CN110343669A - One plant of hybridoma cell strain DNC for secreting anti-Triclabendazole monoclonal antibody and its application - Google Patents
One plant of hybridoma cell strain DNC for secreting anti-Triclabendazole monoclonal antibody and its application Download PDFInfo
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- CN110343669A CN110343669A CN201910653689.0A CN201910653689A CN110343669A CN 110343669 A CN110343669 A CN 110343669A CN 201910653689 A CN201910653689 A CN 201910653689A CN 110343669 A CN110343669 A CN 110343669A
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- triclabendazole
- solution
- monoclonal antibody
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- 229960000323 triclabendazole Drugs 0.000 title claims abstract description 42
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 21
- 230000003248 secreting effect Effects 0.000 title abstract description 3
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 230000028327 secretion Effects 0.000 claims abstract description 9
- 244000005700 microbiome Species 0.000 claims abstract description 6
- 235000013305 food Nutrition 0.000 claims abstract description 5
- 239000000243 solution Substances 0.000 claims description 35
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 27
- 210000004027 cell Anatomy 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 229910001868 water Inorganic materials 0.000 claims description 18
- 150000001875 compounds Chemical class 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 13
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 12
- 239000008346 aqueous phase Substances 0.000 claims description 12
- 150000003839 salts Chemical class 0.000 claims description 12
- 230000015572 biosynthetic process Effects 0.000 claims description 11
- 238000003786 synthesis reaction Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
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- 239000007787 solid Substances 0.000 claims description 9
- 239000000427 antigen Substances 0.000 claims description 8
- 102000036639 antigens Human genes 0.000 claims description 8
- 108091007433 antigens Proteins 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000004321 preservation Methods 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
- SJZRECIVHVDYJC-UHFFFAOYSA-N 4-hydroxybutyric acid Chemical compound OCCCC(O)=O SJZRECIVHVDYJC-UHFFFAOYSA-N 0.000 claims description 4
- 229940006015 4-hydroxybutyric acid Drugs 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 4
- ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 2,3-dimethylbutane Chemical group CC(C)C(C)C ZFFMLCVRJBZUDZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000007832 Na2SO4 Substances 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
- NEHMKBQYUWJMIP-UHFFFAOYSA-N chloromethane Chemical class ClC NEHMKBQYUWJMIP-UHFFFAOYSA-N 0.000 claims description 3
- 238000002242 deionisation method Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 108010045069 keyhole-limpet hemocyanin Proteins 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
- 238000012856 packing Methods 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 238000007788 roughening Methods 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
- 238000003756 stirring Methods 0.000 claims description 3
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 2
- 150000003851 azoles Chemical class 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- UXGNZZKBCMGWAZ-UHFFFAOYSA-N dimethylformamide dmf Chemical compound CN(C)C=O.CN(C)C=O UXGNZZKBCMGWAZ-UHFFFAOYSA-N 0.000 claims description 2
- RELMFMZEBKVZJC-UHFFFAOYSA-N 1,2,3-trichlorobenzene Chemical class ClC1=CC=CC(Cl)=C1Cl RELMFMZEBKVZJC-UHFFFAOYSA-N 0.000 claims 1
- 235000019441 ethanol Nutrition 0.000 claims 1
- 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 abstract description 27
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 241001465754 Metazoa Species 0.000 abstract description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 abstract 1
- 230000036039 immunity Effects 0.000 abstract 1
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- 239000012980 RPMI-1640 medium Substances 0.000 description 5
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- 229920001223 polyethylene glycol Polymers 0.000 description 5
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- 239000007924 injection Substances 0.000 description 4
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- 238000012360 testing method Methods 0.000 description 4
- 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
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000007853 buffer solution Substances 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
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 3
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- 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
- 108010010803 Gelatin Proteins 0.000 description 2
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- 239000002202 Polyethylene glycol Substances 0.000 description 2
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- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 239000012894 fetal calf serum Substances 0.000 description 2
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- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 1
- 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
