Fenitrothion hapten, artificial antigen, specified antibody and use thereof

A technology of fenitrothion and artificial antigen, which is applied in the field of immunology, can solve the problems that there are no research papers or works on the immune recognition mechanism of hapten molecules, and achieve the effect of good affinity and low cross-reaction rate

Inactive Publication Date: 2007-08-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there are no dissertations or works dedicated to the study of hapten molecular design and its immune recognition mechanism

Method used

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  • Fenitrothion hapten, artificial antigen, specified antibody and use thereof
  • Fenitrothion hapten, artificial antigen, specified antibody and use thereof
  • Fenitrothion hapten, artificial antigen, specified antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Preparation of fenitrothion hapten, artificial antigen and antibody thereof

[0024] 1. Synthesis of hapten O, O-dimethyl-O-[3-methyl-4-aminophenyl] phosphorothioate (abbreviated as FNH2)

[0025] The synthesis of the hapten FNH2 is accomplished through the amination of fenitrothion. The synthetic route is as follows:

[0026]

[0027] The specific operation is as follows:

[0028] Take about 2.0 g of fenitrothion, dissolve it in 20 ml of ether, wash twice with 2×10 ml of cold 1% sodium carbonate solution, separate the ether layer, transfer it to a three-necked bottle, add acetic acid / hydrochloric acid (9:1 ) solution 20ml, add 4g zinc powder, reflux for 1.5h, and filter. Filter residue with 30mL CCl 4 Wash three times, then wash CCl with 30 ml of distilled water 4 phase, discarding the aqueous phase. Add 10% NaOH to the filtrate to adjust the pH value to neutral. At this time, a flocculent precipitate appears, and extract with ether 4×25ml. Dieth...

Embodiment 2

[0052] The assembly of embodiment 2 test kits

[0053] The present invention applies the prepared artificial antigen and antibody to the preparation of an immunoassay kit suitable for the detection of fenitrothion residues. In each well of the target plate, the coating antigen capable of specifically binding to the anti-fenitrothion antibody is coated with the coating solution, and blocked with 2% to 5% gelatin.

[0054] Reagents in the box include: washing solution (diluent), substrate diluent, anti-fenitrothion antibody (suitable for indirect ELISA), fenitrothion standard solution, horseradish peroxidase-labeled goat anti-rabbit antibody ( Suitable for indirect competition ELISA) or horseradish peroxidase-labeled anti-fenitrothion rabbit antibody (for direct competition ELISA), substrate, chromogenic substance and reaction termination solution, the preparation method is as follows:

[0055] Washing solution (reaction diluent) 40mL, the composition ratio is 0.1g of potassium...

Embodiment 3

[0062] Preparation of embodiment 3 enzyme-labeled antibody (improved sodium periodate method)

[0063] Weigh 5-10mg of horseradish peroxidase HRP and dissolve it in 1L of distilled water, add 0.2-0.4mL of newly prepared 0.1mol / L NaIO 4 The solution was stirred at room temperature in the dark for 15-30 minutes. Put the above solution into a dialysis bag, dialyze with 1 mmol / L acetate buffer solution, pH 4.4, overnight at 4°C. Use a carbonate buffer solution of pH 9.5 to raise the pH of the above-mentioned hydroformylated HRP solution to 9.0-9.5, then immediately add 1-2 mL of 0.01 mol / L carbonate buffer solution containing 10-20 mg of purified antibody, and keep away from room temperature. Stir lightly for 2 to 3 hours. Add 0.1-0.2mL of freshly prepared 4mg / mL NaBH4 solution, mix well, and place at 4°C for 2-3 hours. The reaction solution was put into a dialysis bag, dialyzed with 0.15 mol / L PBS, pH7.4, overnight at 4°C. Add an equal volume of saturated ammonium sulfate sol...

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Abstract

The invention discloses a O, O-dimethyl-O-[-3-methyl-4-amino phenyl] thionic phosphate (FNH2) as fenitrothion hapten, artificial antigen and specific antibody, which is characterized by the following: reducing nitro in the fenitrothion molecular structure into amino under acid condition with zinc powder; reacting to obtain the hapten molecule in connection with carrier protein; diazotizing to couple hapten and protein to obtain immunogen and peridium; using immune animal with immunogen to make the specific antibody.

Description

technical field [0001] The invention belongs to the field of immunology, and in particular relates to a fenitrothion hapten, an artificial antigen and a specific antibody prepared from the antigen, and also relates to the application of the hapten, the artificial antigen and the specific antibody. Background technique [0002] Due to the continuous development of pesticide use technology and the continuous expansion of the scale of use, the environmental impact caused by pesticide residues and the chronic and long-term effects on human health are increasingly concerned and worried, and the restrictions on pesticide residues in daily consumption and foreign trade are also increasing. It is becoming more and more strict, and puts forward new requirements and higher standards for the analysis and detection of objects, types, quantities, scope, indicators and many other aspects. In terms of the analysis and detection of pesticides and their metabolites residues, traditional anal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/177C07K16/18G01N33/53G01N33/577
Inventor 韩丽君马永强钱传范江树人
Owner CHINA AGRI UNIV
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