CN110343669B - 一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 - Google Patents
一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 Download PDFInfo
- Publication number
- CN110343669B CN110343669B CN201910653689.0A CN201910653689A CN110343669B CN 110343669 B CN110343669 B CN 110343669B CN 201910653689 A CN201910653689 A CN 201910653689A CN 110343669 B CN110343669 B CN 110343669B
- Authority
- CN
- China
- Prior art keywords
- triclabendazole
- solution
- monoclonal antibody
- cell strain
- dnc
- 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
- 229960000323 triclabendazole Drugs 0.000 title claims abstract description 44
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 20
- 230000003248 secreting effect Effects 0.000 title claims abstract description 10
- NQPDXQQQCQDHHW-UHFFFAOYSA-N 6-chloro-5-(2,3-dichlorophenoxy)-2-(methylthio)-1H-benzimidazole Chemical compound ClC=1C=C2NC(SC)=NC2=CC=1OC1=CC=CC(Cl)=C1Cl NQPDXQQQCQDHHW-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000004321 preservation Methods 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 239000012267 brine Substances 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 12
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 10
- 239000008346 aqueous phase Substances 0.000 claims description 9
- 238000001035 drying Methods 0.000 claims description 9
- 230000002163 immunogen Effects 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- NSGDYZCDUPSTQT-UHFFFAOYSA-N N-[5-bromo-1-[(4-fluorophenyl)methyl]-4-methyl-2-oxopyridin-3-yl]cycloheptanecarboxamide Chemical compound Cc1c(Br)cn(Cc2ccc(F)cc2)c(=O)c1NC(=O)C1CCCCCC1 NSGDYZCDUPSTQT-UHFFFAOYSA-N 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
- 229910021641 deionized water Inorganic materials 0.000 claims description 6
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 239000011259 mixed solution Substances 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 6
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 claims description 6
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 5
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 5
- NHQDETIJWKXCTC-UHFFFAOYSA-N 3-chloroperbenzoic acid Chemical compound OOC(=O)C1=CC=CC(Cl)=C1 NHQDETIJWKXCTC-UHFFFAOYSA-N 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 4
