An enzyme-linked immunosorbent assay method for indirect detection of pirimiphos-methyl

An enzyme-linked immunoassay and pirimiphos-methyl technology, which is applied in the field of immunoassay, can solve the problems of cumbersome preparation steps and complex hapten structure, and achieve the effects of convenient use, avoiding the steps of hapten design, synthesis and identification, and high sensitivity

Active Publication Date: 2022-03-11
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • An enzyme-linked immunosorbent assay method for indirect detection of pirimiphos-methyl
  • An enzyme-linked immunosorbent assay method for indirect detection of pirimiphos-methyl
  • An enzyme-linked immunosorbent assay method for indirect detection of 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 method for indirect detection of pirimiphos-methyl. The artificial antigen coupled with bovine serum albumin to 2-diethylamino-6-methyl-4-pyrimidinol is used as the immunogen. The artificial antigen of chicken ovalbumin coupled with 2-diethylamino-6-methyl-4-pyrimidinol was used as the coating source. The experimental animals are immunized with an artificial immunogen to make their bodies produce antibodies specific to the hydrolyzate of pirimiphos-methyl, which can specifically recognize the hydrolyzate of pirimiphos-methyl-2-diethylamino-6-methyl ‑4‑pyrimidinol, used for the indirect detection of pirimiphos-methyl in food after the hydrolysis of pesticide raw materials. The detection limit of the method was 3.2 ng / mL, and the half-inhibitory concentration (IC 50 ) was 23.42ng / mL, the linear detection range was 6.09~90.07ng / mL, and the correlation coefficient of the curve was 0.9996. The method of the invention has strong specificity, high sensitivity, is simple and quick, and has wide application prospects in the detection of pirimiphos-methyl residues in food.

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 Patents(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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