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- ZDPIZLCVJAAHHR-UHFFFAOYSA-N Clopidol Chemical compound CC1=NC(C)=C(Cl)C(O)=C1Cl ZDPIZLCVJAAHHR-UHFFFAOYSA-N 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000204939 Fasciola gigantica Species 0.000 description 1
- 241000242711 Fasciola hepatica Species 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000005662 Paraffin oil Substances 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
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- QXAITBQSYVNQDR-ZIOPAAQOSA-N amitraz Chemical compound C=1C=C(C)C=C(C)C=1/N=C/N(C)\C=N\C1=CC=C(C)C=C1C QXAITBQSYVNQDR-ZIOPAAQOSA-N 0.000 description 1
- 229960002587 amitraz Drugs 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
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
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- 238000012827 research and development Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000926 separation method Methods 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
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
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- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- 231100000419 toxicity Toxicity 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- -1 trichloro-benzenes Azoles Chemical class 0.000 description 1
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
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- 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)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
One plant of hybridoma cell strain DNC for secreting anti-Triclabendazole monoclonal antibody and its application, belong to food safety field of immunodetection.The present invention secretes the hybridoma cell strain DNC of anti-Triclabendazole monoclonal antibody, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, deposit number CGMCC No.17395.The monoclonal antibody that the hybridoma cell strain DNC secretion generates has preferable affinity and higher sensitivity to Triclabendazole, to 50% inhibition concentration IC of Triclabendazole50For 1.77ng/mL, it can be used for preparing the immunity detection reagent and colloidal gold strip of Triclabendazole, the detection for Triclabendazole in animal derived food provides strong detection method and means.
Description
Technical field
The hybridoma cell strain DNC for secreting anti-Triclabendazole monoclonal antibody the present invention relates to one plant and its application belong to
In food safety field of immunodetection.
Background technique
Triclabendazole belongs to novel benzimidazole anthelmintic, to the Fasciola hepatica of each stage of development in ox, sheep body,
Fasciola gigantica and front and back disk fluke have it is good kill effect, and toxicity is smaller.Therefore, it is used extensively in veterinary clinic
To prevent and treat the fluke disease of ruminant domestic animal.
Detection method in relation to Triclabendazole medicament residue in animal tissue, it is existing both at home and abroad to report on a small quantity, mainly
It is with one tandem mass spectrometry of high performance liquid chromatography~fluorescence detector detection method and liquid chromatogram.The method of extraction also respectively has not
Together, there is liquid-liquid extraction, liquid-solid extraction etc..Instrument detection method can carry out quantitative analysis and have lower detection limit, but
It is to usually require expensive instrument and complicated operation, pre-treatment and detection time are long, seriously constrain these detection methods
It promotes.And immunoassay method have the characteristics that it is inexpensive, high-throughput, highly sensitive, low to technical staff's relative requirement therefore suitable
Rapid screening for a large amount of samples.The purpose of the present invention is to provide a kind of pair of Triclabendazoles to have higher affinity and inspection
Survey the preparation method of the monoclonal antibody hybridoma cell strain of sensitivity.For indirect competitive ELISA kit and colloidal gold
The research and development popularization of test strips is laid a good foundation.
Summary of the invention
The object of the present invention is to provide the hybridoma cell strain DNC of one plant of anti-Triclabendazole monoclonal antibody of secretion and its
Using the monoclonal antibody prepared by the cell strain with preferable affinity and sensitivity, can be used to Triclabendazole
Triclabendazole enzyme-linked immune detection method is established, or establishes colloidal gold immuno-chromatography test paper strip rapid detection method.
Technical solution of the present invention, the hybridoma cell strain DNC of one plant of anti-Triclabendazole monoclonal antibody of secretion, has been protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, address BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica, institute, classification naming are monoclonal cell strain, preservation date on March 7th, 2019, are protected
Hide number CGMCC No.17395.
Anti- Triclabendazole monoclonal antibody, the anti-trichloro-benzenes of secretion that it is CGMCC No.17395 by the deposit number
Hybridoma cell strain DNC up to azoles monoclonal antibody secretes generation.
The application of the anti-Triclabendazole monoclonal antibody, for the remaining detection of Triclabendazole in food.