- JUYVXCGKMCYNBN-UHFFFAOYSA-N methyl 4-hydroxybutanoate Chemical compound COC(=O)CCCO JUYVXCGKMCYNBN-UHFFFAOYSA-N 0.000 claims description 4
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 3
- VVWRJUBEIPHGQF-UHFFFAOYSA-N propan-2-yl n-propan-2-yloxycarbonyliminocarbamate Chemical compound CC(C)OC(=O)N=NC(=O)OC(C)C VVWRJUBEIPHGQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- HYHCSLBZRBJJCH-UHFFFAOYSA-M sodium hydrosulfide Chemical compound [Na+].[SH-] HYHCSLBZRBJJCH-UHFFFAOYSA-M 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 2
- 244000005700 microbiome Species 0.000 claims description 2
- 239000012071 phase Substances 0.000 claims 3
- 238000001514 detection method Methods 0.000 abstract description 11
- 230000005764 inhibitory process Effects 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000012360 testing method Methods 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 3
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 3
- 238000003018 immunoassay Methods 0.000 abstract description 3
- 230000003053 immunization Effects 0.000 description 16
- 238000002649 immunization Methods 0.000 description 16
- 238000002965 ELISA Methods 0.000 description 11
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 239000002671 adjuvant Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000012980 RPMI-1640 medium Substances 0.000 description 5
- 230000007910 cell fusion Effects 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 206010003445 Ascites Diseases 0.000 description 4
- 238000011725 BALB/c mouse Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000000683 abdominal cavity Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
- 239000012154 double-distilled water Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000009260 cross reactivity Effects 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- HYZJCKYKOHLVJF-UHFFFAOYSA-N 1H-benzimidazole Chemical compound C1=CC=C2NC=NC2=C1 HYZJCKYKOHLVJF-UHFFFAOYSA-N 0.000 description 1
- JWYUFVNJZUSCSM-UHFFFAOYSA-N 2-aminobenzimidazole Chemical compound C1=CC=C2NC(N)=NC2=C1 JWYUFVNJZUSCSM-UHFFFAOYSA-N 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 239000005500 Clopyralid Substances 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000204939 Fasciola gigantica Species 0.000 description 1
