CN108998425A - One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application - Google Patents
One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application Download PDFInfo
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- CN108998425A CN108998425A CN201811041508.0A CN201811041508A CN108998425A CN 108998425 A CN108998425 A CN 108998425A CN 201811041508 A CN201811041508 A CN 201811041508A CN 108998425 A CN108998425 A CN 108998425A
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- pyridoxol
- solution
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- compound
- monoclonal antibody
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- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 title claims abstract description 60
- 239000011677 pyridoxine Substances 0.000 title claims abstract description 56
- 235000008160 pyridoxine Nutrition 0.000 title claims abstract description 53
- 210000004408 hybridoma Anatomy 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 claims abstract description 19
- 239000000427 antigen Substances 0.000 claims abstract description 13
- 102000036639 antigens Human genes 0.000 claims abstract description 13
- 108091007433 antigens Proteins 0.000 claims abstract description 13
- 238000002156 mixing Methods 0.000 claims abstract description 10
- 235000013305 food Nutrition 0.000 claims abstract description 8
- 230000028327 secretion Effects 0.000 claims abstract description 5
- 235000008452 baby food Nutrition 0.000 claims abstract description 4
- 235000013365 dairy product Nutrition 0.000 claims abstract description 4
- 239000003814 drug Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 48
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 31
- 150000001875 compounds Chemical class 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 24
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 19
- 239000000284 extract Substances 0.000 claims description 17
- 239000000203 mixture Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 13
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- 239000007832 Na2SO4 Substances 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000012267 brine Substances 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 239000011541 reaction mixture Substances 0.000 claims description 9
- 239000000741 silica gel Substances 0.000 claims description 9
- 229910002027 silica gel Inorganic materials 0.000 claims description 9
- 229960001866 silicon dioxide Drugs 0.000 claims description 9
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 9
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 8
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 6
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 6
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims description 6
- WQDUMFSSJAZKTM-UHFFFAOYSA-N Sodium methoxide Chemical compound [Na+].[O-]C WQDUMFSSJAZKTM-UHFFFAOYSA-N 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000000502 dialysis Methods 0.000 claims description 5
- WMFOQBRAJBCJND-UHFFFAOYSA-M lithium hydroxide Inorganic materials [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 5
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 229940126214 compound 3 Drugs 0.000 claims description 4
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- KFAVSLIIWCUXFU-UHFFFAOYSA-N 1-benzyl-4-chloro-2,3-dimethylbenzene Chemical compound C1=C(Cl)C(C)=C(C)C(CC=2C=CC=CC=2)=C1 KFAVSLIIWCUXFU-UHFFFAOYSA-N 0.000 claims description 3
- HEWZVZIVELJPQZ-UHFFFAOYSA-N 2,2-dimethoxypropane Chemical compound COC(C)(C)OC HEWZVZIVELJPQZ-UHFFFAOYSA-N 0.000 claims description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 3
- 239000008346 aqueous phase Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- AGEZXYOZHKGVCM-UHFFFAOYSA-N benzyl bromide Chemical compound BrCC1=CC=CC=C1 AGEZXYOZHKGVCM-UHFFFAOYSA-N 0.000 claims description 3
- 229940125782 compound 2 Drugs 0.000 claims description 3
- 239000012043 crude product Substances 0.000 claims description 3
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- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims 1
- LSNNMFCWUKXFEE-UHFFFAOYSA-M Bisulfite Chemical compound OS([O-])=O LSNNMFCWUKXFEE-UHFFFAOYSA-M 0.000 claims 1
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- 244000005700 microbiome Species 0.000 abstract description 3
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
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- 210000002966 serum Anatomy 0.000 description 3
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- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 2
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- 241001465754 Metazoa Species 0.000 description 2
- 101100203936 Mus musculus Srpra gene Proteins 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
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- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
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- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- LBLYYCQCTBFVLH-UHFFFAOYSA-N 2-Methylbenzenesulfonic acid Chemical compound CC1=CC=CC=C1S(O)(=O)=O LBLYYCQCTBFVLH-UHFFFAOYSA-N 0.000 description 1
- 102000040350 B family Human genes 0.000 description 1
