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Enzyme-linked immunosorbent assay kit for detecting pendimethalin and application thereof

An enzyme-linked immunosorbent reagent, pendimethalinase technology, applied in the direction of biological testing, measuring devices, material inspection products, etc., can solve the problems of high technical level requirements, expensive equipment, unfavorable on-site screening, etc., to achieve detection The method is efficient, the pretreatment process is simple, and the inspection method is convenient and easy to implement

Active Publication Date: 2018-12-21
ZHENGZHOU TOBACCO RES INST OF CNTC +2
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the expensive equipment and high technical level requirements of these analytical methods, the samples need to be purified, which is not conducive to on-site screening.

Method used

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  • Enzyme-linked immunosorbent assay kit for detecting pendimethalin and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting pendimethalin and application thereof
  • Enzyme-linked immunosorbent assay kit for detecting pendimethalin and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Embodiment 1 Preparation of kit components

[0033] 1. Synthesis of pendimethalin hapten (see attached figure 1 ) and identification

[0034] Take 1.00 g of N-(1-ethylpropyl)-3,4-dimethylaniline, add 80 mL of acetone to dissolve, add 0.35 g of potassium hydroxide, add 1.21 g of ethyl 4-bromobutyrate, and heat to reflux for reaction 3 h, TLC detection, the reaction of all the raw materials is complete, stop the reaction, cool to room temperature, rotary evaporate, remove acetone, add 60 mL of water, add 50 mL of dichloromethane for extraction, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness and concentrate, and Silica gel column, eluted and separated by petroleum ether / ethyl acetate with a volume ratio of 10:1, to obtain 1.51 g of intermediate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, yield 94.97%;

[0035] Take 1.50 g of the intermediate alkylated N-(1-ethylpropyl)-3,4-dimethylaniline, add 5 mL of concentrated sulfuric acid to dissolv...

Embodiment 2

[0068] Example 2 Establishment of an enzyme-linked immunosorbent assay kit for detecting pendimethalin

[0069] An enzyme-linked immunosorbent assay kit for detecting pendimethalin was set up to include the following components:

[0070] 1) A microtiter plate coated with pendimethalin-conjugated antigen;

[0071] 2) Pendimethalin standard solution, the concentrations are 0 μg / L, 3 μg / L, 9 μg / L, 27 μg / L, 81 μg / L, 243 μg / L;

[0072] 3) pendimethalin monoclonal antibody working solution;

[0073] 4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0074] 5) The substrate chromogenic solution is composed of A liquid and B liquid, the substrate chromogenic liquid A is carbamide peroxide, and the substrate chromogenic liquid B is tetramethylbenzidine;

[0075] 6) The stop solution is 2 mol / L sulfuric acid buffer;

[0076] 7) The washing solution is a 0.02 mol / L phosphate buffer solution with a pH value of 7.4, containing 0.05% Tween-20 and 0.01‰thimerosal pre...

Embodiment 3

[0078] Example 3 Detection of Pendimethalin Residues in Tobacco Leaf Samples

[0079] 1. Sample pretreatment

[0080] Weigh 1.0±0.05 g of tobacco leaf sample into a 10 mL polystyrene centrifuge tube, add 5 mL of methanol, vortex for 3 min, centrifuge at room temperature (20-25 °C) at 6000 rpm for 5 min; pipette 10 μL of supernatant and add 990 μL complex solution, mix well; take 50 μL for analysis.

[0081] 2. Detection with kit

[0082]Add 50 μL / well of pendimethalin standard solution or pretreated sample solution to the microwells of the microtiter plate coated with pendimethalin-conjugated antigen, and then add pendimethalin monoclonal antibody working solution 50 μL / well. μL / well, oscillate gently to mix well, cover the plate with a cover film and place it in a light-proof environment at 25°C for 30 min; pour out the liquid in the well, add 250 μL washing solution to each well and wash fully 4~5 times, each time At intervals of 10 s, pat dry with absorbent paper; then a...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting pendimethalin and application thereof. The enzyme-linked immunosorbent assay kit comprises elisa plates coated with dox-ova, pendimethalinspecific antibodies, enzyme markers, a pendimethalin standardized products solution, a substrate developing solution, a stop solution, a scrubbing solution and a reconstitution solution; and the dox-ova is a pendimethalin coupled antigen, the pendimethalin specific antibodies are prepared with the pendimethalin coupled antigen as immunogen, pendimethalinhatpin is prepared through the steps that N-(1-ethyl propyl ether)-3,4-methyl toluidine reacts with 4-ethyl 4-bromobutyrate to generate hydrocarbylation N-(1-ethyl propyl ether)-3,4-methyl toluidine, then the hydrocarbylation N-(1-ethyl propyl ether)-3,4-methyl toluidine reacts with concentrated nitric acid to generatenitro hydrocarbonylation N-(1-ethyl propyl ether)-3,4-methyl toluidine, and then the nitro hydrocarbonylation N-(1-ethyl propyl ether)-3,4-methyl toluidine reacts with potassium hydroxide. The enzyme linked immunosorbent assay kit provided by the invention can be used for detecting the residual quantity ofthe pendimethalin in tobaccos, is easy and convenient to operate, low in cost and high in sensitivity, can achieve on-site monitoring, and is suitable for screening of lots of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunoassay kit for detecting pendimethalin, which is particularly suitable for detecting the residual amount of pendimethalin in tobacco leaves. Background technique [0002] Pendimethalin belongs to dinitroaniline herbicides, and its mechanism of action is to inhibit meristem cell division. The part of pendimethalin absorbed by dicotyledons is hypocotyl, and the part absorbed by monocots is sprout. Pendimethalin is suitable for corn, soybean, wheat, peanut, cotton and other dryland crops to control annual grass and broad-leaved weeds such as crabgrass, foxtail and purslane. Tobacco is mainly used for chemical bud suppression and tobacco weed control. According to the classification standard of acute toxicity of pesticides in China, pendimethalin is classified as a low toxicity compound. According to the toxic classification standard of the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/543G01N33/535
CPCG01N33/535G01N33/543G01N33/581
Inventor 陈黎范子彦吴小胜鲁亚辉刘惠民唐纲岭颜权平潘立宁刘二战
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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