- 241000242711 Fasciola hepatica Species 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000018297 Immunoglobulin subtype Human genes 0.000 description 1
- 108050007411 Immunoglobulin subtype Proteins 0.000 description 1
- 239000005662 Paraffin oil Substances 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- 241000242541 Trematoda Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 230000000507 anthelmentic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- HUBANNPOLNYSAD-UHFFFAOYSA-N clopyralid Chemical compound OC(=O)C1=NC(Cl)=CC=C1Cl HUBANNPOLNYSAD-UHFFFAOYSA-N 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 238000001172 liquid--solid extraction Methods 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000002609 medium Substances 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
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- IEHKWSGCTWLXFU-IIBYNOLFSA-N tadalafil Chemical compound C1=C2OCOC2=CC([C@@H]2C3=C([C]4C=CC=CC4=N3)C[C@H]3N2C(=O)CN(C3=O)C)=C1 IEHKWSGCTWLXFU-IIBYNOLFSA-N 0.000 description 1
- 229960000835 tadalafil Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- 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
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
- G01N2430/10—Insecticides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC及其应用,属于食品安全免疫检测领域。本发明分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,保藏编号CGMCC No.17395。所述杂交瘤细胞株DNC分泌产生的单克隆抗体对三氯苯达唑具有较好亲和力和较高灵敏度,对三氯苯达唑的50%抑制浓度IC50为1.77ng/mL,可用于制备三氯苯达唑的免疫检测试剂盒以及胶体金试纸条,为动物源性食品中三氯苯达唑的检测提供有力的检测方法及手段。
Description
技术领域
本发明涉及一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC及其应用,属于食品安全免疫检测领域。
背景技术
三氯苯达唑属于新型的苯并咪唑类驱虫药,对牛、羊体内各发育阶段的肝片吸虫、大片形吸虫及前后盘吸虫均有良好的驱杀作用,且毒性较小。因此,在兽医临床上被广泛用来防治反刍家畜的吸虫病。
有关三氯苯达唑药物残留在动物组织中的检测方法,国内外已有少量报道,主要是用高效液相色谱~荧光检测器检测法及液相色谱一串联质谱法。提取的方法也各有不同,有液—液萃取、液—固萃取等。仪器检测方法可进行定量分析并具有较低的检测限,但是通常需要昂贵的仪器和复杂的操作,前处理及检测时间长,严重制约了这些检测方法的推广。而免疫分析方法具有低成本、高通量、高灵敏、对技术人员相对要求低等特点,因此适用于大量样品的快速筛查。本发明的目的在于提供一种对三氯苯达唑具有较高亲和力和检测灵敏度的单克隆抗体杂交瘤细胞株的制备方法。为间接竞争 ELISA 试剂盒以及胶体金试纸条的研发推广奠定了基础。
发明内容
本发明的目的是提供一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC及其应用,由该细胞株制备的单克隆抗体对三氯苯达唑具有较好的亲和力和灵敏度,可以用来建立三氯苯达唑酶联免疫检测方法,或建立胶体金免疫层析试纸条快速检测方法。
本发明的技术方案,一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCC No.17395。
抗三氯苯达唑单克隆抗体,它由所述保藏编号为CGMCC No.17395的分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC分泌产生。