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- JIGUQPWFLRLWPJ-UHFFFAOYSA-N Ethyl acrylate Chemical compound CCOC(=O)C=C JIGUQPWFLRLWPJ-UHFFFAOYSA-N 0.000 description 1
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- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- YDVNLQGCLLPHAH-UHFFFAOYSA-N dichloromethane;hydrate Chemical compound O.ClCCl YDVNLQGCLLPHAH-UHFFFAOYSA-N 0.000 description 1
- 235000015872 dietary supplement Nutrition 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
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 239000008363 phosphate buffer 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
- 238000002203 pretreatment Methods 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- -1 propylene Acetoacetic ester Chemical compound 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 1
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 1
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 1
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 1
- 229960001327 pyridoxal phosphate Drugs 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
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- 239000006152 selective media Substances 0.000 description 1
- 208000031162 sideroblastic anemia Diseases 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 208000003265 stomatitis Diseases 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- VUYXVWGKCKTUMF-UHFFFAOYSA-N tetratriacontaethylene glycol monomethyl ether Chemical compound COCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO VUYXVWGKCKTUMF-UHFFFAOYSA-N 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
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Classifications
-
- 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
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D213/62—Oxygen or sulfur atoms
- C07D213/63—One oxygen atom
-
- 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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
One plant of pyridoxol monoclonal antibody hybridoma cell strain and its application, belong to food safety field of immunodetection.The present invention is prepared for pyridoxol comlete antigen, and it is it is complete with equivalent Freund's adjuvant mixing and emulsifying, BALB/c mouse is immunized by dorsal sc injection, obtain one plant of pyridoxol monoclonal antibody hybridoma cell strain SS0708, it is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.14687, and the deposit date is on September 5th, 2017.The monoclonal antibody of this cell strain secretion has preferable specificity and detection sensitivity (IC to pyridoxol50For 250 ng/mL).Achievement of the present invention can be used for establishing infant food, in dairy products and special medicine purposes food pyridoxol content immunologic detection method, there is practical application value.
Description
Technical field
The present invention relates to one plant of pyridoxol monoclonal antibody hybridoma cell strain and its applications, belong to the immune inspection of food safety
Survey field.
Background technique
Pyridoxol belongs to B family vitamin, is one of the substance with vitamin B6 effect, also referred to as adermin or dimension life
Plain B6 is widely present in food and can be used as dietary supplements.Pyridoxol is easily become in vivo as coenzyme
Pyridoxal 5-phosphate participates in body metabolism.Lack the animal of pyridoxol, protein is changed into carbohydrate and fat will
Suppressed, also will appear rhinitis, stomatitis and dermatitis.Furthermore the B6 that is deficient in vitamin can also cause the artery sclerosis of various internal organs
Disease.Since pyridoxol plays an important role in central nervous system, spasm can be caused by lacking pyridoxol, given pyrrole and trembled
Alcohol can restore quickly.Pyridoxol is insane for treating and preventing pyridoxine deficiency, sideroblastic anemia, pyridoxine dependency
Epilepsy and certain metabolic disorders, thus in food pyridoxol control it is particularly important.
Pyridoxol content analysis method has gas chromatography (GC), high performance liquid chromatography (HPLC), gas chromatography mass spectrometry at present
(GC-MS), the instrumental methods such as LC-MS (LC-MS), but these methods need expensive instrument, professional operator, and
Sample pre-treatments are complicated, and at high cost, the time is long, can not achieve the quick detection of a large amount of samples, therefore establish fast and convenient pyrrole
Alcohol detection method of trembling is of great significance.Enzyme-linked immunization (ELISA) is a kind of extremely efficient, sensitive, quick detection method,
Field quick detection not high and easy to operate to the purity requirement of sample when detection, suitable for great amount of samples.It establishes efficient
Immunological detection method, the monoclonal antibody for screening high specific is important prerequisite.
Summary of the invention
The object of the present invention is to provide one plant of pyridoxol monoclonal antibody hybridoma cell strain SS0708 and its applications, by this
The antibody of cell strain preparation has preferable specificity and detection sensitivity to pyridoxol, can be used to establish the immune of pyridoxol
Learn detection method.
