Enzyme-linked immunosorbent assay for indirectly detecting pirimiphos-methyl

An enzyme-linked immunoassay, pirimiphos-methyl technology, applied in the field of immunoassay, can solve the problems of cumbersome preparation steps and complex hapten structure

Active Publication Date: 2020-10-27
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CN201910890291.9 discloses a preparation method of pirimiphos-methyl monoclonal antibody, but the hapten prepared by it has complex structure and cumbersome preparation steps

Method used

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  • Enzyme-linked immunosorbent assay for indirectly detecting pirimiphos-methyl
  • Enzyme-linked immunosorbent assay for indirectly detecting pirimiphos-methyl
  • Enzyme-linked immunosorbent assay for indirectly detecting pirimiphos-methyl

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Embodiment 1 Preparation and Identification of Immunogen and Coating Gen

[0043] The preparation of the immunogen and the coating source is realized by using different carrier proteins. The immunogen carrier protein is bovine serum albumin (BSA), the coating original carrier protein is chicken ovalbumin (OVA), and the coupling method adopted is N,N-carbonyldiimidazole method. The specific implementation scheme is as follows:

[0044] Dissolve 0.03mmol of hapten formula I (2-diethylamino-6-methyl-4-pyrimidinol) in 250μL of DMF, add 8.11mg of N,N-carbonyldiimidazole, stir at 37°C for 2h, this is Liquid A. Weigh 10 mg of carrier protein (BSA or OVA) and dissolve it in 2 mL of coating solution, which is B solution. Slowly add solution A to solution B dropwise, stir and react for 72 hours at 4°C. After the reaction, put it into a dialysis bag boiled for 10 minutes in advance, dialyze with PBS for 3 days, and change the dialysate 3 times a day. The obtained complete ant...

Embodiment 2

[0048] Preparation and Identification of Embodiment 2 Antibodies

[0049] 1. Preparation of Polyclonal Antiserum

[0050] Mix the immunogen Pr-OH-BSA prepared in Example 1 with an equal dose of Freund's adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent immunizations), fully emulsify, and use the back, The mice were immunized by multi-point injections such as abdomen, subcutaneous, and foot pads. Booster immunizations were given every two weeks after the first immunization. Tail blood was collected every other week after the third booster immunization, and the serum titer was determined by indirect competition ELISA. When the titer does not rise any more, intraperitoneal injection is used to boost the immunization once, and the eyeballs are picked out one week later to take blood. Let stand at room temperature for 0.5-1 hour, centrifuge at 12,000 r / min at 4°C for 10 minutes, take the supernatant and put it in a c...

Embodiment 3

[0054] Example 3 Establishment of an ELISA method for indirect determination of pirimiphos-methyl

[0055] The antiserum prepared in Example 2 was used to draw an indirect competition ELISA standard curve. Use phosphate buffered saline (PBS, 0.1mol / L, pH7.4) as the diluent and reaction buffer of all samples; Use Pr-OH-OVA as the coating former, specific examples are as follows:

[0056] (1) Coat 1 ng / mL of the original Pr-OH-OVA coating on the ELISA microtiter plate. After coating overnight at 37 ° C, add 120 μ L of 5% skimmed milk powder solution per well, block at 37 ° C for 3 hours, and then spin dry. Dry in an oven at 37°C for later use.

[0057] (2) adding sodium hydroxide solution to the sample treatment solution, adjusting the pH of the solution to 11, and shaking at 50° C. for 2 hours to obtain a hydrolyzed sample solution;

[0058] (3) Add 50 μL of a series of concentrations (0.128, 0.64, 3.2, 16, 80, 400, 2000ng / mL) 2-diethylamino-6-methyl-4-pyrimidinol standard so...

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Abstract

The invention discloses an enzyme-linked immunosorbent assay for indirectly detecting pirimiphos-methyl. An artificial antigen obtained by coupling bovine serum albumins with 2-diethylamino-6-methyl-4-pyrimidinol is used as an immunogen, and an artificial antigen obtained by coupling chicken ovalbumins with 2-diethylamino-6-methyl-4-pyrimidinol is used as a coating antigen. An experimental animalis immunized by virtue of an artificial immunogen, and an antibody specifically aiming at pirimiphos-methyl hydrolysate is generated by the body of the animal and is capable of specifically recognizing the pirimiphos-methyl hydrolysate, namely the 2-diethylamino-6-methyl-4-pyrimidinol and can be used for indirectly determining the content of the pirimiphos-methyl in foods after hydrolysis of raw farm chemicals. According to the method, the detection limit is 3.2ng/mL, the 50% inhibiting concentration (IC50) is 23.42ng/mL, the linear detection range is 6.09ng/mL-90.07ng/mL, and the curve correlation coefficient is 0.9996. The method is high in specificity, high in sensitivity, simple and convenient and has a wide application prospect in residue detection of the pirimiphos-methyl in foods.

Description

technical field [0001] The invention relates to the technical field of immunoassay, and more specifically relates to an enzyme-linked immunosorbent assay method for indirect detection of pirimiphos-methyl. Background technique [0002] Pirimipho-methyl, also known as O-(2-diethylamino-6-methyl-4-pyrimidinyl)-O,O-dimethyl phosphorothioate, trade name is also known as Andrew. Molecular weight 305, yellow liquid, can be hydrolyzed by strong acid and strong base, unstable in case of light, soluble in most organic solvents, solubility in water is about 5mg / L. Pirimiphos-methyl is a fast-acting and low-toxic insecticide and acaricide, and has contact and fumigation effects. It has good efficacy against stored grain beetles, weevils, moths and mites, and can be used not only The pest control of field crops and commercial crops can also be used to control fruit, vegetable and household pests. It is worth pointing out that it has a particularly good control effect on storage pests ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/765C07K14/77C07K16/44G01N33/53G01N33/58G01N33/543
CPCC07K14/765C07K14/77C07K16/44G01N33/5308G01N33/581G01N33/54393G01N2430/10
Inventor 肖治理李莉萍田巍孙远明徐振林沈玉栋罗林柳心梅谢波
Owner SOUTH CHINA AGRI UNIV
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