所述抗三氯苯达唑单克隆抗体的应用,用于食品中三氯苯达唑残留的检测。
本发明提供的分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC的制备步骤如下:
(1)半抗原的合成:通过3-氯过苯甲酸,4-羟基丁酸甲酯与三氯苯达唑反应从而衍生出羧基,以便于连接载体蛋白;
(2)免疫原的制备与鉴定:以三氯苯达唑衍生物(TCD-H)为原料,通过活化酯法与蛋白载体的氨基相连,反应结束后,通过透析分离完全抗原和未偶联的小分子半抗原,完全抗原通过紫外吸收扫描方法鉴定;
(3)小鼠的免疫:选取6~8周龄的BALB/c小鼠进行免疫。将免疫原与福氏佐剂乳化完全后,通过皮下多点注射免疫小鼠,首次免疫采用福氏完全佐剂,加强免疫使用福氏不完全佐剂,冲刺免疫时免疫剂量为前一次免疫剂量的一半,与生理盐水混合均匀后直接进行腹腔注射;各次免疫间隔为三周。第三次免疫后,间隔一周采血检测血清效价和抑制;
(4)细胞融合与细胞株建立:通过聚乙二醇(PEG 2000)法,使小鼠脾细胞和小鼠骨髓瘤细胞融合,通过HAT培养基培养,利用间接ELISA检测阳性细胞孔,并进一步利用间接竞争ELISA法测定阳性细胞孔的抑制效果,通过有限稀释法对有最好抑制的阳性细胞孔进行三次亚克隆,最终筛选获得杂交瘤细胞株DNC;
(5)杂交瘤细胞株性质的鉴定:采用小鼠单抗Ig类/亚类鉴定用酶标二抗套装测定;IC50值、交叉反应率和亲和力的测定通过ELISA法。
所述杂交瘤细胞株DNC采用的完全抗原的制备过程,具体步骤如下所示:
(1)半抗原的合成:
首先将5g TCD溶于100mL二氯甲烷/四氢呋喃(v/v, 1:1)中,加入4g 3-氯过苯甲酸,所得混合溶液在室温下搅拌3小时。然后将100mL 1M Na2S2O3溶液加入上述溶液中,三次用二氯甲烷萃取水相。结合有机部分用200mL盐水冲洗,减压浓缩,得到化合物C1,沉淀为黄色固体;
将5g化合物C1溶于100mL DMF中,加入4g NaHS,90℃搅拌过夜。反应结束后,加入300mL去离子水,用二氯甲烷三次萃取水相。结合有机部分用200mL盐水冲洗,减压浓缩得到粗化合物C2为白色固体;
4.0g化合物C2、0.6 g 4-羟基丁酸甲酯、6.4g三苯基膦溶于N、N-二甲基甲酰胺50mL中,冷却至0℃。滴加2 g偶氮二甲酸二异丙酯。反应混合物在0℃下搅拌2小时,加入去离子水淬硬。用二氯甲烷三次萃取水相。结合有机部分用200mL盐水冲洗,MgSO4上干燥,真空浓缩,得到化合物C3,为棕色液体;
将1.0g化合物C3溶于50%四氢呋喃水溶液20mL中,加入0.5g LiOH-H2O, 室温搅拌1h。反应结束后,溶液在真空中浓缩,混合溶液的pH值调整为3,加入1M盐酸溶液,然后用20mL乙醇三次萃取水相。将组合后的有机部分用200mL盐水冲洗,在Na2SO4上干燥,减压浓缩得到TCD-H半抗原,为白色固体;
(2)完全抗原的合成:取4.5mg上述半抗原,再加入5.0 mg EDC,即1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和3.7mg N-羟基琥珀酰亚胺NHS,使用N,N-二甲基甲酰胺DMF溶解,室温搅拌,活化6h;另取15mg钥孔血蓝蛋白KLH溶解于3mL、0.05M、pH9.6的碳酸盐缓冲溶液CB中;将上述活化液逐滴加入KLH溶液中,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
本发明的有益效果:本发明细胞株获得的抗三氯苯达唑单克隆抗体,对三氯苯达唑有较好的检测灵敏度和亲和力;本发明还提供了一种新的合成三氯苯达唑半抗原和免疫原的方法,合成步骤更加简化,有效,为今后人们的研究提供了合成免疫原的思路与方法。
生物材料样品保藏:一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCC No.17395。
附图说明
图1 是三氯苯达唑半抗原的合成路径。
图2是免疫原的紫外吸收光谱表征。
图3是三氯苯达唑单克隆抗体的标准抑制曲线。
具体实施方式
本发明下面的实施例仅作为本发明内容的进一步说明,不能作为本发明的限定内容或范围。下面通过实施例对本发明作进一步说明。
本发明通过将三氯苯达唑完全抗原免疫小鼠,通过细胞融合,HAT选择性培养基培养,通过间接ELISA和间接竞争ELISA筛选细胞上清,最终得到了对三氯苯达唑有较好亲和力和灵敏度的单克隆抗体杂交瘤细胞株。
实施例1:抗三氯苯达唑单克隆抗体杂交瘤细胞株DNC的制备
1、半抗原的合成:合成路径如图1所示
首先将5g TCD溶于100mL二氯甲烷/四氢呋喃(v/v, 1:1)中,加入4g 3-氯过苯甲酸,所得混合溶液在室温下搅拌3小时。然后将100mL 1M Na2S2O3溶液加入上述溶液中,三次用二氯甲烷萃取水相。结合有机部分用200mL盐水冲洗,减压浓缩,得到化合物C1,沉淀为黄色固体;
将5g化合物C1溶于100mL DMF中,加入4g NaHS,90℃搅拌过夜。反应结束后,加入300mL去离子水,用二氯甲烷三次萃取水相。结合有机部分用200mL盐水冲洗,减压浓缩得到粗化合物C2为白色固体;