Technical solution of the present invention, one plant of pyridoxol monoclonal antibody hybridoma cell strain, is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, deposit number are CGMCC No.14687, and the deposit date is Septembers 5 in 2017
Day.
Pyridoxol monoclonal antibody is CGMCC No.14687, the hybridization of pyridoxol monoclonal antibody by the deposit number
Tumor cell strain SS0708 secretion generates.
The application of the pyridoxol monoclonal antibody is applied for establishing the immunologic detection method of pyridoxol content
In the detection of residual pyridoxol.
The detection field is in infant food, dairy products or special medicine purposes food.
The preparation method of the pyridoxol immunogene Pyr-KLH, mainly comprises the steps that
(1) preparation of haptens Pyr: pyridoxol (20g, 118mmol) is dissolved in 200mL acetone, and 200mL 2,2- is added
Dimethoxy propane and p-methyl benzenesulfonic acid (81.4g, 473mmol), 130 DEG C are stirred overnight.Reaction mixture is poured into ice water
Cooling simultaneously extracts water phase with DCM.Mixing organic phase is simultaneously washed with brine, Na2SO4Dry concentration.It is produced obtained by Diethyl ether recrystallization
Object.Mixture is filtered, compound as white solid 2 is obtained；
Under 0 DEG C of nitrogen, by NaH(6.88 g, 287 mmol, 60% organic phase) solution be added in 70 mL THF.It is added molten
Compound 2(15 g, 71.7 mmols of the solution in 200 mLTHF).Flow back 30min, and after being cooled to room temperature, benzyl bromide is added
(24.5g, 143mmol), flow back 4h again, is then extracted with dichloromethane.Organic extract is collected, is washed with brine, it is dry
Concentration, obtains dark brown oil.With silicagel column purification residues, brown oil 3 is obtained；
Compound 3(12g, 40.1mmol) aqueous solution in 24 mL, 98% formic acid is added.50 DEG C of 24 h of stirring, then with saturation
NaHCO3Solution is adjusted to pH 7.After neutralization, reaction mixture is repeatedly extracted with methylene chloride.Organic extract is dry
Concentration, obtains dark brown solid, with purified by flash chromatography crude product, obtains compound 4；
Under 0 DEG C of nitrogen, benzyl dimethyl Phenyl chloride (7.60g, 26.6mmol) is dissolved in 30mL anhydrous methanol, by this
Solution is added in sodium methoxide (1.80g, 34mmol) solution.Then 60mL compound 4(4.50g is added into mixture,
In methanol solution 17.4mmol).20min is stirred at room temperature, and evaporation concentrated mixture is simultaneously added it to containing 500 milliliters of heat
In the round-bottomed flask of toluene.After reacting 30min, mixture is concentrated.After reaction, oil residue is dissolved in saturated ammonium chloride solution
In, then it is extracted with dichloromethane.By the dry concentration of organic extract.By organic extract through silica gel column purification, chemical combination is obtained
Object 5；
5(2.60g containing compound, 169mmol) 60mL toluene solution be added CS2CO3(3.70g114mmol) and acrylic acid
Ethyl ester (2.80g, 22.6mmol), 85 DEG C are stirred overnight.Reaction mixture is cooled and poured into water, then with HCl by solution
It is adjusted to pH7, with EA aqueous phase extracted.Mixing organic phase is washed with brine, and uses Na2SO4It is dried and concentrated.It is remained with silica gel column purification
Object obtains compound 6；
6(1.60g containing compound, 3.50mmol) THF/H2O(16.0/4.00mL) LiOH H is added in solution2O(34.7mg,
890 mmol), it is stirred overnight at room temperature.Then solution is adjusted to pH 7 with HCl, extracts water layer with EA.Organic layer is mixed to use
Salt water washing, through Na2SO4Dry concentration, obtains compound 7；
Compound 7(1 g, 2.30 mmol) 20mL MeOH solution in 100 mg Pd/C are added, be stirred overnight at room temperature.It will
Mixture is filtered and is concentrated, and obtains compound as white solid 8 i.e. Pyr.
