCR product 70 ng each, pMD19-T (sKPCle) vector 1. Mu.L, solution I ligation reaction 5. Mu.L; ddH 2 O was made up to 10. Mu.L and was attached overnight at 4 ℃.
The ligation product was transformed into E.coli DH 5. Alpha. Competent bacteria, after overnight incubation at 37℃individual colonies were picked up, and after shaking for 2 hours at 37℃bacterial solution PCR identification was performed, using cDNA corresponding to the antibody as positive control. The reaction system (25. Mu.L) was prepared as follows:
bacterial liquid: 1 μl, upstream primer (10 μM): 1. mu L; downstream primer (10. Mu.M): 1 μl; dNTP mix (2.5. 2.5 Mm each) 2. Mu.L; taq DNA polymerase (5U/. Mu.L): 0.5 Mu L;10 xTaq Buffer (Mg 2+ plus): 2.5 Mu L; the water was made up to 25. Mu.L. The reaction conditions are the same as before.
The PCR positive clones were selected for amplification culture, positive clone plasmids were extracted with a plasmid extraction kit (Takara Co.), and were subjected to sequencing. At least 5 clone samples were sent for each chain of each antibody, and at least three samples were sequenced until the results were identical. The heavy chain and light chain variable region sequences of the antibodies 1HF9 and 1GE10 are successfully cloned, and the sequences are aligned to meet the characteristic of the typical antibody variable region sequences.
Example 4: glutamate dehydrogenase detection card prepared by using colloidal gold method
The preparation method of the immune colloidal gold test strip by the double-antibody sandwich method comprises the following steps:
step one adding 0.1. 0.1M K to the colloidal gold solution while stirring 2 CO 3 Solution, after regulating pH value, adding anti-glutamate dehydrogenase monoclonal antibody 1GE10, stirring, adding 10% bovine serum albumin solution and 2% PEG 20000 Centrifuging at low speed after stirring to obtain supernatant, centrifuging at high speed to obtain precipitate, and fixing volume with colloid Jin Chong suspension to form gold-labeled antibody;
spraying the gold-labeled antibody on a glass cellulose membrane, and drying to prepare a gold-labeled pad;
adding a 1% sulfur Liu Gongna solution into an anti-glutamate dehydrogenase monoclonal antibody 1GE10, uniformly mixing to form a detection line coating solution, adding PBS and a 1% sulfur Liu Gongna solution into goat anti-mouse IgG, uniformly mixing to form a quality control line coating solution, marking the quality control line coating solution and the detection line coating solution on a nitrocellulose membrane, and drying to obtain a coating membrane;
and fourthly, attaching a coating film on a bottom plate, overlapping a gold-labeled pad and water absorbing paper with the coating film, laminating, and cutting to obtain the colloidal gold method glutamate dehydrogenase detection card.
And detecting the blank sample, the positive sample, the negative sample and the sample to be detected by using the prepared detection card, wherein the results are shown in figures 3-4. The left side of the figure 3 is a blank sample detection result, the right side is a sample detection result to be detected, the left side of the figure 4 is a positive sample detection result, and the right side is a negative sample detection result, so that whether the sample contains glutamate dehydrogenase or not can be effectively presented by the prepared colloidal gold method glutamate dehydrogenase detection card, and the detection is effective.
The foregoing describes the embodiments of the present invention in detail, but the description is only a preferred embodiment of the present invention and should not be construed as limiting the scope of the invention. All equivalent changes and modifications within the scope of the present invention are intended to be covered by the present invention.
Claims (10)
1. A murine anti-GDH hybridoma cell line characterized by: named as 1HF9, the preservation number is CGMCC No.45517 or named as 1GE10, and the preservation number is CGMCC No.45516.
2. A murine anti-GDH monoclonal antibody, characterized in that: antibody 1HF9 comprises a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO. 1, CDRH2 as shown in SEQ ID NO. 2 and CDRH3 as shown in SEQ ID NO. 3, and a light chain variable region comprising CDRL1 as shown in SEQ ID NO.4, CDRL2 as shown in SEQ ID NO. 5 and CDRL3 as shown in SEQ ID NO. 6;
or,
antibody 1GE10 comprises a heavy chain variable region comprising CDRH1 as shown in SEQ ID NO. 11, CDRH2 as shown in SEQ ID NO. 12 and CDRH3 as shown in SEQ ID NO. 13, and a light chain variable region comprising CDRL1 as shown in SEQ ID NO. 14, CDRL2 as shown in SEQ ID NO. 15 and CDRL3 as shown in SEQ ID NO. 16.
3. The murine anti-GDH monoclonal antibody of claim 2, wherein: the amino acid sequence of the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 9;
or,
the amino acid sequence of the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 17, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 19.
4. The murine anti-GDH monoclonal antibody of claim 2 or 3, wherein: antibody 1HF9 is produced by a mouse anti-GDH hybridoma cell strain with a preservation number of CGMCC No.45517;
antibody 1GE10 was produced by a murine anti-GDH hybridoma cell line with a accession number of CGMCC No.45516.
5. A nucleic acid molecule characterized in that: a polynucleotide encoding the murine anti-GDH monoclonal antibody of claim 2 or 3.
6. The nucleic acid molecule of claim 5, wherein: the nucleotide sequence of the heavy chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 8, and the nucleotide sequence of the light chain variable region of the antibody 1HF9 is shown as SEQ ID NO. 10;
or,
the nucleotide sequence of the heavy chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 18, and the nucleotide sequence of the light chain variable region of the antibody 1GE10 is shown as SEQ ID NO. 20.
7. Use of the murine anti-GDH monoclonal antibody of any one of claims 2-4 in the preparation of a reagent for detecting glutamate dehydrogenase antigen.
