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ELISA kit for detecting chlopyrifos residue and method of use thereof

An enzyme-linked immunosorbent reagent, chlorpyrifos technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of time-consuming analysis, poor stability, and high cost

Active Publication Date: 2008-08-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, the conventional methods for detecting chlorpyrifos residues include gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Although these methods are sensitive and accurate, they require expensive professional instruments, and the analysis is time-consuming and costly.
The enzyme-linked immunoassay (ELISA) rapid detection technology has become a commonly used screening method due to its low cost, simple operation, fast speed, large sample size for one test, and low degree of instrumentation. There are still many problems, such as poor stability, cumbersome operation steps, and low detection sensitivity, which are difficult to apply to the actual detection of chlorpyrifos

Method used

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  • ELISA kit for detecting chlopyrifos residue and method of use thereof
  • ELISA kit for detecting chlopyrifos residue and method of use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0079] The preparation of embodiment 1 antigen

[0080] Preparation of chlorpyrifos antigen:

[0081] a. Dissolve 50 μmol / L chlorpyrifos hapten in 1 mL of DMF, then add equimolar DCC and NHS to the solution, and allow it to react overnight at room temperature;

[0082] b. Centrifuge, take 800 μL of supernatant, slowly add to 4 mL of 15 mg / mL BSA or OVA carrier protein carbonic acid buffer solution, and then react for 4 hours under magnetic stirring;

[0083] c. After the reaction is completed, put it into a dialysis bag, first dialyze twice with distilled water, and then dialyze with 0.8% normal saline to obtain the product;

[0084] d. Determine the binding ratio by using the UV scanning method disclosed by Chen Xinjian et al. in 1998. Finally, the antigen is concentrated and stored or freeze-dried to obtain the chlorpyrifos immunogen and the coating agent, which are stored in a refrigerator at -20°C.

Embodiment 2

[0085] The preparation of embodiment 2 antibody

[0086] Chlorpyrifos mouse monoclonal antibody preparation:

[0087] Animal immunization procedure: Balb / c mice were used as immunized animals, the chlorpyrifos hapten and bovine serum albumin conjugate was used as the immunogen, the immunization dose was 60 μg / mouse, and the immunogen was mixed with the same amount of Freund's complete The adjuvant was mixed to make an emulsifier, injected intraperitoneally, and the same dose of immunogen plus an equal amount of Freund's incomplete adjuvant was mixed and emulsified at intervals of 3 weeks, and the booster immunization was given once.

[0088] Cell fusion and cloning: Splenocytes from immunized Balb / c mice were fused with SP2 / 0 myeloma cells at a ratio of 4:1, and the cell supernatant was measured by indirect competitive enzyme-linked immunosorbent assay, and positive wells were screened. The positive wells were cloned by microcloning until a hybridoma cell line stably secretin...

Embodiment 3

[0091] The extraction of embodiment 3 horseradish peroxidase or alkaline phosphatase

[0092] 1. Extraction of horseradish peroxidase

[0093] a. Water extraction: take by weighing 20 kilograms of washed fresh horseradish or horseradish skin, cut into small pieces, and mince in a pulverizer. Add 10 kg of water to the crushed slag slurry, stir and extract at low temperature for 8 hours, centrifuge at 3000 rpm for 10 minutes, and collect the supernatant.

[0094]b. Fractional separation of ammonium sulfate: add 226 grams of ammonium sulfate powder per liter of filtrate, stir while adding, put overnight at room temperature. The next day, draw the supernatant, and then add 258 grams of ammonium sulfate powder per liter of supernatant, and stir as you add it. After the ammonium sulfate is completely dissolved, put it in a cold room overnight. The next day, the supernatant was sucked off, and the precipitated part was centrifuged at 13,000 rpm for 20 minutes in a refrigerated cent...

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Abstract

The present invention discloses enzyme-linked immune kits for detecting chlorpyrifos residue in pesticide including antigenic enzyme label plate coated with antibody, enzyme labeled chlorpyrifos antibody working fluid, standard chlorpyrifos solution, substrate solution, substrate buffer solution, reaction termination liquid, condense washing liquid and sample diluted concentrated liquid. The present invention also discloses using method for detecting chlorpyrifos by said kits including steps of pretreatment of sample, detecting with kits, result process and analysis and so on. The kits provided by present invention employ directly competing enzyme-linked immunoadsorption analysis has merits of high sensitivity and good stability, is suitable for detecting chlorpyrifos residue in environment and farm products, and has importance practical significance.

Description

technical field [0001] The invention relates to the technical field of enzyme-linked immunoassay detection, in particular to an enzyme-linked immunoassay kit for detecting pesticide chlorpyrifos residues and a use method thereof. Background technique [0002] Chlorpyrifos, also known as Lesiben, English name chlorpyrifos, chemical name O, O-diethyl-O (3,5,6-trichloro-2-pyridyl) phosphorothioate, is produced by Dow Chemical Company ( The organophosphate insecticide developed by Dow Chemical Co. in 1965 is one of the most widely used insecticides in the world (Manclus, 1996). my country also regards it as an alternative product of highly toxic organophosphorus pesticides, and its production and usage are increasing year by year. [0003] Chlorpyrifos affects brain development and has an effect on the thyroid system, resulting in a decrease in blood thyroxine concentration. Excessive intake may cause convulsions and dizziness, especially harmful to children's health. Over the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/543G01N33/535G01N33/577
Inventor 孙远明杨金易雷红涛吴青王弘肖治理沈玉栋
Owner SOUTH CHINA AGRI UNIV
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