(2) preparation of comlete antigen:
The preparation of immunogene Pyr-KLH: weighing haptens Pyr, and 1- ethyl-carbodiimide hydrochloride and N- hydroxysuccinimidyl acyl are sub-
Amine is dissolved to obtain A liquid with anhydrous n,N-Dimethylformamide；Keyhole limpet hemocyanin KLH is taken, dissolves to obtain with borate buffer solution
B liquid；In room temperature condition, A liquid is added in B liquid dropwise, room temperature reaction passes through overnight to get conjugate Pyr-KLH mixed liquor
Dialysis separation comlete antigen and the small haptens not being coupled obtain immunogene Pyr-KLH.
The screening technique for preparing the pyridoxol monoclonal antibody hybridoma cell strain is provided, is mainly comprised the steps that
(1) mouse is immune: after immunogene Pyr-KLH and equivalent Freund's adjuvant mixing and emulsifying, being exempted from by dorsal sc injection
Epidemic disease BALB/c mouse；First immunisation complete Freund's adjuvant, multiple booster immunization cannot be used up full Freund's adjuvant, first immunisation and the
It is spaced 28 days between secondary booster immunization, is spaced between multiple booster immunization 21 days, uses Pyr-KLH comlete antigen for the last time
(being free of adjuvant) spurt is immune；Serum titer and inhibition are detected by Indirect cELISA (ic-ELISA)；
(2) cell fusion and cell strain are established: by polyethylene glycol (PEG 4000) method by mouse boosting cell and mouse myeloma
Cell fusion detects positive cell hole using Indirect cELISA (ic-ELISA) by HAT culture medium culture, and
Further using the inhibitory effect of ic-ELISA measurement positive cell hole, by limiting dilution assay to the sun for having best inhibitory effect
Property cell hole be subcloned three times, finally screen obtain the strain of pyridoxol monoclonal antibody hybridoma cell；
(3) identification of hybridoma cell strain property: pass through ic-ELISA measurement sensitivity and specificity.
Beneficial effects of the present invention: the monoclonal of pyridoxol monoclonal antibody hybridoma cell strain secretion provided by the invention
Antibody has preferable specificity and detection sensitivity (IC to pyridoxol50Value be 250 ng/mL), for detection infant food,
Pyridoxol content provides immunological method in dairy products and special medicine purposes food.Pyridoxol monoclonal provided by the invention is anti-
Body hybridoma cell strain and its monoclonal antibody of secretion can be made into the kit for detecting pyridoxol, have practical application valence
Value.
Biological material specimens preservation: one plant of pyridoxol monoclonal antibody hybridoma cell strain SS0708, the hybridoma are thin
Born of the same parents' strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, Institute of Microorganism, Academia Sinica, deposit number are CGMCC No.14687, classification naming monoclonal
Cell strain, the deposit date is on September 5th, 2017.
Detailed description of the invention
Fig. 1 is inhibition standard curve of the pyridoxol monoclonal antibody to pyridoxol.
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 is by being immunized mouse for pyridoxol comlete antigen, by cell fusion, HAT selective medium culture,
Cell conditioned medium is screened by ic-ELISA, having finally obtained has preferably specificity and the monoclonal antibody of sensitivity miscellaneous pyridoxol
Hand over tumor cell strain.
