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Monoclonal antibodies against three organophosphorus pesticides and their application
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A monoclonal antibody, organophosphorus pesticide technology, applied in the biological field, can solve the problems of high cost, long time, complicated processing and so on
Inactive Publication Date: 2016-06-29
NANJING AGRICULTURAL UNIVERSITY
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However, due to the need for expensive instruments, dedicated operators, and the complexity, high cost, and long time of sample pretreatment, it cannot better meet the requirements of quick and easy on-site detection
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[0038] ① Synthesis of O-methyl-O-(2-isopropoxycarbonylphenyl)phosphoryl thiochloride
[0039] Add 2.47g (15mmol) O-methyl phosphorothioate dichloride, 4.27g (30mmol) finely ground K 2 CO 3 and 0.8ml of acetonitrile, stirred, cooled to below 10°C, added dropwise a solution of acetonitrile (0.4ml) containing 1.8g (10mmol) of isopropyl salicylate, stirred at room temperature for 30 minutes, evaporated the solvent under reduced pressure, and the silica gel column After separation by chromatography (2:1 n-hexane-benzene), 2.85 g of product (a) was obtained as a colorless liquid (92.5% yield).
[0040] ② Synthesis of O-methyl-O-(2-isopropoxycarbonylphenyl)-N-(3-carboxypropyl)thiophosphoramide (H1)
[0041] Add 0.65g (2.1mmol) O-methyl-O-(2-isopropoxycarbonylphenyl)phosphoryl thiochloride (a) and 3ml methanol to the reaction flask, cool in an ice-water bath, dropwise add 0.32g ( 5.72mmol) KOH, 0.268g (2.6mmol) 4-aminobutyric acid and ...
Embodiment 2
[0084] The hybridoma cell FQ-6E with the preservation number CCTCCNO: C2013105 was cryopreserved in a liquid nitrogen tank for one week, half a month, one month, and half a year after resuscitation, all of them could secrete antibodies stably, and the titer did not see a significant drop, indicating that the Hybridoma cells have good stability.
Embodiment 3
[0085] The detection of embodiment 3 isocarbophos, isofenphos-methyl, isofenphos-ethyl standard sample
[0086] The indirect competitive ELISA method was used to conduct a preliminary exploration of the detection curve. Methods as below:
[0087](1) Coating: Dilute the coated antigen to an appropriate concentration with coating buffer (0.05mol / L, pH9.6), add enzymelabel
[0088] plate, 50 μl per well, and incubated in a 37°C incubator for 2h.
[0089] (2) Plate washing: Pour off the residual liquid, wash 5 times with washing liquid PBST on the plate washer, and pat dry with absorbent paper.
[0090] (3) Sealing: add 110 μl of 1% gelatin to each well, and incubate in a 37°C incubator for 1.5h.
[0091] (4) Plate washing: Pour off the residual liquid, wash 5 times with washing solution PBST on the plate washer, and pat dry with absorbent paper.
[0092] (5) Add primary antibody: The hybridoma cell culture supernatant is diluted with PBST to an appropriate concentration, and...
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Abstract
The invention belongs to the field of biotechnology, and relates to monoclonal antibodies against three organophosphorus pesticides and applications thereof. The present invention chemically synthesizes the artificial hapten against the public structure of the above-mentioned pesticide, and then prepares the complete antigen, immunizes Balb / C mice, prepares a highly specific monoclonalantibody through hybridoma technology, and uses it in the above-mentioned three kinds of agricultural products and agricultural production environments. Highly sensitive and rapid detection of pesticide residues. The preparation technology of the antibody of the present invention is feasible, the whole preparation process of the antigen does not need special instruments and equipment, and is easy for large-scale production.
Description
technical field [0001] The invention belongs to the field of biotechnology, and relates to monoclonal antibodies against three organophosphorus pesticides and applications thereof. Background technique [0002] In the past 20 years, with the rapid development of agricultural production, the output and usage of organophosphorus pesticides have been increasing. Extensive use or even improper use of organophosphorus pesticides has made the problem of organophosphorus pesticide residues increasingly prominent. This not only makes the environment safe and people's health more likely to be threatened by organophosphorus pesticides, but also the maximum residue limit of pesticides has become an important technical trade barrier for the import and export of Chinese agricultural products around the world. Therefore, the development of rapid, simple and sensitive detection methods for organophosphorus pesticide residues plays a decisive role in the environment, food safety, people's ...
Claims
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