8. The use according to claim 7, characterized in that: the mouse anti-GDH monoclonal antibody is used for an in vitro diagnosis kit, in particular for a colloidal gold immunoassay kit, a chemiluminescent kit, a radioimmunoassay kit, an enzyme linked immunoassay kit, a fluorescent immunoassay kit or a microfluidic chip.
9. A kit for detecting glutamate dehydrogenase antigen, characterized in that: comprising the murine anti-GDH monoclonal antibody according to any one of the claims 2-4.
10. The kit for detecting glutamate dehydrogenase antigen according to claim 9, wherein: comprises a double-antibody sandwich immune colloidal gold test strip, wherein an antibody 1HF9 is a coated antibody, and an antibody 1GE10 is a gold-labeled antibody;
or antibody 1GE10 is a coated antibody, and antibody 1HF9 is a gold-labeled antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311030863.9A CN116769040B (en) | 2023-08-16 | 2023-08-16 | Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311030863.9A CN116769040B (en) | 2023-08-16 | 2023-08-16 | Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116769040A CN116769040A (en) | 2023-09-19 |
| CN116769040B true CN116769040B (en) | 2023-11-10 |
Family
ID=88013722
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311030863.9A Active CN116769040B (en) | 2023-08-16 | 2023-08-16 | Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116769040B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR970001373A (en) * | 1996-10-28 | 1997-01-24 | 최수영 | Monoclonal Antibody Against Glutamic Acid Dehydrogenase |
| CN101805723A (en) * | 2009-10-20 | 2010-08-18 | 中国农业科学院哈尔滨兽医研究所 | Anti-glutamate dehydrogenase protein monoclonal antibody, preparation method thereof and use thereof |
| CN109336979A (en) * | 2018-11-16 | 2019-02-15 | 宁夏医科大学 | Nano antibody, coded sequence and its screening technique of a kind of clostridium difficile glutamte dehydrogenase and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10577665B2 (en) * | 2017-09-05 | 2020-03-03 | Mcmaster University | Aptamers for clostridium difficile detection |
-
2023
- 2023-08-16 CN CN202311030863.9A patent/CN116769040B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR970001373A (en) * | 1996-10-28 | 1997-01-24 | 최수영 | Monoclonal Antibody Against Glutamic Acid Dehydrogenase |
| CN101805723A (en) * | 2009-10-20 | 2010-08-18 | 中国农业科学院哈尔滨兽医研究所 | Anti-glutamate dehydrogenase protein monoclonal antibody, preparation method thereof and use thereof |
| CN109336979A (en) * | 2018-11-16 | 2019-02-15 | 宁夏医科大学 | Nano antibody, coded sequence and its screening technique of a kind of clostridium difficile glutamte dehydrogenase and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116769040A (en) | 2023-09-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112500489B (en) | anti-OXA-23 type carbapenemase hybridoma cell strain, monoclonal antibody and application | |
| CN111153991A (en) | Human SARS-CoV-2 monoclonal antibody and its preparation method and use | |
| CN112501131B (en) | anti-IMP (anti-IMP) carbapenemase hybridoma cell strain, monoclonal antibody and application | |
| CN112980803B (en) | VIM-resistant carbapenemase hybridoma cell strain, monoclonal antibody and application | |
| EP4170021B1 (en) | Mouse anti-mcr-1 protein hybridoma cell strain, monoclonal antibody, and application | |
| EP4265717A1 (en) | Mouse anti-mcr-1/mcr-2 protein hybridoma cell strain, monoclonal antibody and use | |
| CN116042532B (en) | Mouse anti-NDM carbapenemase hybridoma cell strain, monoclonal antibody and application | |
| WO2022237138A1 (en) | Anti-kpc type carbapenemase hybridoma cell line, monoclonal antibody and application thereof | |
| CN114317454B (en) | Mouse anti-OXA-48 type carbapenemase hybridoma cell strain, monoclonal antibody and application | |
| CN116769023A (en) | Mouse anti-marneffei basket mannoprotein hybridoma cell strain, monoclonal antibody and application | |
| CN114317455B (en) | Mouse anti-MCR-1 protein hybridoma cell strain, monoclonal antibody and application | |
| CN116925218B (en) | Antibody of small heat shock protein HSPB1, antibody composition, hybridoma cell strain and application thereof | |
| CN111793130A (en) | Haemophilus parasuis CdtB hybridoma cell and application of monoclonal antibody | |
| CN116462758B (en) | Anti-human PTPRZ1 monoclonal antibody and application thereof in cell flow | |
| CN116769040B (en) | Mouse anti-GDH hybridoma cell strain, monoclonal antibody and application | |
| CN110540966B (en) | Human haemophilus influenzae surface protein monoclonal antibody and antigen capture ELISA kit | |
| CN113980126B (en) | Pasteurella multocida toxin monoclonal antibody and blocking ELISA kit thereof | |
| CN116925219B (en) | Antibody of small heat shock protein HSPB1, hybridoma cell strain and application thereof | |
| CN116731186B (en) | Anti-human IgG-Fc rabbit monoclonal antibody and preparation method thereof, polynucleotide molecule, expression vector and host cell | |
| CN115948346A (en) | Hybridoma cell strain capable of stably secreting anti-conotoxin mu-KIIIA-CTX monoclonal antibody | |
| CN118620042A (en) | SARS-CoV-2 coronavirus envelope surface S protein antigen epitope and its monoclonal antibody preparation screening method and application | |
| CN115724953A (en) | Full-molecular IgG antibody and preparation method and application thereof | |
| CN118725096A (en) | NS1 protein monoclonal antibody and base application |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |