CN103589688A - Monoclonal antibody resistant to three organophosphorus pesticides and application thereof - Google Patents
Monoclonal antibody resistant to three organophosphorus pesticides and application thereof Download PDFInfo
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- CN103589688A CN103589688A CN201310365944.4A CN201310365944A CN103589688A CN 103589688 A CN103589688 A CN 103589688A CN 201310365944 A CN201310365944 A CN 201310365944A CN 103589688 A CN103589688 A CN 103589688A
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- Prior art keywords
- methyl
- isofenphos
- hybridoma
- monoclonal antibody
- isocarbophos
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- 239000000575 pesticide Substances 0.000 title claims abstract description 13
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 36
- 239000000427 antigen Substances 0.000 claims abstract description 19
- 102000036639 antigens Human genes 0.000 claims abstract description 19
- 108091007433 antigens Proteins 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 13
- 238000005516 engineering process Methods 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 30
- 238000000034 method Methods 0.000 claims description 25
- YFVOXLJXJBQDEF-UHFFFAOYSA-N isocarbophos Chemical compound COP(N)(=S)OC1=CC=CC=C1C(=O)OC(C)C YFVOXLJXJBQDEF-UHFFFAOYSA-N 0.000 claims description 22
- -1 ethyl isofenphos Chemical compound 0.000 claims description 21
- IXTOWLKEARFCCP-UHFFFAOYSA-N propan-2-yl 2-[methoxy-(propan-2-ylamino)phosphinothioyl]oxybenzoate Chemical group CC(C)NP(=S)(OC)OC1=CC=CC=C1C(=O)OC(C)C IXTOWLKEARFCCP-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 16
- 210000004027 cell Anatomy 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 13
- CFEGKPLFISCOOB-UHFFFAOYSA-N CC(C)OC(C(C=CC=C1)=C1P(OC)(Cl)=S)=O Chemical compound CC(C)OC(C(C=CC=C1)=C1P(OC)(Cl)=S)=O CFEGKPLFISCOOB-UHFFFAOYSA-N 0.000 claims description 12
- 238000010790 dilution Methods 0.000 claims description 12
- 239000012895 dilution Substances 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 210000002966 serum Anatomy 0.000 claims description 12
- 238000002965 ELISA Methods 0.000 claims description 11
- 230000002163 immunogen Effects 0.000 claims description 10
- RQTVIKMRXYJTDX-UHFFFAOYSA-N 1-(4-methylphenyl)sulfonyl-4-phenylpiperidine-4-carbonitrile Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N1CCC(C=2C=CC=CC=2)(C#N)CC1 RQTVIKMRXYJTDX-UHFFFAOYSA-N 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 230000036039 immunity Effects 0.000 claims description 8
- 239000011248 coating agent Substances 0.000 claims description 7
- 238000000576 coating method Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 7
- 108010010803 Gelatin Proteins 0.000 claims description 6
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 6
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 6
- 239000008273 gelatin Substances 0.000 claims description 6
- 229920000159 gelatin Polymers 0.000 claims description 6
- 235000019322 gelatine Nutrition 0.000 claims description 6
- 235000011852 gelatine desserts Nutrition 0.000 claims description 6
- 239000012263 liquid product Substances 0.000 claims description 6
- 201000000050 myeloid neoplasm Diseases 0.000 claims description 6
- 238000007789 sealing Methods 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000010025 steaming Methods 0.000 claims description 6
- 239000003905 agrochemical Substances 0.000 claims description 5
- 230000002860 competitive effect Effects 0.000 claims description 5
- 150000002148 esters Chemical class 0.000 claims description 5
- 230000028327 secretion Effects 0.000 claims description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Substances ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- 238000004440 column chromatography Methods 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 4
- 230000024835 cytogamy Effects 0.000 claims description 4
- 239000013642 negative control Substances 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- 229960001196 thiotepa Drugs 0.000 claims description 4
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 3
- 238000003556 assay Methods 0.000 claims description 3
- 238000010241 blood sampling Methods 0.000 claims description 3
- 230000006957 competitive inhibition Effects 0.000 claims description 3
- 239000003636 conditioned culture medium Substances 0.000 claims description 3
- 239000012228 culture supernatant Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims description 3
- 239000005457 ice water Substances 0.000 claims description 3
- 230000000670 limiting effect Effects 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 239000011541 reaction mixture Substances 0.000 claims description 3
- 230000000717 retained effect Effects 0.000 claims description 3
- 210000000952 spleen Anatomy 0.000 claims description 3
- 210000004988 splenocyte Anatomy 0.000 claims description 3
- 210000003462 vein Anatomy 0.000 claims description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000003986 organophosphate insecticide Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 17
- 241000699670 Mus sp. Species 0.000 abstract 1
- 238000012271 agricultural production Methods 0.000 abstract 1
- 230000003053 immunization Effects 0.000 abstract 1
- 238000005406 washing Methods 0.000 description 36
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 18
- 239000003987 organophosphate pesticide Substances 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108010058846 Ovalbumin Proteins 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000000502 dialysis Methods 0.000 description 4
- 230000031700 light absorption Effects 0.000 description 4
- 240000003291 Armoracia rusticana Species 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 108010078791 Carrier Proteins Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 3
- 238000011481 absorbance measurement Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 229960001701 chloroform Drugs 0.000 description 3
- 238000013016 damping Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 229940005654 nitrite ion Drugs 0.000 description 3
- 229940092253 ovalbumin Drugs 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003760 magnetic stirring Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010898 silica gel chromatography Methods 0.000 description 2
- 239000005558 Fluroxypyr Substances 0.000 description 1
- 231100000703 Maximum Residue Limit Toxicity 0.000 description 1
- 101100460719 Mus musculus Noto gene Proteins 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SLUNEGLMXGHOLY-UHFFFAOYSA-N benzene;hexane Chemical compound CCCCCC.C1=CC=CC=C1 SLUNEGLMXGHOLY-UHFFFAOYSA-N 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- MEFQWPUMEMWTJP-UHFFFAOYSA-N fluroxypyr Chemical compound NC1=C(Cl)C(F)=NC(OCC(O)=O)=C1Cl MEFQWPUMEMWTJP-UHFFFAOYSA-N 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003822 preparative gas chromatography Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the field of biotechnology, and relates to a monoclonal antibody resistant to three organophosphorus pesticides and an application thereof. According to the invention, an artificial hapten for common structure of the pesticides is synthesized chemically, and thus, a complete antigen can be prepared for immunizing Balb/C mice, a strong-specificity monoclonal antibody is prepared by a hybridoma technology, and the monoclonal antibody can be used for high-sensitivity rapid detection on agricultural products and residuals of the three pesticides in agricultural production environment. The preparation technology of the monoclonal antibody is feasible, the whole preparation process of the antigen has no need of special instrument and equipment, and the antigen is easy to produce in a large scale.
Description
Technical field
The invention belongs to biological technical field, relate to monoclonal antibody and the application thereof of anti-three kinds of organophosphorus pesticides.
Background technology
Over nearly 20 years, along with the development rapidly of agriculture production, output and the usage quantity of organophosphorus pesticide constantly rise.A large amount of use, the even not proper organophosphorus pesticide that uses, becomes increasingly conspicuous organophosphorus pesticide problem.This not only makes environmental safety, and the healthy probability that threatened by organophosphorus pesticide of people increases, and maximum residue limits for pesticide also becomes the important technology trade barrier that countries in the world are imported and exported for China's Agricultural.Therefore, quick, easy, the sensitive detection method of development organophosphorus pesticide is to environment, food safety, and people are healthy and promote socialist society's Economic development to have very important effect.
At present, the residue detection of organophosphorus pesticide adopts the methods such as tlc, vapor-phase chromatography, high performance liquid chromatography more.These methods and resultses are reliable, highly sensitive, and existing relevant technical bid will definitely be for reference.But due to the instrument of needs costliness, special operator, and sample pre-treatments is complicated, cost is high, the time is long, therefore can not meet better fast and convenient Site Detection requirement.
The immunologic detection method of organophosphorus pesticide has advantages of fast, cheap, easy, special, can be portable and carry out field monitoring.Overcome the shortcoming of traditional detection method.By the haptens of chemosynthesis organophosphorus pesticide, obtain complete antigen with carrier protein couplet, preparation, for the monoclonal antibody of organophosphorus pesticide isocarbophos, isofenphos_methyl and ethyl isofenphos in environment and agricultural-food, is set up above-mentioned three kinds of organophosphorus pesticide immunological detection methods.Completing of the method, by gordian techniquies such as solution are synthetic for haptens in the malicious organophosphorus pesticide isocarbophos of height, isofenphos_methyl, ethyl isofenphos tachysynthesis detection method, artificial antigen synthetic, prepared by monoclonal antibody, and set up the immune rapid detection system of above-mentioned three kinds of agricultural chemicals.This patent is not only food safety detection, and is that the entry and exit of agricultural products in China etc. detect, and the water area monitoring of environmental monitoring department provides a kind of new technique means and detection method.The Sustainable Healthy Development of agricultural products in China, solution food-safety problem are had important practical significance and important society, economic worth.
Summary of the invention
The above-mentioned deficiency that the object of this invention is to provide pin prior art, provides a kind of monoclonal antibody that resists three kinds of organophosphorus pesticides.
Another object of the present invention is to provide the application of this monoclonal antibody.
Object of the present invention can be achieved through the following technical solutions:
The hybridoma FQ-6E of the monoclonal antibody of the anti-three kinds of organophosphorus pesticides of secretion, on August 13rd, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:C2013105, and three kinds of described organophosphorus pesticides are isocarbophos, isofenphos_methyl and ethyl isofenphos.
A monoclonal antibody for anti-isocarbophos, isofenphos_methyl and ethyl isofenphos, the hybridoma FQ-6E that is CCTCC NO:C2013105 by deposit number secretes.
The preparation method of three kinds of organophosphorus pesticide monoclonal antibodies such as isocarbophos of the present invention, comprises the steps:
(1) for the synthetic artificial semiantigen of public structural chemistry of isocarbophos, isofenphos_methyl and ethyl isofenphos, synthetic route is as follows:
(2) artificial antigen preparation:
Described artificial semiantigen is coupled to respectively to carrier proteins bovine serum albumin BSA to employing active ester method and ovalbumin OVA is upper, obtains respectively immunogen H1-BSA and coating antigen H1-OVA;
(3) mouse immune:
Using the H1-BSA preparing as immunogen immune Balb/C mouse, caudal vein blood sampling detects the specificity of serum antibody by indirect non-competing ELISA method: with blank, return to zero; Using without immune serum as negative control, if treat, gaging hole OD450 value is more than or equal to 2.1 times of negative hole values and is decided to be the positive;
(4) cytogamy:
By positive value screening Balb/C mouse, the high Balb/C mouse of positive value is directly used H1-BSA immunity, after three days, gets spleen, and the Sp2/0 myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology;
(5) filtering hybridoma:
Coating antigen H1-OVA coated elisa plate with 1000 times of dilutions, 1% gelatin sealing, get the culture supernatant of hybridoma, adopt non-competing indirect elisa method tentatively to screen, wherein primary antibodie is hybridoma supernatant, with the positive contrast of serum of immune mouse, with the negative contrast of myeloma cell's supernatant, choose the cell hole being positive, cell conditioned medium is retained for next step and is detected, if the hole of being negative is still negative after repeating once, can give up; The cell hole being positive for previous step adopts indirect competitive ELISA method further to confirm, the hole that agricultural chemicals isocarbophos is occurred to obvious competitive inhibition reaction is the positive hybridoma cell that produces anti-organophosphorus insecticide isocarbophos antibody, deliver the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC NO:C2013105; Deposit number is that the hybridoma of CCTCC NO:C2013105 carries out subclone by limiting dilution assay, and the supernatant of secretion is prepared monoclonal antibody.
Described artificial semiantigen is preparation by the following method preferably:
1. O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride is synthetic
O-methyl thio-phosphoryl dichloride, K
2cO
3stir with acetonitrile, be cooled to below 10 ℃, drip the acetonitrile solution containing isopropyl salicylate, at 20 ℃～28 ℃, continue to stir 30～45min, pressure reducing and steaming solvent, column chromatography for separation, obtains colorless liquid product O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride; O-methyl thio-phosphoryl dichloride: K wherein
2cO
3: acetonitrile=(18～20) mol:(35～40) mol:1L; Concentration containing isopropyl salicylate in the acetonitrile solution of isopropyl salicylate is 22～28mol/L, isopropyl salicylate and K
2cO
3mol ratio be 1:2～4;
2. artificial semiantigen: O-methyl-O-(2-isopropoxy carbonyl phenyl)-N-(3-carboxyl propyl group) thio-phosphamide synthetic
O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride and methyl alcohol are according to 1mol:(1.4～1.5) L mixing, ice-water bath is cooling, dropping by KOH, 4-Aminobutanoicacid and methyl alcohol according to (3.2～3.5) mol:(1.5～1.6) mixing solutions that forms of mol:1L, at 20 ℃～28 ℃, continue to stir 10～15min, 1N HCl-chloroform extraction for reaction mixture, extraction liquid is after anhydrous sodium sulfate drying, pressure reducing and steaming solvent, column chromatography for separation, obtains colorless liquid product O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride; Wherein the mol ratio of O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride and KOH is 1:2.5～3.0.
The application of hybridoma FQ-6E of the present invention in detecting isocarbophos, isofenphos_methyl and/or ethyl isofenphos.
The application of monoclonal antibody of the present invention in detecting isocarbophos, isofenphos_methyl and/or ethyl isofenphos.
, isofenphos_methyl and an ethyl isofenphos haptens, structure is as follows:
The application of haptens of the present invention in the monoclonal antibody of preparation anti-isocarbophos, isofenphos_methyl and ethyl isofenphos.
Beneficial effect:
This antibody by synthetic H1 ?BSA artificial antigen immunity Blab/C, by hybridoma technology, obtain the monoclonal antibody of specific recognition isocarbophos, isofenphos_methyl and ethyl isofenphos, belong in the world at home pioneering, this monoclonal antibody can be used for the high-sensitivity rapid detection of isocarbophos in agricultural-food and agriculture production environment, isofenphos_methyl and ethyl isofenphos.
Biomaterial preservation information
Hybridoma cell strain FQ-6E, on August 13rd, 2013 is preserved in Chinese Typical Representative culture collection center, and address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:C2013105.
embodiment
Embodiment 1
1.1 is haptenic synthetic:
1. O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride is synthetic
In reaction flask, add 2.47g(15mmol) O-methyl thio-phosphoryl dichloride, 4.27g(30mmol) K of porphyrize
2cO
3with 0.8ml acetonitrile, stir, be cooled to below 10 ℃, drip containing 1.8g(10mmol) acetonitrile (0.4ml) solution of isopropyl salicylate, under room temperature, continue to stir 30 minutes, pressure reducing and steaming solvent, silica gel column chromatography separated (2:1 normal hexane-benzene), obtaining colorless liquid product (a) 2.85g(productive rate is 92.5%).
2. O-methyl-O-(2-isopropoxy carbonyl phenyl)-N-(3-carboxyl propyl group) thio-phosphamide (H1) is synthetic
In reaction flask, add 0.65g(2.1mmol) O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride (a) and 3ml methyl alcohol, ice-water bath is cooling, dropping is by 0.32g(5.72mmol) KOH, the mixing solutions that 0.268g(2.6mmol) 4-Aminobutanoicacid and 1.7ml methyl alcohol form, under room temperature, continue to stir 10 minutes, 1N HCl-chloroform extraction for reaction mixture, extraction liquid is after anhydrous sodium sulfate drying, pressure reducing and steaming solvent, separated (trichloromethane/ethyl acetate/the fluroxypyr of silica gel column chromatography, 65:35:1), obtaining colorless liquid product O-methyl-O-(2-isopropoxy carbonyl phenyl)-N-(3-carboxyl propyl group) thio-phosphamide (H1) 0.6g(productive rate is 76%), (H1) haptens just needing for us.
The nuclear magnetic resonance map data of above-mentioned two kinds of resultants are as follows:
（a）
1H?NMR(300M?Hz,CDCl
3)ppm:7.92(1H,m?Ar
H),7.53(1H,m?Ar
H),7.43(1H,m?Ar
H),7.31(1H,m?Ar
H),5.24(1H,hept,J=6.3Hz,C
H(CH
3)
2),4.03(3H,d,J=16.5Hz,C
H 3OP),1.37(6H,d,J=6.3Hz,C
H(CH
3)
2).
H1:
1H?NMR(300M?Hz,CDCl
3)ppm:7.79(1H,m,Ar
H),7.48(2H,m,Ar
H),7.18(1H,m,Ar
H),5.21(1H,hept,J=6.3Hz,C
H(CH
3)
2),4.02(1H,m,N
H),3.77(3H,d,J=14.1Hz,C
H 3OP),3.12(2H,m,NHC
H 2)2.33(2H,t,J=7.5Hz,C
H 2COOH),1.76(2H,m,CH
2C
H 2CH
2),1.35(6H,d,J=6.3Hz,CH(C
H 3)
2)
1.2 artificial antigen preparations:
Adopt active ester method (DCC/NHS) that haptens is coupled to respectively on carrier proteins bovine serum albumin (BSA) and ovalbumin (OVA), operate as follows: take the about 0.075g of haptens 0.2mmol() and the about 0.023g of NHS0.2mmol(), with 400 μ lDMF, be dissolved in reaction unit; Take again the about 0.0495g of 0.24mmolDCC(), with 600 μ lDMF, dissolve, DCC/DMF is added drop-wise in above-mentioned reaction unit.Reaction is under magnetic stirring apparatus, and room temp is carried out 7 hours (spending the night).
The whole liquid of the reaction of spending the night is put to 4 ℃ of refrigerators cooling more than 2 hours, through 12000rpm centrifugal 5 minutes, get supernatant liquor, i.e. active ester, this active ester liquid is divided into two parts, is added drop-wise to respectively the BSA solution of 5ml6mg/ml and the OVA solution of 5ml6mg/ml (dropping process is very slow).Reaction buffer is the phosphoric acid buffer (PB liquid) of 0.02mol/L pH8.0.Reaction room temp under magnetic stirring apparatus is carried out 4 hours (from application of sample, finishing to start timing).The whole liquid of reaction is put in dialysis tubing, and in the PB of 0.02mol/L pH6.8 liquid, 4 ℃ are stirred dialysis, within every 4～8 hours, change dialyzate one time, dialyse altogether 60 hours.After dialysis finishes, the white milk sap in dialysis tubing be respectively immunogen H1-BSA and coating antigen H1-OVA.Immunogen and coating antigen are sub-packed in respectively in some 1.5ml centrifuge tubes, in-20 ℃ of Refrigerator stores.