The preparation step of the hybridoma cell strain DNC of the anti-Triclabendazole monoclonal antibody of secretion provided by the invention is such as
Under:
(1) synthesis of haptens: by 3- chlorine benzylhydroperoxide, 4 hydroxybutyric acid methyl esters reacts to derive with Triclabendazole
Carboxyl, in order to connect carrier protein；
(2) preparation and identification of immunogene: with Triclabendazole derivative (TCD-H) for raw material, pass through active ester method and albumen
The amino of carrier is connected, and after reaction, passes through small haptens dialysis separation comlete antigen and be not coupled, comlete antigen
It is identified by UV absorption scan method；
(3) mouse is immune: the BALB/c mouse for choosing 6~8 week old is immunized.Immunogene and freund adjuvant emulsification is complete
Afterwards, mouse is immunized by subcutaneous multi-point injection, first immunisation uses Freund's complete adjuvant, and booster immunization is not exclusively helped using Fu Shi
Agent, immunizing dose is the half of a preceding immunizing dose when spurt is immune, directly carries out abdominal cavity after mixing with physiological saline
Injection；Each secondary immunization interval is three weeks.After third time is immune, interval blood sampling in one week detection serum titer and inhibition；
(4) cell fusion and cell strain are established: by polyethylene glycol (PEG 2000) method, making mouse boosting cell and mouse myeloma
Cell fusion detects positive cell hole using indirect ELISA, and further utilize indirect competition by HAT culture medium culture
ELISA method measure positive cell hole inhibitory effect, by limiting dilution assay to have the positive cell hole preferably inhibited carry out three
Secondary subclone finally screens and obtains hybridoma cell strain DNC；
(5) it the identification of hybridoma cell strain property: is set with and is measured with ELIAS secondary antibody using mouse monoclonal Ig class/subgroup identification；
IC50The measurement of value, cross reacting rate and affinity passes through ELISA method.
The preparation process for the comlete antigen that the hybridoma cell strain DNC is used, it is shown that specific step is as follows:
(1) synthesis of haptens:
5g TCD is dissolved in 100mL methylene chloride/tetrahydrofuran (v/v, 1:1) first, 4g 3- chlorine benzylhydroperoxide, institute is added
Mixed solution is obtained to be stirred at room temperature 3 hours.Then by 100mL 1M Na2S2O3Solution is added in above-mentioned solution, three times with two
Chloromethanes aqueous phase extracted.It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure, obtains compound C1, it is solid to be precipitated as yellow
Body；
5g compound C1 is dissolved in 100mL DMF, 4g NaHS is added, 90 DEG C are stirred overnight.After reaction, 300mL is added
Deionized water, with methylene chloride aqueous phase extracted three times.It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure to give roughening
Conjunction object C2 is white solid；
4.0g compound C2,0.6 g 4 hydroxybutyric acid methyl esters, 6.4g triphenylphosphine are dissolved in N, dinethylformamide 50mL,
It is cooled to 0 DEG C.2 g diisopropyl azodiformates are added dropwise.Reaction mixture stirs 2 hours at 0 DEG C, and deionization water quenching is added
Firmly.With methylene chloride aqueous phase extracted three times.It is rinsed in conjunction with organic moiety 200mL salt water, MgSO4Upper drying, vacuum concentration, obtains
It is brown liquid to compound C3；
1.0g compound C3 is dissolved in 50% aqueous tetrahydrofuran solution 20mL, 0.5g LiOH-H is added21h is stirred at room temperature in O.
After reaction, solution is concentrated in a vacuum, and the pH value of mixed solution is adjusted to 3, and 1M hydrochloric acid solution is added, and then uses 20mL second
Alcohol aqueous phase extracted three times.Organic moiety after combination 200mL salt water is rinsed, in Na2SO4Upper drying, is concentrated under reduced pressure to give
TCD-H haptens is white solid；
(2) synthesis of comlete antigen: taking the above-mentioned haptens of 4.5mg, adds 5.0 mg EDC, i.e. 1- (3- dimethylamino third
Base) -3- ethyl-carbodiimide hydrochloride and 3.7mg n-hydroxysuccinimide NHS, it is molten using n,N-Dimethylformamide DMF
Solution, is stirred at room temperature, and activates 6h；The carbonate buffer for separately 15mg keyhole limpet hemocyanin KLH being taken to be dissolved in 3mL, 0.05M, pH9.6 is molten
In liquid CB；Above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, immunogene PBS dialysis 3 is taken out
It, -20 DEG C of packing save.