4.0g化合物C2、0.6 g 4-羟基丁酸甲酯、6.4g三苯基膦溶于N、N-二甲基甲酰胺50mL中,冷却至0℃。滴加2 g偶氮二甲酸二异丙酯。反应混合物在0℃下搅拌2小时,加入去离子水淬硬。用二氯甲烷三次萃取水相。结合有机部分用200mL盐水冲洗,MgSO4上干燥,真空浓缩,得到化合物C3,为棕色液体;
将1.0g化合物C3溶于50%四氢呋喃水溶液20mL中,加入0.5g LiOH-H2O, 室温搅拌1h。反应结束后,溶液在真空中浓缩,混合溶液的pH值调整为3,加入1M盐酸溶液,然后用20mL乙醇三次萃取水相。将组合后的有机部分用200mL盐水冲洗,在Na2SO4上干燥,减压浓缩得到TCD-H半抗原,为白色固体。
2、完全抗原的合成:取4.5mg上述半抗原,再加入5.0 mg EDC(1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐)和3.7mg NHS(N-羟基琥珀酰亚胺),使用DMF(N,N-二甲基甲酰胺)溶解,室温搅拌,活化6h;另取15mg KLH(钥孔血蓝蛋白)溶解于3mL、0.05M、pH9.6的CB(碳酸盐缓冲溶液)溶液中;将上述活化液逐滴加入KLH溶液中,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
3、动物免疫:选择健康的6~8周龄的BALB/c小鼠进行免疫。取三氯苯达唑完全抗原(1mg/mL)与等量福氏佐剂乳化均匀后,通过皮下多点注射免疫BALB/c小鼠,每只100μL。首次免疫采用福氏完全佐剂,加强免疫使用福氏不完全佐剂,冲刺免疫时免疫剂量为前一次免疫剂量的一半,与生理盐水混合均匀后直接进行腹腔注射;各次免疫间隔为三周。第三次免疫后,间隔一周采血检测血清效价和抑制;选择抑制最好的小鼠,在五免后18天冲刺免疫,准备融合。
4、细胞融合:在冲刺免疫三天后,按照常规PEG(聚乙二醇,分子量为2000)方法进行细胞融合,具体步骤如下:
(1)无菌取小鼠脾脏,研磨并通过200目细胞筛网得到脾细胞悬液,并进行细胞计数;
(2)收集SP2/0细胞,悬浮于RPMI-1640基础培养液中,进行细胞计数;
(3)将脾细胞和SP2/0细胞按照2~10:1的计数比例混合,离心后用PEG融合,时间1min,之后按照从慢到快,加入RPMI-1640基础培养液,离心后悬浮于含20% 胎牛血清、2%的50×HAT的RPMI-1640筛选培养液中,加到96孔细胞培养板,置于37℃、5% CO2的培养箱中培养。
5、细胞筛选与细胞株建立:在细胞融合的第3天对融合细胞进行RPMI-1640筛选培养液半换液,第5天进行用含20%胎牛血清、1%的100×HT的RPMI-1640过渡培养液进行全换液,在第7天取细胞上清进行筛选。筛选分两步:第一步先用间接ELISA筛选出阳性细胞孔,第二步选用三氯苯达唑为标准品,用间接竞争ELISA对阳性细胞进行抑制效果测定。选择对三氯苯达唑有较好抑制的细胞孔,采用有限稀释法进行亚克隆,用同样的方法进行检测。重复三次,获得细胞株DNC。
6、单克隆抗体的制备与鉴定:取8~10周龄BALB/c小鼠,每只小鼠腹腔注射石蜡油1mL;7天后每只小鼠腹腔注射1×106杂交瘤细胞,从第7天开始收集腹水,将腹水通过辛酸-饱和硫酸铵法纯化,获得的单抗置于-20℃保存。
使用小鼠单抗亚型鉴定试剂盒对腹水纯化获得的单克隆抗体进行免疫球蛋白亚型鉴定,其亚型为IgG2b型,具体如表1所示。
表1. 三氯苯达唑单克隆抗体的亚型鉴定
使用间接竞争ELISA法,测定单克隆抗体对三氯苯达唑的IC50为1.77μg/L,并验证了其对他达拉非等的IC50及交叉反应率,具体如表2所示。
表2三氯苯达唑单克隆抗体对三氯苯达唑、氨基苯并咪唑、氯羟吡啶、双甲脒的IC50及交叉反应率
7、抗体应用:将杂交瘤细胞株DNC通过体内腹水制备的单克隆抗体应用于三氯苯达唑ELISA添加回收试验,具体步骤如下:
(1)用碳酸盐缓冲液(CBS)稀释好的0.1μg/mL的三氯苯达唑包被作为包被原包被96孔酶标板,每孔100μL,37℃包被2 h后,用PBST洗液洗板三次,每次每孔200μL,每次3min,拍干;
(2)用含0.2%明胶的CBS进行封闭,每孔200μL,37℃封闭2 h,用PBST洗液洗板三次,每次每孔200μL,每次3 min,拍干;
(3)用磷酸盐缓冲液(PBS)分别配置0,0.02,0.05,0.1,0.2,0.5,1,2μg/L的三氯苯达唑标准溶液。将标准溶液以及待检测样品提取液,分别加入到已经封闭好的酶标板中,每孔50μL,每个样品重复3个孔,再每孔加入50μL 1:16000稀释的抗三氯苯达唑单克隆抗体,37℃反应半小时后,洗板拍干;
(4)每孔加入100μL 用含0.1% 明胶的PBS 1:3000稀释的HRP标记的羊抗鼠IgG二抗,37℃反应半小时后,洗板拍干;
(5)每孔加入100μL TMB显色液,37℃显色15min后,每孔加入50μL 2M H2SO4终止液,450nm测吸光值;
(6)添加回收及样品前处理:取新鲜或回温(冷藏保存)的羊肉5g,添加三个不同剂量的三氯苯达唑标准品,分别为5 ng、10ng、20ng。将其置于50mL离心管中,缓慢滴入50%氢氧化钾溶液1mL,在旋涡混合器上充分振荡,缓慢滴入乙酸乙酯20mL,在旋涡混合器上振荡10min,然后放入离心器中以3000 r/min离心5min。移取4mL上清液于另一支离心管中,氮气吹干,加入1mL 含有10%甲醇的PBS 复溶,取50μL 用于检测。采用间接竞争ELISA进行添加回收试验,其回收率分别为91.2%、102.1%、99.4%。