The preparation of 1 hybridoma cell strain SS0708 of embodiment
(1) preparation of comlete antigen:
A, hapten synthesis route is as follows:
Pyridoxol (20 g, 118 mmol) is dissolved in 200 mL acetone, 200 mL 2,2-dimethoxypropanes and right are added
Toluenesulfonic acid (81.4g, 473 mmol), 130 DEG C are stirred overnight.Reaction mixture is poured into ice water and cools down and is extracted with DCM
Water phase.Mixing organic phase is simultaneously washed with brine, Na2SO4Dry concentration.With Diethyl ether recrystallization required product.Mixture is filtered,
Obtain compound as white solid 2；
Under 0 DEG C of nitrogen, by NaH(6.88 g, 287 mmol, 60% oil) solution be added in 70mL THF.Addition is dissolved in
Compound 2(15g, 71.7mmol in 200 mLTHF).Flow back 30min, after being cooled to room temperature, addition benzyl bromide (24.5 g,
143 mmol), flow back 4h again, is then extracted with dichloromethane.Organic extract is collected, is washed with brine, dry, concentration,
Obtain dark brown oil.With silicagel column purification residues, brown oil 3 is obtained；
Compound 3(12 g, 40.1mmol) aqueous solution in 24 mL98% formic acid are added.50 DEG C of 24 h of stirring, then with saturation
NaHCO3Solution is adjusted to pH 7 by solution.After neutralization, reaction mixture is repeatedly extracted with methylene chloride.Organic extract is done
Dry concentration, obtains dark brown solid, with purified by flash chromatography crude product, obtains compound 4；
Under 0 DEG C of nitrogen, benzyl dimethyl Phenyl chloride (7.60 g, 26.6 mmol) is dissolved in 30mL anhydrous methanol, it will
The solution is added in sodium methoxide (1.80 g, 34 mmol) solution.Then 60mL compound 4(4.50 is added into mixture
G, 17.4 mmol) methanol solution in.20 min are stirred at room temperature, and evaporation concentrated mixture is simultaneously added it to containing 500 millis
In the round-bottomed flask for rising hot toluene.After reacting 30 min, mixture is concentrated.After reaction, oil residue is dissolved in saturated ammonium chloride
In solution, then it is extracted with dichloromethane.By the dry concentration of organic extract.By organic extract through silica gel column purification, obtain
Compound 5；
5(2.60 containing compound g, 169 mmol) 60 mL toluene solutions be added CS2CO3(3.70g114mmol) and propylene
Acetoacetic ester (2.80g, 22.6mmol), 85 DEG C are stirred overnight.Reaction mixture is cooled and poured into water, it then will be molten with HCl
Liquid is adjusted to pH7, with EA aqueous phase extracted.Mixing organic phase is washed with brine, and uses Na2SO4It is dried and concentrated.It is residual with silica gel column purification
Object is stayed, compound 6 is obtained；
6(1.60 containing compound g, 3.50 mmol) THF/H2O(16.0/4.00mL) LiOH H is added in solution2O(34.7mg,
890 mmol), it is stirred overnight at room temperature.Then solution is adjusted to pH 7 with HCl, extracts water layer with EA.Organic layer is mixed to use
Salt water washing, through Na2SO4Dry concentration, obtains compound 7；
100 mg Pd/C are added into the 20mL MeOH solution of compound 7(1 g, 2.30 mmol), are stirred overnight at room temperature.
Mixture is filtered and is concentrated, compound as white solid 8, i.e. haptens Pyr are obtained.
B, 2.7 mg Pyr, 1- ethyl-carbodiimide hydrochloride 7.6mg, n-hydroxysuccinimide 4.6mg are weighed, are used
The anhydrous n,N-Dimethylformamide dissolution of 400 μ L, obtains A1 liquid, anti-6-8h is stirred at room temperature.Take keyhole limpet hemocyanin KLH 10mg
(Pyr and KLH molar ratio are respectively 2000:1,4000:1 and 6000:1), is dissolved with 6mL borate buffer solution, obtains B1 liquid,
A1 liquid is added in B1 liquid by room temperature condition dropwise, and room temperature reaction is overnight to get conjugate Pyr-KLH(2000:1,4000:1
And 6000:1) mixed liquor by dialysis separation comlete antigen and the small haptens that are not coupled obtains conjugate Pyr-KLH
(2000:1), Pyr-KLH(4000:1) and Pyr-KLH(6000:1).
(2) preparation of coating antigen Pyr-OVA:
1.6 mg Pyr, 1- ethyl-carbodiimide hydrochloride 3.8 mg, 2.3 mg of n-hydroxysuccinimide are weighed, with 300 μ L
Anhydrous n,N-Dimethylformamide dissolution, obtains A2 liquid, anti-6-8h is stirred at room temperature.Weigh 5mg chicken ovalbumin OVA(Pyr with
OVA molar ratio is 60:1), it is dissolved in 2mL borate buffer solution, obtains B2 solution, at room temperature, dropwise add A2 liquid
Enter into B2 liquid, room temperature reaction is not coupled by dialysis separation coating antigen and overnight to get conjugate Pyr-OVA mixed liquor
Small haptens.Detection of the coating antigen for mouse serum titer and inhibition in monoclonal antibody preparation process, is not used directly for small
Mouse is necessary to prepare monoclonal antibody.