1.3 mouse immunes:
Using the H1-BSA preparing as immunogen immune 6-8 age in week female Balb/C mouse, the immunogen consumption 100 μ g of each every mouse, immunity is four times altogether, immunity for the first time adopts by isopyknic immunogen and Freund's complete adjuvant emulsification, abdominal injection.Immune interval is after three weeks for the first time, and 2 weeks, four each immune intervals of for the second time to the, adopt isopyknic immunogen and Freund's incomplete adjuvant emulsification;
From for the third time, after each immunity one week, the specificity of indirect non-competing ELISA method detection serum antibody for caudal vein blood sampling: return to zero with blank; Using without immune serum as negative control, if treat, gaging hole OD450 value is more than or equal to 2.1 times of negative hole values and is decided to be the positive.
Indirect non-competing ELISA elementary operation flow process is as follows:
(1) coated: with coated damping fluid (0.05mol/L, pH9.6) dilution envelope antigen, to suitable concn, to add enzyme mark
Plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators.
(2) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(3) sealing: every hole adds 1% gelatin 110 μ l, hatches 1.5h in 37 ℃ of incubators.
(4) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(5) add primary antibodie (mouse resisting anteserum to be checked or Hybridoma Cell Culture liquid): mouse resisting anteserum to be checked is diluted to a series of suitable gradient concentrations through PBST, adds enzyme plate, every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(6) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(7) add enzyme labelled antibody: 20000 times of PBST solution dilutions for horseradish peroxidase-goat anti-mouse igg, every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(8) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(9) colour developing: every hole adds freshly prepared nitrite ion 50 μ l, and lucifuge is put 37 ℃ of incubator 10～15min.
(10) stop: every hole adds the H of 25 μ l2mol/L
2sO
4termination reaction.
(11) absorbance measurement: each hole light absorption value of measuring 450nm wavelength place in microplate reader.
(12) result judgement: with blank zeroing, using the serum of immune mouse not as negative control, to treat that gaging hole OD450 value is more than or equal to 2.1 times of negative hole and is decided to be the positive.
By enzyme linked immunosorbent assay (ELISA), measure the serum titer of mouse, thereby can grasp the level that in antiserum(antisera), antibody produces.As mouse, tire not high enoughly, will continue immunity, detection, until obtain suitable antibody horizontal.
Before fusion, the serum titer of all immune mouses is carried out to one-time detection, choose tire the highest, compete best mouse for cytogamy.
1.4 cytogamy
The highest Balb/C mouse of the positive value of previous step is directly used H1-BSA immunity, after three days, get spleen, the Sp2/0 myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology, 37 ℃, 5% CO2gas incubator was cultivated after 10-12 days, by detection, merged hole supernatant screening hybridoma;
1.5 hybridoma screenings:
Coating antigen H1-OVA coated elisa plate with 1000 times of dilutions, 1% gelatin sealing, get the culture supernatant of hybridoma, adopt non-competing indirect elisa method tentatively to screen that (as mentioned above, wherein primary antibodie is hybridoma supernatant to method, with the positive contrast of serum of immune mouse, with the negative contrast of myeloma cell's supernatant), choose the cell hole being positive, cell conditioned medium is retained for next step and is detected, if the hole of being negative is still negative after repeating once, can give up.
The cell hole being positive for previous step adopts indirect competitive ELISA method further to confirm, the elementary operation flow process of indirect competitive ELISA is as follows:
(1) coated: with coated damping fluid (0.05mol/L, pH9.6) dilution envelope antigen, to suitable concn, to add enzyme mark
Plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators.
(2) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(3) sealing: every hole adds 1% gelatin 110 μ l, hatches 1.5h in 37 ℃ of incubators.
(4) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(5) add primary antibodie: Hybridoma Cell Culture liquid supernatant is diluted to suitable concn through PBST, and add therein isocarbophos, isofenphos_methyl and the ethyl isofenphos of different concns, after mixing, add enzyme plate, every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(6) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(7) add enzyme labelled antibody: add the horseradish peroxidase-goat anti-mouse igg with 20000 times of PBST solution dilutions,
Every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(8) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(9) colour developing: every hole adds freshly prepared nitrite ion 50 μ l, and lucifuge is put 37 ℃ of incubator 10～15min.
(10) stop: every hole adds the H of 25 μ l2mol/L
2sO
4termination reaction.
(11) absorbance measurement: each hole light absorption value of measuring 450nm wavelength place in microplate reader.
The hole that agricultural chemicals isocarbophos is occurred to obvious competitive inhibition reaction is the positive hole that produces anti-organophosphorus pesticide isocarbophos antibody, the hybridoma in positive hole carries out subclone by limiting dilution assay, and the supernatant of positive hole secretion is prepared monoclonal antibody.The hybridoma cell strain FQ-6E in positive hole is preserved in Chinese Typical Representative culture collection center on August 13rd, 2013, and deposit number is CCTCC NO:C2013105.
Embodiment 2
By deposit number, be that the hybridoma FQ-6E of CCTCC NO:C2013105 is frozen in liquid nitrogen container behind one week, two weeks, one month, recovery half a year, equal energy stably excreting antibody, and tire and have no obvious decline, illustrate that this hybridoma has satisfactory stability.
The detection of embodiment 3 isocarbophoses, isofenphos_methyl, ethyl isofenphos standard model
Adopt indirect competitive ELISA method, detection curve is carried out to desk study.Method is as follows:
(1) coated: with coated damping fluid (0.05mol/L, pH9.6) dilution envelope antigen, to suitable concn, to add enzyme mark
Plate, every hole 50 μ l, hatch 2h in 37 ℃ of incubators.
(2) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(3) sealing: every hole adds 1% gelatin 110 μ l, hatches 1.5h in 37 ℃ of incubators.
(4) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(5) add primary antibodie: Hybridoma Cell Culture liquid supernatant is diluted to suitable concn through PBST, and adds therein the agricultural chemicals of different sorts different concns, after mixing, adds enzyme plate, every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(6) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(7) add enzyme labelled antibody: add the horseradish peroxidase-goat anti-mouse igg with 20000 times of PBST solution dilutions,
Every hole 50 μ l, hatch 0.5h in 37 ℃ of incubators.
(8) wash plate: remove raffinate, washing on plate machine with washings PBST washing 5 times, thieving paper pats dry.
(9) colour developing: every hole adds freshly prepared nitrite ion 50 μ l, and lucifuge is put 37 ℃ of incubator 10～15min.
(10) stop: every hole adds the H of 25 μ l2mol/L
2sO
4termination reaction.
(11) absorbance measurement: each hole light absorption value of measuring 450nm wavelength place in microplate reader.
By microplate reader (Thermo CO.), measure each hole light absorption value at 450nm wavelength place, and calculate inhibiting rate.The IC of this monoclonal antibody
50be respectively 23.39ng/ml (isocarbophos), 70.79ng/ml(isofenphos_methyl), 62.52ng/ml(ethyl isofenphos).Result shows, the above-mentioned three kinds high malicious organophosphorus pesticides of the good identification of antibody capable of preparation, and there is higher sensitivity.
Claims (8)
1. secretion resists the hybridoma FQ-6E of the monoclonal antibody of three kinds of organophosphorus pesticides, on August 13rd, 2013, be preserved in Chinese Typical Representative culture collection center, deposit number is CCTCC NO:C2013105, and three kinds of described organophosphorus pesticides are isocarbophos, isofenphos_methyl and ethyl isofenphos.
2. a monoclonal antibody for anti-isocarbophos, isofenphos_methyl and ethyl isofenphos, is characterized in that being secreted by hybridoma claimed in claim 1.
