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

An enzyme-linked immunosorbent reagent, the technology of zhongdingling, is applied in the field of enzyme-linked immunosorbent assay, which can solve the problems of inability to carry out large-scale on-site detection, cumbersome processing, poor timeliness, etc., and achieves a convenient and easy-to-implement testing method with high sensitivity and accuracy. high effect

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

AI Technical Summary

Problems solved by technology

[0003] At present, the research on the detection of Zhongbuling residues is mostly concentrated on gas chromatography, liquid chromatography, gas chromatography tandem mass spectrometry, liquid chromatography tandem mass spectrometry and other chromatography mass spectrometry methods. Although the instrument method has high detection sensitivity and specificity Advantages, but the sample pretreatment is cumbersome and time-consuming, requires expensive large-scale instruments and equipment, and is equipped with professional testing technicians for operation and management. It is impossible to conduct large-scale on-site testing, and the timeliness is poor.

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Embodiment 1 Preparation of kit components

[0029] 1. Synthesis of Zhongbutaline hapten (see the appendix for the synthetic route figure 1 ) and identification

[0030] Take 1.00 g of 4-chloro-3,5-dinitrophenylacetic acid, add 50 mL of ethanol to dissolve, add 0.45 g of sodium carbonate, stir, add 0.31 g of butylamine, heat in an oil bath at 80°C, stir for 3 h, and detect the raw material The basic reaction is complete; stop the reaction, cool to room temperature, rotary evaporate, remove ethanol, add 80 mL of water, adjust the pH value to 4 with 1 mol / L hydrochloric acid, there is a lot of turbidity, add 80 mL of ethyl acetate to extract, wash with water, and apply silica gel The column was eluted and separated with dichloromethane / methanol with a volume ratio of 5:1 to obtain 1.05 g of the hapten product of sec-butyrin, with a yield of 92.11%.

[0031] NMR identification 1 H NMR (CDCl 3 , 300MHz) δ: 11.01 (1H, d, J=0.000), 8.281 (1H, d, J=0.000), 4.011 (1H, tq, J...

Embodiment 2

[0064] Example 2 The formation of an enzyme-linked immunosorbent assay kit for detecting Zhongbutaline

[0065] Set up the enzyme-linked immunosorbent assay kit that detects Zhongbulin, so that it includes the following components:

[0066] 1) A microtiter plate coated with secbutaline-coupled antigen;

[0067] 2) Zhongbuling standard solution, the concentrations are 0 μg / L, 10 μg / L, 30 μg / L, 90 μg / L, 270 μg / L, 810 μg / L;

[0068] 3) Zhongbuling monoclonal antibody working solution;

[0069] 4) Goat anti-mouse anti-antibody labeled with horseradish peroxidase;

[0070] 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;

[0071] 6) The stop solution is 2 mol / L sulfuric acid buffer;

[0072] 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 preser...

Embodiment 3

[0074] Example 3 The detection of secbutaline residues in tobacco leaf samples

[0075] 1. Sample pretreatment

[0076] After crushing the tobacco leaf sample, weigh 1.0±0.05 g of the sample into a 50 mL polystyrene centrifuge tube, add 10 mL of acetonitrile, vortex for 2 min with a vortex mixer, and mix well; ) and centrifuge for 5 min, take 100 μL of the supernatant to a 2 mL polystyrene centrifuge tube, add 900 μL of reconstitution solution, and vortex for 20 s; take 50 μL for analysis.

[0077] 2. Detection with kit

[0078] Add 50 μL / well of the standard solution of secbutaline or the pretreated sample solution to the microwells of the microplate of the microplate coated with secbutaline-conjugated antigen, and then add 50 μL / well of secbutaline monoclonal antibody working solution Gently oscillate and mix well, cover the plate with a cover film, and place it in a dark environment at 25°C for 30 min; pour out the liquid in the well, add 250 μL of washing solution to eac...

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Abstract

The invention provides an enzyme-linked immunosorbent assay kit for detecting butralin and application thereof. The enzyme-linked immunosorbent assay kit is characterized in that the enzyme-linked immunosorbent assay kit comprises an elisa plate wrapped with a butralin coupled antigen, a butralin monoclonal antibody, an enzyme-labelled antiantibody, a butralin standard product solution, a substrate developing solution, a stop solution, a washing solution and a redissolving solution. The butralin coupled antigen is obtained by coupling of a butralin hapten with a carrier protein. The butralin hapten is obtained in the mode of reacting of 4-chlorine-3,5-binitro phenylacetic acid with butyl amine. The invention further provides a method for detecting the residual quantity of the butralin through the butralin hapten. The method comprises the steps that firstly, sample pretreatment is conducted, then detecting is conducted through the assay kit, and finally, the detecting result is analyzed. The enzyme-linked immunosorbent assay kit provided by the invention can be used for detecting the residual quantity of the butralin in tobacco leaves, and is easy and convenient to operate, low in cost, high in sensitivity, capable of conducting monitoring on site and suitable for screening of a lot of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunosorbent detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting secbutaline and its application, which is particularly suitable for detecting secbutaline residues in tobacco and tobacco products. Background technique [0002] Butralin (Butralin) is also known as dimethamine, bibutralin, nifediamine, bidarin, and budling. The chemical name is N-sec-butyl-4-tert-butyl-2,6-dinitro Aniline, the molecular formula is C 14 h 21 N 3 o 4 . Zhongbuling is a dinitroaniline herbicide. The pure product is an orange-yellow crystal. It is easily soluble in organic solvents but hardly soluble in water. It is volatile and easy to decompose when exposed to light. It has low toxicity to humans and animals. The drug is a selective preemergent herbicide, and its effect is similar to that of trifluralin. After the medicament enters the plant, it mainly inhibits the cell division of t...

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 陈黎范子彦鲁亚辉刘玉梅刘惠民唐纲岭颜权平潘立宁申梁
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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