(3) animal immune: the BALB/c mouse of 6~8 week old of health is selected to be immunized.Take three kinds of different mol ratios
After pyridoxol comlete antigen and equivalent Freund's adjuvant mixing and emulsifying, BALB/c mouse is immunized by dorsal sc injection respectively.The
Primary immunization complete Freund's adjuvant all cannots be used up full Freund's adjuvant later.Between between first immunisation and second of booster immunization
It is spaced 21 days between 28 days, multiple booster immunization.(mouse docking blood sampling+995 uL of 5 uL of blood sampling in 7 days after third time is immune
Antibody diluent=antiserum), mice serum potency and inhibition are measured using ic-ELISA, the mouse for selecting potency height to inhibit,
Spurt in 21 days is immune after the fifth immunization, intraperitoneal injection, it is desirable that punching is exempted from dosage and halved and without any adjuvant.
(4) cell fusion: after spurt immune three days, according to conventional PEG(polyethylene glycol, molecular weight 4000) method into
Row cell fusion, the specific steps are as follows:
A, it plucks eyeball and takes blood, after cervical dislocation puts to death mouse, be immediately placed in 75% alcohol and sterilize, impregnate 5 min or so, nothing
The spleen of mouse is taken out in bacterium operation, is moderately ground with the rubber head of syringe and obtains splenocyte suspension by 200 mesh cell screen clothes,
It collects, is centrifuged (1200 rpm, 8 min), washs splenocyte three times with RPMI-1640 culture medium, after last time is centrifuged, by spleen
Cell is diluted to certain volume, counts, spare；
B, collect SP2/0 cell: 7-10 days before fusion, SP2/0 oncocyte being used and contains 10% FBS(fetal calf serum) RPMI-1640
Culture medium is in 5% CO2In incubator.SP2/0 oncocyte quantity is required to reach 1 ~ 4 × 10 before fusion7, guarantee SP2/0 before merging
Oncocyte is in logarithmic growth phase.When fusion, oncocyte is collected, is suspended in RPMI-1640 basic culture solution, carries out cytometer
Number；
C, 7 min of fusion process.The PEG 1500 of 1 mL is added drop-wise in cell by the 1st min from slow to fast；2nd min, it is quiet
It sets.1 mL RPMI-1640 culture medium is added dropwise in 3rd min and the 4th min in 1 min；5th min and the 6th min, 1
2 mL RPMI-1640 culture mediums are added dropwise in min；The RPMI-1640 culture medium of 1 mL is added dropwise in 7th min, every 10 s.Then 37
DEG C 5 min of warm bath.It is centrifuged (800 rpm, 8 min), abandons supernatant, be resuspended into containing 20% fetal calf serum, 2% 50 × HAT's
In RPMI-1640 screening and culturing liquid, 96 porocyte plates are added to according to 200 holes μ L/, are placed in 37 DEG C, 5% CO2It is cultivated in incubator.
(5) cell screening and cell strain are established: carrying out RPMI-1640 screening to fused cell within the 3rd day in cell fusion
Culture solution partly changes liquid, carries out within the 5th day being carried out with the RPMI-1640 transition culture solution containing 20% fetal calf serum, 1% 100 × HT complete
Liquid is changed, 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 ic-ELISA,
It is standard items that second step, which selects pyridoxol, carries out inhibitory effect measurement to positive cell with ic-ELISA.Selection is to pyridoxol mark
Quasi- product have the cell hole preferably inhibited, are subcloned using limiting dilution assay, are detected with same method.Repeat three
It is secondary, obtain cell strain 2F9.