Beneficial effects of the present invention: the anti-Triclabendazole monoclonal antibody that cell strain of the present invention obtains reaches trichloro-benzenes
Azoles has preferable detection sensitivity and affinity；The present invention also provides a kind of new synthesis Triclabendazole haptens and it is immunized
Former method, synthesis step more simplify, and effectively, provide the thinking and method of synthetic immunogen for the research of people from now on.
Biological material specimens preservation: the hybridoma cell strain DNC of one plant of anti-Triclabendazole monoclonal antibody of secretion has been protected
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center CGMCC, address BeiChen West Road, Chaoyang District, BeiJing City 1
Number No. 3 Institute of Microorganism, Academia Sinica, institute, classification naming are monoclonal cell strain, preservation date on March 7th, 2019, are protected
Hide number CGMCC No.17395.
Detailed description of the invention
Fig. 1 is the synthesis path of Triclabendazole haptens.
Fig. 2 is the ultra-violet absorption spectrum characterization of immunogene.
Fig. 3 is the standard suppression curve of Triclabendazole monoclonal antibody.
Specific embodiment
The following examples of the present invention are only used as the further explanation of the content of present invention, not as a limitation of the present invention interior
Perhaps range.Below by embodiment, the invention will be further described.
The present invention passes through cell fusion, the training of HAT selective medium by the way that mouse is immunized in Triclabendazole comlete antigen
Support, cell conditioned medium screened by indirect ELISA and indirect competitive ELISA, finally obtained have to Triclabendazole it is preferably affine
The monoclonal antibody hybridoma cell strain of power and sensitivity.
Embodiment 1: the preparation of anti-Triclabendazole monoclonal antibody hybridoma cell strain DNC
1, the synthesis of haptens: synthesis path is as shown in Figure 1
5g TCD is dissolved in 100mL methylene chloride/tetrahydrofuran (v/v, 1:1) first, 4g 3- chlorine benzylhydroperoxide, institute is added
Mixed solution is obtained to be stirred at room temperature 3 hours.Then by 100mL 1M Na2S2O3Solution is added in above-mentioned solution, three times with two
Chloromethanes aqueous phase extracted.It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure, obtains compound C1, it is solid to be precipitated as yellow
Body；
5g compound C1 is dissolved in 100mL DMF, 4g NaHS is added, 90 DEG C are stirred overnight.After reaction, 300mL is added
Deionized water, with methylene chloride aqueous phase extracted three times.It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure to give roughening
Conjunction object C2 is white solid；
4.0g compound C2,0.6 g 4 hydroxybutyric acid methyl esters, 6.4g triphenylphosphine are dissolved in N, dinethylformamide 50mL,
It is cooled to 0 DEG C.2 g diisopropyl azodiformates are added dropwise.Reaction mixture stirs 2 hours at 0 DEG C, and deionization water quenching is added
Firmly.With methylene chloride aqueous phase extracted three times.It is rinsed in conjunction with organic moiety 200mL salt water, MgSO4Upper drying, vacuum concentration, obtains
It is brown liquid to compound C3；
1.0g compound C3 is dissolved in 50% aqueous tetrahydrofuran solution 20mL, 0.5g LiOH-H is added21h is stirred at room temperature in O.
After reaction, solution is concentrated in a vacuum, and the pH value of mixed solution is adjusted to 3, and 1M hydrochloric acid solution is added, and then uses 20mL second
Alcohol aqueous phase extracted three times.Organic moiety after combination 200mL salt water is rinsed, in Na2SO4Upper drying, is concentrated under reduced pressure to give
TCD-H haptens is white solid.
2, the synthesis of comlete antigen: taking the above-mentioned haptens of 4.5mg, adds 5.0 mg EDC(1- (3- dimethylaminos third
Base) -3- ethyl-carbodiimide hydrochloride) and 3.7mg NHS(N- HOSu NHS), use DMF(N, N- dimethyl formyl
Amine) dissolution, it is stirred at room temperature, activates 6h；Separately take 15mg KLH(keyhole limpet hemocyanin) it is dissolved in the CB of 3mL, 0.05M, pH9.6
In (carbonate buffer solution) solution；Above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, takes out
Immunogene PBS dialyses 3 days, and -20 DEG C of packing save.
3, animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Take Triclabendazole completely anti-
After former (1mg/mL) and the emulsification uniformly of equivalent freund adjuvant, BALB/c mouse, every 100 μ L are immunized by subcutaneous multi-point injection.
First immunisation uses Freund's complete adjuvant, and booster immunization uses freund 's incomplete adjuvant, and immunizing dose is previous when spurt is immune
The half of secondary immunizing dose is directly injected intraperitoneally after mixing with physiological saline；Each secondary immunization interval is three weeks.Third
It is secondary it is immune after, interval blood sampling in one week detects serum titer and inhibition；Selection inhibits best mouse, and spurt in 18 days is exempted from after exempting from five
Epidemic disease prepares fusion.
4, cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 2000) method into
Row cell fusion, the specific steps are as follows:
(1) sterile to take mouse spleen, it grinds and obtains splenocyte suspension by 200 mesh cell screen clothes, and carry out cell count；
(2) SP2/0 cell is collected, is suspended in RPMI-1640 basic culture solution, cell count is carried out；
(3) splenocyte and SP2/0 cell are mixed according to the counting ratio of 2~10:1, is merged after centrifugation with PEG, the time 1
RPMI-1640 basic culture solution is added later according to from slowly to fast in min, is suspended in after centrifugation containing 20% fetal calf serum, 2%
In the RPMI-1640 screening and culturing liquid of 50 × HAT, 96 porocyte culture plates are added to, are placed in 37 DEG C, 5% CO2Incubator in train
It supports.
5, cell screening and cell strain are established: carrying out RPMI-1640 screening training to fused cell within the 3rd day in cell fusion
Nutrient solution partly changes liquid, carries out within the 5th day being changed entirely with the RPMI-1640 transition culture solution of 100 × HT containing 20% fetal calf serum, 1%
Liquid took cell conditioned medium to be screened at the 7th day.Screen in two steps: the first step first filters out positive cell hole with indirect ELISA,
It is standard items that second step, which selects Triclabendazole, carries out inhibitory effect measurement to positive cell with indirect competitive ELISA.Selection pair
Triclabendazole has the cell hole preferably inhibited, is subcloned using limiting dilution assay, is detected with same method.Weight
Again three times, cell strain DNC is obtained.
6, the preparation and identification of monoclonal antibody: taking 8~10 week old BALB/c mouses, and every mouse peritoneal injects paraffin oil
1mL；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma collected ascites since the 7th day, and ascites is passed through octanoic acid-
The purifying of saturated ammonium sulfate method, the monoclonal antibody of acquisition are placed in -20 DEG C of preservations.
It is sub- that immunoglobulin is carried out using the monoclonal antibody that mouse monoclonal subtype identification kit obtains ascites purifying
Type identification, hypotype are IgG2b type, specific as shown in table 1.
The subtype identification of 1. Triclabendazole monoclonal antibody of table
Using indirect competitive ELISA method, monoclonal antibody is measured to the IC of Triclabendazole50For 1.77 μ g/L, and demonstrate it
To the IC of Tadalafei etc.50And cross reacting rate, it is specific as shown in table 2.
