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Broadspectrum specificity polyclone antibody of methoxyl organophosphorus pesticide and uses thereof

A polyclonal antibody, organophosphorus pesticide technology, applied in specific peptides, instruments, analytical materials, etc.

Inactive Publication Date: 2008-12-31
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem that most current pesticide residue immunological detection methods are only applicable to single pesticide residue detection, and to save the detection time and detection cost of pesticide residues, the present invention provides a method that can simultaneously detect multiple methoxyl A broad-spectrum specific polyclonal antibody based on organophosphorus pesticide residues was successfully applied to the detection of vegetable samples

Method used

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  • Broadspectrum specificity polyclone antibody of methoxyl organophosphorus pesticide and uses thereof
  • Broadspectrum specificity polyclone antibody of methoxyl organophosphorus pesticide and uses thereof
  • Broadspectrum specificity polyclone antibody of methoxyl organophosphorus pesticide and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Synthesis of Haptens

[0042] 0.58 g of acrylonitrile and 18 mL of 6% potassium hydroxide aqueous solution were added to 4 g of 45% O, O-dimethyl sodium thiophosphate aqueous solution, and the mixture was refluxed in an oil bath for 4 h. Cool to room temperature after the reaction, and acidify the reaction solution through 732 strong ionic resin. The solvent was evaporated under vacuum, and the residue was separated by column chromatography to obtain a yellow oil as the target product. The substance was subjected to thin-layer chromatography TLC, and the Rf value obtained by using ethyl acetate:petroleum ether=1:2 as a developing solvent was 0.31.

[0043] The spectrogram in Figure 1 is parsed as: 1 H NMR (DMSO-d 6 )δ: 2.18(t, J=6.3Hz, 2H, CH 2 CO), 2.31(t, J=6.6Hz, 2H, SCH 2 ), 3.35 (d, J=14.1Hz, 6H, CH 3 O); Fig. 2 spectrogram resolves as 13 C NMR (DMSO-d 6 )δ: 37.99 (CH 2 CO), 51.19 (d, J=2.7Hz, SCH 2 ), 57.45 (d, J=30.9, CH 3 O), 173.55 (COOH);...

Embodiment 2

[0044] Synthesis of embodiment 2 antigen and coated antigen

[0045] antigen:

[0046] Active ester method Weigh 0.05mmol (equivalent to 10.8mg) hapten and 0.15mmol (equivalent to 15.5mg) N-hydroxysuccinimide NHS, dissolve in 0.4mL DMF, weigh 0.1mmol (equivalent to 20.6mg ) Cyclodihexylcarboimide DCC was dissolved in 0.4mL of DMF, the above two solutions were combined, stirred and reacted at room temperature for 4h, and the above reaction solution was centrifuged to remove the precipitated dicyclohexyl urea and the supernatant; 40mg BSA was weighed, Dissolve in 10 mL of carbonate buffer solution to form the reaction solution; place the reaction solution on a magnetic stirrer, add the supernatant solution dropwise to the reaction solution at 4°C, and magnetically stir overnight. After the reaction, the reaction solution was put into a dialysis bag, and dialyzed in ultrapure water at 4° C. for three days, and the water was changed every four hours. After dialysis, aliquots wer...

Embodiment 3

[0054] Example 3 Preparation of polyclonal antibody

[0055] Three New Zealand white rabbits were immunized with the two antigens prepared according to the method of Example 2, and the specific immunization scheme is shown in Table 1.

[0056] Table 1 Animal immunization scheme for preparing broad-spectrum specific antibody

[0057] Number of immunizations

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Abstract

The invention relates to a methoxy-organophosphorus pesticide broad-spectrum specific polyclonal antibody and the application; the invention is characterized in that the antibody is obtained through the following steps: phosphate is taken as the semi-antigen and coupled with bovine serum albumin BSA to prepare an antigen, and immunise New Zealand white rabbits to prepare the polyclonal antibody. The polyclonal antibody is used to detect the methoxy-organophosphorus pesticide residue through indirect competitive ELISA; the coating antigen used in the detection is a conjugate between hapten and OVA through active ester or mixed anhydride method. The methoxy-organophosphorus pesticide broad-spectrum specific polyclonal antibody has the advantages that the broad-spectrum specific antibody can detect a variety of methoxy-organophosphorus pesticide in vegetables and other foods at the same time so as to significantly save the sample testing time and cost, thereby fully demonstrating the rapid and low-cost characteristics of immunoassay technology.

Description

Technical field: [0001] The invention relates to a broad-spectrum specific polyclonal antibody for the detection of various methoxyorganophosphorus pesticide residues and its application in the detection of vegetable pesticide residues. Background technique: [0002] Organophosphorus pesticides have the characteristics of broad insecticidal spectrum, high activity, and low residue. They have been widely used in agriculture, forestry, and health industries. Their residues in agricultural products have serious consequences for consumers' food safety. At present, organophosphorus pesticide residues have become one of the most prominent problems in food safety in my country, and it is urgent to establish a fast and efficient detection method. [0003] The analysis methods of organophosphorus pesticide residues mainly use high performance liquid chromatography (HPLC) or gas chromatography (GC). Although these methods have the advantages of reliability and high sensitivity, the co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/44G01N33/543
Inventor 梁颖刘贤金刘媛余向阳
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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