(6) preparation and identification of monoclonal antibody: taking 8-10 week old BALB/c mouse, and every mouse peritoneal injects sterile stone
1 mL of wax oil；Every mouse peritoneal injection 1 × 10 after 7 days6Hybridoma collects ascites since the 7th day, ascites is led to
Cross caprylic acid-ammonium purifying.Under the conditions of meta-acid, caprylic acid can be precipitated in ascites except ultrawhite other of IgG immune globulin
Foreign protein is then centrifuged for, and abandons precipitating；Again with the monoclonal antibody of the ammonium sulfate precipitating IgG type of equivalent saturation degree, it is centrifuged,
Supernatant is abandoned, after 0.01M PBS solution (pH 7.4) dissolution, dialysis desalting, the monoclonal antibody for finally obtaining after purification is placed in-
20 DEG C of preservations.
The IC of 2 pyridoxol monoclonal antibody of embodiment50Measurement
Carbonate buffer solution (CBS): Na is weighed2CO31.59 g, NaHCO32.93 g are mixed after being dissolved in a small amount of distilled water respectively
It closes, adds distilled water to mix to about 800 mL, adjust pH value to 9.6, add distilled water to be settled to 1000 mL, 4 DEG C of storages are spare.
Phosphate buffer (PBS): 8.0 g NaCl, 0. 2g KCl, 0.2g KH2PO4, 2.9 g Na2HPO4•12
H2O is dissolved in 800 mL pure water, with NaOH or HCl tune pH to 7.2~7.4, is settled to 1000 mL；
Cleaning solution (PBST): the Tween -20 of 0.5 mL is added in the PBS solution of 0.01 mol/LpH7.4 of 1000 mL；
PBST: the PBS containing 0.05 % Tween -20；
Antibody diluent: the washing buffer containing 0.1% gelatin；
TMB developing solution: A liquid: Na2HPO4.12H218.43 g of O, 9.33 g of citric acid, pure water are settled to 1000 mL；B liquid: 60
Mg TMB is dissolved in 100 mL ethylene glycol.A, B liquid 1:5 mixing by volume is TMB
Developing solution, it is current existing mixed.
(1) be coated with: by coating antigen Pyr-OVA, with 0.05M pH9.6 carbonate buffer solution, the multiple proportions since 1 μ g/mL is dilute
It releases, 100 holes μ L/, 37 DEG C of 2 h of reaction.
(2) it washs: solution in plate is inclined, and wash 3 times with cleaning solution, every time 3 min.
(3) it closes: after patting dry, 200 hole μ L/ confining liquids, 37 DEG C of 2 h of reaction is added.It is dried for standby after washing.
(4) be loaded: by antiserum (after mouse docking blood sampling, i.e. antiserum being diluted after corresponding multiple with antibody diluent) from
1:1000 starts doubling dilution, and is added in the coating hole of each dilution, 100 holes μ L/, 37 DEG C of 30 min of reaction；Sufficiently wash
After washing, the diluted HRP- sheep anti-mouse igg of 1:3000,100 holes μ L/, 37 DEG C of 30 min of reaction are added.
(5) it develops the color: ELISA Plate is taken out, sufficiently after washing, the TMB developing solution of 100 μ L is added in every hole, and 37 DEG C are protected from light instead
Answer 15 min.
(6) terminate and measure: 50 μ L terminate liquids are added to terminate reaction in every hole, and the OD450 in each hole is then measured with microplate reader
Value.
With the IC of ic-ELISA measurement monoclonal antibody pyridoxol50For 250 ng/mL, illustrate there is good spirit to pyridoxol
Sensitivity can be used for the detection of pyridoxol immunoassay.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (5)
1. one plant of pyridoxol monoclonal antibody hybridoma cell strain, it is common to be preserved in China Committee for Culture Collection of Microorganisms
Microorganism center, deposit number are CGMCC No.14687, and the deposit date is on September 5th, 2017.
2. pyridoxol monoclonal antibody, which is characterized in that the deposit number as described in claim 1 is CGMCC No.14687, pyrrole
The alcohol monoclonal antibody hybridoma cell strain SS0708 that trembles secretion generates.
3. the application of pyridoxol monoclonal antibody described in claim 2, it is characterized in that: the immune inspection for establishing pyridoxol content
Survey method, applied in the detection for remaining pyridoxol.
4. the application of pyridoxol monoclonal antibody according to claim 3, it is characterized in that: the detection field is infant's food
In product, dairy products or special medicine purposes food.