2 Triclabendazole monoclonal antibody of table to Triclabendazole, aminobenzimidazole, clopidol, Amitraz IC50
And cross reacting rate
7, hybridoma cell strain DNC antibody application: is applied to Triclabendazole by monoclonal antibody prepared by internal ascites
ELISA adds recovery test, the specific steps are as follows:
(1) Triclabendazole for the 0.1 μ g/mL for using carbonate buffer solution (CBS) to dilute is coated with as coating 96 hole of primordial covering
ELISA Plate, after every hole 100 μ L, 37 DEG C of 2 h of coating, three times with PBST washing lotion board-washing, each every 200 μ L of hole, 3 min, is clapped every time
It is dry；
(2) it is closed with the CBS containing 0.2% gelatin, every hole 200 μ L, 37 DEG C of 2 h of closing, three times with PBST washing lotion board-washing, often
Secondary 200 μ L of every hole, 3 min, pats dry every time；
(3) Triclabendazole of 0,0.02,0.05,0.1,0.2,0.5,1,2 μ g/L is respectively configured with phosphate buffer (PBS)
Standard solution.Standard solution and sample to be tested extracting solution are added separately in the ELISA Plate closed, every hole 50
μ L, 3 holes of each sample repetition, then every hole addition diluted anti-Triclabendazole monoclonal antibody of 50 μ L 1:16000,37 DEG C
After reacting half an hour, board-washing is patted dry；
(4) 100 μ L of the every hole addition sheep anti-mouse igg secondary antibody of the diluted HRP label of the PBS 1:3000 containing 0.1% gelatin, 37
DEG C reaction half an hour after, board-washing pats dry；
(5) every hole is added 100 μ L TMB developing solutions, after 37 DEG C of colour developing 15min, 50 μ L 2M H of every hole addition2SO4Terminate liquid,
450nm surveys light absorption value；
(6) addition recycling and sample pre-treatments: taking fresh or the mutton 5g of (stored refrigerated) of rising again, and adds three various doses
Triclabendazole standard items, respectively 5 ng, 10ng, 20ng.It places it in 50mL centrifuge tube, is slowly dropped into 50% hydroxide
Potassium solution 1mL, sufficiently vibrates on vortex mixer, is slowly dropped into ethyl acetate 20mL, vibrates on vortex mixer
10min is then placed in centrifuge and is centrifuged 5min with 3000 r/min.4mL supernatant is pipetted in another centrifuge tube, nitrogen
Drying is added the PBS that 1mL contains 10% methanol and redissolves, takes 50 μ L for detecting.It is added using indirect competitive ELISA
Recovery test, the rate of recovery are respectively 91.2%, 102.1%, 99.4%.
The configuration of solution:
Carbonate buffer solution (CBS): Na is weighed2CO31.59g NaHCO32.93g is mixed after being dissolved in a small amount of distilled water respectively,
Add distilled water to mix to about 800mL, adjusts pH value to 9.6, add distilled water to be settled to 1000mL, 4 DEG C of storages are spare；
Phosphate buffer (PBS): 8.00g NaCl, 0.2g KCl, 0.24g KH2PO4, 3.62g Na2HPO4·12 H2O,
It is dissolved in 800mL pure water, with NaOH or HCl tune pH to 7.2~7.4, is settled to 1000mL；
PBST: the PBS containing 0.05% Tween20；
TMB developing solution: A liquid: Na2HPO4·12H2O 18.43g, citric acid 9.33g, pure water are settled to 1000mL；B liquid: 60mg
TMB is dissolved in 100 mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB developing solution, current existing mixed.
It is in summary only presently preferred embodiments of the present invention, practical range not for the purpose of limiting the invention.It is i.e. all
Equivalent changes and modifications made by content according to the present patent application range all should be technology scope of the invention.
Claims (4)
1. the hybridoma cell strain DNC of one plant of anti-Triclabendazole monoclonal antibody of secretion, has been preserved in Chinese microorganism strain
Preservation administration committee common micro-organisms center CGMCC, the address Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Institute of microbiology, classification naming are monoclonal cell strain, preservation date on March 7th, 2019, deposit number CGMCC
No.17395。
2. anti-Triclabendazole monoclonal antibody, it is characterised in that: its deposit number as described in claim 1 is CGMCC
The hybridoma cell strain DNC of the anti-Triclabendazole monoclonal antibody of secretion of No.17395, which secretes, to be generated.
3. the application of anti-Triclabendazole monoclonal antibody described in claim 2, it is characterised in that: reached for trichloro-benzenes in food
The remaining detection of azoles.