3. the preparation method of monoclonal antibody claimed in claim 2, is characterized in that mainly comprising following steps:
(1) for the synthetic artificial semiantigen of public structural chemistry of isocarbophos, isofenphos_methyl and ethyl isofenphos, synthetic route is as follows:
(2) artificial antigen preparation:
Described artificial semiantigen is coupled to respectively to carrier proteins bovine serum albumin BSA to employing active ester method and ovalbumin OVA is upper, obtains respectively immunogen H1-BSA and coating antigen H1-OVA;
(3) mouse immune:
Using the H1-BSA preparing as immunogen immune Balb/C mouse, caudal vein blood sampling detects the specificity of serum antibody by indirect non-competing ELISA method: with blank, return to zero; Using without immune serum as negative control, if treat, gaging hole OD450 value is more than or equal to 2.1 times of negative hole values and is decided to be the positive;
(4) cytogamy:
By positive value screening Balb/C mouse, the high Balb/C mouse of positive value is directly used H1-BSA immunity, after three days, gets spleen, and the Sp2/0 myeloma cell of splenocyte and logarithmic phase prepares hybridoma by hybridoma technology;
(5) filtering hybridoma:
Coating antigen H1-OVA coated elisa plate with 1000 times of dilutions, 1% gelatin sealing, get the culture supernatant of hybridoma, adopt non-competing indirect elisa method tentatively to screen, wherein primary antibodie is hybridoma supernatant, with the positive contrast of serum of immune mouse, with the negative contrast of myeloma cell's supernatant, choose the cell hole being positive, cell conditioned medium is retained for next step and is detected, if the hole of being negative is still negative after repeating once, can give up; The cell hole being positive for previous step adopts indirect competitive ELISA method further to confirm, the hole that agricultural chemicals isocarbophos is occurred to obvious competitive inhibition reaction is the positive hybridoma cell that produces anti-organophosphorus insecticide isocarbophos antibody, deliver the center preservation of Chinese Typical Representative culture collection, deposit number is CCTCC NO:C2013105; Deposit number is that the hybridoma of CCTCC NO:C2013105 carries out subclone by limiting dilution assay, and the supernatant of secretion is prepared monoclonal antibody.
4. the preparation method of monoclonal antibody according to claim 3, is characterized in that described artificial semiantigen prepared by the following method:
1. O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride is synthetic
O-methyl thio-phosphoryl dichloride, K
2cO
3stir with acetonitrile, be cooled to below 10 ℃, drip the acetonitrile solution containing isopropyl salicylate, at 20 ℃～28 ℃, continue to stir 30～45min, pressure reducing and steaming solvent, column chromatography for separation, obtains colorless liquid product O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride; O-methyl thio-phosphoryl dichloride: K wherein
2cO
3: acetonitrile=(18～20) mol:(35～40) mol:1L; Concentration containing isopropyl salicylate in the acetonitrile solution of isopropyl salicylate is 22～28mol/L, isopropyl salicylate and K
2cO
3mol ratio be 1:2～4;
2. artificial semiantigen: O-methyl-O-(2-isopropoxy carbonyl phenyl)-N-(3-carboxyl propyl group) thio-phosphamide synthetic
O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride and methyl alcohol are according to 1mol:(1.4～1.5) L mixing, ice-water bath is cooling, dropping by KOH, 4-Aminobutanoicacid and methyl alcohol according to (3.2～3.5) mol:(1.5～1.6) mixing solutions that forms of mol:1L, at 20 ℃～28 ℃, continue to stir 10～15min, 1N HCl-chloroform extraction for reaction mixture, extraction liquid is after anhydrous sodium sulfate drying, pressure reducing and steaming solvent, column chromatography for separation, obtains colorless liquid product O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride; Wherein the mol ratio of O-methyl-O-(2-isopropoxy carbonyl phenyl) thiophosphoryl chloride and KOH is 1:2.5～3.0.
5. the application of hybridoma claimed in claim 1 in detecting isocarbophos, isofenphos_methyl and/or ethyl isofenphos.
6. the application of monoclonal antibody claimed in claim 2 in detecting isocarbophos, isofenphos_methyl and/or ethyl isofenphos.
7. isocarbophos, isofenphos_methyl and an ethyl isofenphos haptens, is characterized in that structure is as follows:
8. the application of haptens claimed in claim 5 in the monoclonal antibody of preparation anti-isocarbophos, isofenphos_methyl and ethyl isofenphos.
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