5. being used to prepare the immunogene Pyr-KLH of pyridoxol monoclonal antibody hybridoma cell strain SS0708 described in claim 1
Preparation method, it is characterized in that steps are as follows:
(1) preparation of haptens Pyr:
A, pyridoxol 20g, 118mmol are dissolved in 200mL acetone, 200mL 2,2-dimethoxypropane are added and to toluene
81.4g, 473mmol, 130 DEG C of sulfonic acid are stirred overnight；Reaction mixture is poured into ice water and cools down and extracts water phase with DCM, is mixed
It closes organic phase and is washed with brine, Na2SO4Dry concentration, with Diethyl ether recrystallization products therefrom, mixture is filtered, obtains white
Solid chemical compound 2；
B, under 0 DEG C of nitrogen, by 6.88 g of NaH, 287 mmol, the solution of 60% organic phase is added in 70 mL THF, is added molten
15 g compound 2,71.7 mmol of the solution in 200 mL THF；Flow back 30min, and after being cooled to room temperature, benzyl bromide is added
24.5g, 143mmol, flow back 4h again, is then extracted with dichloromethane；Organic extract is collected, is washed with brine, drying is dense
Contracting, obtains dark brown oil, with silicagel column purification residues, obtains brown oil compound 3；
C, 24 mL, 98% formic acid, 50 DEG C of 24 h of stirring, then with saturation are added in the aqueous solution of 12g, 40.1mmol compound 3
NaHCO3Solution is adjusted to pH 7；After neutralization, reaction mixture is repeatedly extracted with methylene chloride, organic extract is dry
Concentration, obtains dark brown solid, with purified by flash chromatography crude product, obtains compound 4；
D, under 0 DEG C of nitrogen, benzyl dimethyl Phenyl chloride 7.60g, 26.6mmol are dissolved in 30mL anhydrous methanol, by this
Solution is added to sodium methoxide 1.80g, in 34mmol solution, is then added into mixture and contains 4.50g, 17.4mmol compound 4
60mL methanol solution in；20min is stirred at room temperature, and evaporation concentrated mixture is simultaneously added it to containing 500mL hot toluene
In round-bottomed flask, after reacting 30min, mixture is concentrated；After reaction, oil residue is dissolved in saturated ammonium chloride solution, then
It is extracted with dichloromethane, by the dry concentration of organic extract, by organic extract through silica gel column purification, obtains compound 5；
E, contain 2.60g, CS is added in the 60mL toluene solution of 169mmol compound 52CO33.70g114mmol and acrylic acid second
2.80g, 22.6mmol, 85 DEG C of ester are stirred overnight；Reaction mixture is cooled and poured into water, is then adjusted to solution with HCl
PH7, with EA aqueous phase extracted；Mixing organic phase is washed with brine, and uses Na2SO4It is dried and concentrated；With silicagel column purification residues, obtain
To compound 6；
D, contain 1.60g, the THF/H of 3.50mmol compound 62LiOH H is added in O 16.0/4.00mL meter, solution2O 34.7mg,
890mmol is stirred overnight at room temperature；Then solution is adjusted to pH7 with HCl, extracts water layer with EA；Mix organic layer salt water
Washing, through Na2SO4Dry concentration, obtains compound 7；
Containing 1g, 100mg Pd/C is added in the 20mL MeOH solution of 2.30mmol compound 7, is stirred overnight at room temperature；It will mixing
Object is filtered and is concentrated, and obtains compound as white solid 8 i.e. Pyr；
(2) haptens Pyr, 1- ethyl-carbodiimide hydrochloride and N- hydroxysuccinimidyl the preparation of immunogene Pyr-KLH: are weighed
Acid imide is dissolved to obtain A liquid with anhydrous n,N-Dimethylformamide；Keyhole limpet hemocyanin KLH is taken, is dissolved with borate buffer solution
Obtain B liquid；In room temperature condition, A liquid is added in B liquid dropwise, room temperature reaction is stayed overnight to get conjugate Pyr-KLH mixed liquor,
Immunogene Pyr-KLH is obtained by dialysis separation comlete antigen and the small haptens not being coupled.
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