Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit

An enzyme-linked immunosorbent reagent, chlorpyrifos technology, applied in the detection of chlorpyrifos content in fruits and tea, enzyme-linked immunosorbent immunoassay kits, and vegetables, can solve the problem that it is difficult to meet the needs of a large number of samples and rapid detection of on-site samples, and the processing process is complex and expensive. Time and other issues, to achieve the effect of simple structure, low pre-processing requirements, and convenient use

Active Publication Date: 2017-01-11
BEIJING KWINBON BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The use of these analytical methods requires expensive instruments and specialized technicians. The sample pretreatment process is complex, costly, and time-consuming, and it is difficult to meet the needs of a large number of samples and rapid detection of on-site samples.

Method used

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  • Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit
  • Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit
  • Enzyme-linked immunoassay kit for detecting chlorpyrifos and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Preparation of kit components

[0030] 1. Synthesis of chlorpyrifos hapten (see attached for the synthetic route figure 1 ) and identification

[0031] 1.0g of chlorpyrifos original medicine was dissolved in 30mL of ethanol, 0.41g of sodium hydroxide was added, 0.35g of aminobutyric acid was added, and the reaction was refluxed for 3h. After testing, all the raw materials were reacted. Ethanol was evaporated to dryness, water-ethyl acetate was added for extraction, the aqueous phase was adjusted to pH 3, extracted with ethyl acetate, the organic phase was evaporated to dryness, and recrystallized with dichloromethane and n-hexane to obtain the chlorpyrifos hapten product.

[0032] The above haptens were identified by H NMR spectroscopy, and the results are shown in the attached figure 2 . The carboxyl signal peaks at the chemical shifts of 11.0ppm and the methylene signal peaks at 2.30ppm, 1.89ppm and 3.35ppm in the spectrum prove that the hapten structure...

Embodiment 2

[0064] Embodiment 2 The formation of the enzyme-linked immunosorbent assay kit for detecting chlorpyrifos

[0065] An ELISA kit for the detection of chlorpyrifos was constructed to include the following components:

[0066] (1) ELISA plate coated with chlorpyrifos-conjugated antigen;

[0067] (2) 6 bottles of chlorpyrifos standard solution with concentrations of 0 μg / L, 0.6 μg / L, 1.8 μg / L, 5.4 μg / L, 16.2 μg / L and 48.6 μg / L;

[0068] (3) chlorpyrifos monoclonal antibody working solution;

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

[0070] (5) The substrate color developing solution is composed of A solution and B solution, A solution is carbamide peroxide, and B solution is tetramethylbenzidine;

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

[0072] (7) The washing solution is pH 7.4, 0.1-0.3mol / L phosphate buffer containing 0.5%-1.0% Tween-20, 0.01‰-0.03‰ sodium azide;

[0073] (8) The reconstituted solution is a 0.1 mol / L ph...

Embodiment 3

[0074] Embodiment 3 Detection of chlorpyrifos in vegetable, fruit and tea samples

[0075] 1. Sample pretreatment

[0076] Weigh 1.0g±0.05g sample into a 10mL polystyrene centrifuge tube, add 5mL methanol, vortex with a vortex mixer for 2min, and mix well; centrifuge at 3000g at room temperature (20-25℃) for 5min, take 50μL of supernatant to In a 2mL polystyrene centrifuge tube, add 950μL of reconstituted solution, vortex for 20s; take 50μL for analysis.

[0077] 2. Test with a kit

[0078] Add 50 μL / well of chlorpyrifos standard solution or pretreated sample solution to the microwells of the ELISA plate coated with chlorpyrifos-conjugated antigen, then add 50 μL / well of chlorpyrifos monoclonal antibody working solution, shake and mix gently, and use Cover the plate with a cover plate and place it at 25°C for 30 minutes in the dark; pour out the liquid in the well, add 250 μL of washing solution to each well, and wash thoroughly for 4 to 5 times, every 10 s, and pat dry with...

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Abstract

The invention provides an enzyme-linked immunoassay kit for detecting chlorpyrifos. The kit comprises an enzyme label board coated with chlorpyrifos coupling antigen, a chlorpyrifos monoclonal antibody, an enzyme labeling antiantibody, a chlorpyrifos standard product solution, a substrate developing solution, TMAH, a cleaning solution and a combination solution. The invention further discloses a method for detecting chlorpyrifos by means of the enzyme-linked immunoassay kit. The method includes the steps that firstly, sample pretreatment is conducted; secondly, detection is conducted by means of the kit; finally, a detection result is analyzed. The enzyme-linked immunoassay kit can be used for detecting the content of chlorpyrifos in vegetables, fruits and tea leaves, is easy to operate, low in cost, high in sensitivity, capable of achieving in-situ monitoring and suitable for screening a lot of samples.

Description

technical field [0001] The invention relates to an enzyme-linked immunoassay detection technology, in particular to an enzyme-linked immunosorbent assay kit for detecting chlorpyrifos, which is particularly suitable for detecting the content of chlorpyrifos in vegetables, fruits and tea. Background technique [0002] Chlorpyrifos, the chemical name is O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl) phosphorothioate, the trade name is chlorpyrifos, Lesben, Termites, etc., It is a new high-efficiency and low-toxic alternative to high-efficiency pesticides such as methamidophos and methyl parathion. It is a broad-spectrum organophosphate insecticide and can be used for pest control of grains, vegetables, fruits and economic crops. Chlorpyrifos is one of the important targets for monitoring pesticide residues in agricultural products and food. Not only is the residue in crops becoming more and more serious, but it can also be transferred to the environment through metabolism and other...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543
CPCG01N33/54306G01N2430/10
Inventor 万宇平何方洋朱亮亮贾芳芳罗晓琴崔海峰冯才伟杨昌松冯月君徐念琴
Owner BEIJING KWINBON BIOTECH
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