溶液的配置:
碳酸盐缓冲液(CBS):称取Na2CO3 1.59g,NaHCO3 2.93g,分别溶于少量双蒸水后混合,加双蒸水至约800mL混匀,调pH值至9.6,加双蒸水定容至1000mL,4℃贮存备用;
磷酸盐缓冲液(PBS):8.00g NaCl,0.2g KCl,0.24g KH2PO4,3.62g Na2HPO4·12H2O,溶于800mL纯水中,用NaOH或HCl调pH到7.2~7.4,定容至1000mL;
PBST:含0.05% Tween20的PBS;
TMB显色液:A液:Na2HPO4·12H2O 18.43g,柠檬酸9.33g,纯水定容至1000mL;B液:60mg TMB 溶于100 mL乙二醇中。A、B液按体积比1:5混合即为TMB显色液,现用现混。
综上所述仅为本发明的较佳实施例而已,并非用来限定本发明的实施范围。即凡依本发明申请范围的内容所作的等效变化与修饰,都应为本发明的技术范畴。
Claims (4)
1.一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC,已保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC,地址北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,分类命名为单克隆细胞株,保藏日期2019年3月7日,保藏编号CGMCCNo.17395。
2.抗三氯苯达唑单克隆抗体,其特征在于:它由权利要求1所述保藏编号为CGMCCNo.17395的分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株DNC分泌产生。
3.权利要求2所述抗三氯苯达唑单克隆抗体的应用,其特征在于:用于食品中三氯苯达唑残留的检测。
4.权利要求1所述杂交瘤细胞株DNC采用的完全抗原的制备方法,其特征在于具体步骤如下所示:
(1)半抗原的合成:
首先将5g TCD溶于100mL二氯甲烷/四氢呋喃(v/v, 1:1)中,加入4g 3-氯过苯甲酸,所得混合溶液在室温下搅拌3小时;然后将100mL 1M Na2S2O3溶液加入上述溶液中,三次用二氯甲烷萃取水相;结合有机部分用200mL盐水冲洗,减压浓缩,得到化合物C1,沉淀为黄色固体;
将5g化合物C1溶于100mL DMF中,加入4g NaHS,90℃搅拌过夜;反应结束后,加入300mL去离子水,用二氯甲烷三次萃取水相;结合有机部分用200mL盐水冲洗,减压浓缩得到粗化合物C2为白色固体;
4.0g化合物C2、0.6 g 4-羟基丁酸甲酯、6.4g三苯基膦溶于N、N-二甲基甲酰胺50mL中,冷却至0℃;滴加2 g偶氮二甲酸二异丙酯;反应混合物在0℃下搅拌2小时,加入去离子水淬硬;用二氯甲烷三次萃取水相;结合有机部分用200mL盐水冲洗,MgSO4上干燥,真空浓缩,得到化合物C3,为棕色液体;
将1.0g化合物C3溶于50%四氢呋喃水溶液20mL中,加入0.5g LiOH-H2O, 室温搅拌1h;反应结束后,溶液在真空中浓缩,混合溶液的pH值调整为3,加入1M盐酸溶液,然后用20mL乙醇三次萃取水相;将组合后的有机部分用200mL盐水冲洗,在Na2SO4上干燥,减压浓缩得到TCD-H半抗原,为白色固体;
(2)完全抗原的合成:取4.5mg上述半抗原,再加入5.0 mg EDC,即1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和3.7mg N-羟基琥珀酰亚胺NHS,使用N,N-二甲基甲酰胺DMF溶解,室温搅拌,活化6h;另取15mg钥孔血蓝蛋白KLH溶解于3mL、0.05M、pH9.6的碳酸盐缓冲溶液CB中;将上述活化液逐滴加入KLH溶液中,室温搅拌反应过夜后,取出免疫原PBS透析3天,-20℃分装保存。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910653689.0A CN110343669B (zh) | 2019-07-19 | 2019-07-19 | 一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910653689.0A CN110343669B (zh) | 2019-07-19 | 2019-07-19 | 一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110343669A CN110343669A (zh) | 2019-10-18 |
CN110343669B true CN110343669B (zh) | 2021-10-29 |
Family
ID=68179202
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910653689.0A Active CN110343669B (zh) | 2019-07-19 | 2019-07-19 | 一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110343669B (zh) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112462047B (zh) * | 2020-11-13 | 2023-02-07 | 北京元恩生物技术有限公司 | 一种尼卡巴嗪检测试剂盒及其用途 |