4. the preparation method for the comlete antigen that hybridoma cell strain DNC described in claim 1 is used, it is characterised in that specific steps
It is as follows:
(1) synthesis of haptens:
5g TCD is dissolved in 100mL methylene chloride/tetrahydrofuran (v/v, 1:1) first, 4g 3- chlorine benzylhydroperoxide, institute is added
Mixed solution is obtained to be stirred at room temperature 3 hours；Then by 100mL 1M Na2S2O3Solution is added in above-mentioned solution, three times with two
Chloromethanes aqueous phase extracted；It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure, obtains compound C1, it is solid to be precipitated as yellow
Body；
5g compound C1 is dissolved in 100mL DMF, 4g NaHS is added, 90 DEG C are stirred overnight；After reaction, 300mL is added
Deionized water, with methylene chloride aqueous phase extracted three times；It is rinsed in conjunction with organic moiety 200mL salt water, is concentrated under reduced pressure to give roughening
Conjunction object C2 is white solid；
4.0g compound C2,0.6 g 4 hydroxybutyric acid methyl esters, 6.4g triphenylphosphine are dissolved in N, dinethylformamide 50mL,
It is cooled to 0 DEG C；2 g diisopropyl azodiformates are added dropwise；Reaction mixture stirs 2 hours at 0 DEG C, and deionization water quenching is added
Firmly；With methylene chloride aqueous phase extracted three times；It is rinsed in conjunction with organic moiety 200mL salt water, MgSO4Upper drying, vacuum concentration, obtains
It is brown liquid to compound C3；
1.0g compound C3 is dissolved in 50% aqueous tetrahydrofuran solution 20mL, 0.5g LiOH-H is added21h is stirred at room temperature in O；Instead
After answering, solution is concentrated in a vacuum, and the pH value of mixed solution is adjusted to 3, and 1M hydrochloric acid solution is added, and then uses 20mL ethyl alcohol
Aqueous phase extracted three times；Organic moiety after combination 200mL salt water is rinsed, in Na2SO4Upper drying, is concentrated under reduced pressure to give TCD-
H haptens is white solid；
(2) synthesis of comlete antigen: taking the above-mentioned haptens of 4.5mg, adds 5.0 mg EDC, i.e. 1- (3- dimethylamino third
Base) -3- ethyl-carbodiimide hydrochloride and 3.7mg n-hydroxysuccinimide NHS, it is molten using n,N-Dimethylformamide DMF
Solution, is stirred at room temperature, and activates 6h；The carbonate buffer for separately 15mg keyhole limpet hemocyanin KLH being taken to be dissolved in 3mL, 0.05M, pH9.6 is molten
In liquid CB；Above-mentioned activating solution is added dropwise in KLH solution, is stirred at room temperature after reaction overnight, immunogene PBS dialysis 3 is taken out
It, -20 DEG C of packing save.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462047A (en) * | 2020-11-13 | 2021-03-09 | 北京元恩生物技术有限公司 | Nicarbazin detection kit and application thereof |
| CN114317448A (en) * | 2021-12-28 | 2022-04-12 | 无锡迪腾敏生物科技有限公司 | Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203465267U (en) * | 2013-09-18 | 2014-03-05 | 杭州天迈生物科技有限公司 | Triclabendazole enzyme-linked immunosorbent assay kit |
| CN109001334A (en) * | 2018-09-11 | 2018-12-14 | 广东出入境检验检疫局检验检疫技术中心 | The measuring method of benzimidazoles residues residual quantity in a kind of chicken tissues |
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2019
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN203465267U (en) * | 2013-09-18 | 2014-03-05 | 杭州天迈生物科技有限公司 | Triclabendazole enzyme-linked immunosorbent assay kit |
| CN109001334A (en) * | 2018-09-11 | 2018-12-14 | 广东出入境检验检疫局检验检疫技术中心 | The measuring method of benzimidazoles residues residual quantity in a kind of chicken tissues |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112462047A (en) * | 2020-11-13 | 2021-03-09 | 北京元恩生物技术有限公司 | Nicarbazin detection kit and application thereof |
| CN114317448A (en) * | 2021-12-28 | 2022-04-12 | 无锡迪腾敏生物科技有限公司 | Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof |
| CN114317448B (en) * | 2021-12-28 | 2023-11-28 | 无锡迪腾敏生物科技有限公司 | Hybridoma cell strain secreting anti-etoxazole monoclonal antibody and application thereof |
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