CN114317448B (zh) * | 2021-12-28 | 2023-11-28 | 无锡迪腾敏生物科技有限公司 | 一株分泌抗乙螨唑单克隆抗体杂交瘤细胞株及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN203465267U (zh) * | 2013-09-18 | 2014-03-05 | 杭州天迈生物科技有限公司 | 一种三氯苯哒唑酶联免疫检测试剂盒 |
CN109001334A (zh) * | 2018-09-11 | 2018-12-14 | 广东出入境检验检疫局检验检疫技术中心 | 一种鸡组织中苯并咪唑类药物残留量的测定方法 |
-
2019
- 2019-07-19 CN CN201910653689.0A patent/CN110343669B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN203465267U (zh) * | 2013-09-18 | 2014-03-05 | 杭州天迈生物科技有限公司 | 一种三氯苯哒唑酶联免疫检测试剂盒 |
CN109001334A (zh) * | 2018-09-11 | 2018-12-14 | 广东出入境检验检疫局检验检疫技术中心 | 一种鸡组织中苯并咪唑类药物残留量的测定方法 |
Also Published As
Publication number | Publication date |
---|---|
CN110343669A (zh) | 2019-10-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN113637081B (zh) | 一株分泌抗二甲戊灵单克隆抗体的杂交瘤细胞株及其应用 | |
CN110343669B (zh) | 一株分泌抗三氯苯达唑单克隆抗体的杂交瘤细胞株dnc及其应用 | |
CN112877296A (zh) | 一种抗非那西丁单克隆抗体杂交瘤细胞株ad及其制备方法与应用 | |
CN107119022B (zh) | 一株异菌脲单克隆抗体杂交瘤细胞株zxl-2及其应用 | |
CN104312978B (zh) | 一种妥布霉素单克隆抗体及其制备方法与应用 | |
CN112574956B (zh) | 一株分泌霜霉威单克隆抗体的杂交瘤细胞株及其应用 | |
CN105838681A (zh) | 一株抗***特异性单克隆抗体杂交瘤细胞株c3及其应用 | |
US20220010029A1 (en) | Hybridoma cell strain that secrets anti-dinitolmide monoclonal antibodies and the application of hybridoma cell strain | |
CN110950962B (zh) | 一株分泌双咪苯脲单克隆抗体的杂交瘤细胞株a11s及其应用 | |
CN111334479A (zh) | 一株氯羟吡啶单克隆抗体杂交瘤细胞株tyl及其应用 | |
CN110724192A (zh) | 一株分泌蛇形菌素单克隆抗体的杂交瘤细胞株及其应用 | |
CN106929479B (zh) | 一株维生素b2单克隆抗体杂交瘤细胞株gz-4及其应用 | |
CN111454912B (zh) | 一株赛拉嗪单克隆抗体杂交瘤细胞株及其应用 | |
CN109705220A (zh) | 一株分泌抗氯丙嗪单克隆抗体的杂交瘤细胞株及其应用 | |
CN109022366A (zh) | 一株分泌抗左旋咪唑单克隆抗体的杂交瘤细胞株及其应用 | |
CN111748528B (zh) | 一株分泌抗氟虫腈及其代谢物单克隆抗体的杂交瘤细胞株及其应用 | |
CN114752568A (zh) | 呋塞米单克隆抗体、杂交瘤细胞株及应用 | |
CN106929477B (zh) | 一株抗***素F2α的特异性单克隆抗体杂交瘤细胞株WXX-2及其应用 | |
US10696748B2 (en) | Hybridoma cell line of secreting clarithromycin monoclonal antibodies and preparation method thereof | |
CN113637642A (zh) | 一株分泌三氯杀螨醇单克隆抗体的杂交瘤细胞株及其应用 | |
CN114989144B (zh) | 一种苯醚甲环唑半抗原、完全抗原、抗体及其制备方法和应用 | |
CN106701686B (zh) | 一种杂交瘤细胞株1a5、分泌的抗舒巴坦单克隆抗体及应用 | |
CN112813034B (zh) | 一株分泌恶霜灵单克隆抗体的杂交瘤细胞株及其应用 | |
CN114480295B (zh) | 一株分泌抗仲丁灵单克隆抗体杂交瘤细胞株及其应用 | |
CN107267464B (zh) | 一株分泌抗他达拉非特异性单克隆抗体的杂交瘤细胞株cit1f2及其应用 |
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 |