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Preparation method and application of fenitrothion antibody

A technology of fenitrothion and antibody, applied in the field of immunoassay

Active Publication Date: 2019-05-10
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is no enzyme-linked immunoassay method for fenitrothion

Method used

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  • Preparation method and application of fenitrothion antibody
  • Preparation method and application of fenitrothion antibody
  • Preparation method and application of fenitrothion antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 3

[0123] Example 3 Preparation of anti-fenitrothion monoclonal antibody

[0124] 1. Experimental method

[0125] Take 6-8 week-old Bal b / c mice (Guangdong Provincial Experimental Animal Center), and prepare the concentration of 1 mg / mL fenitrothion complete antigen shown in formula (III-1) to immunize the antigen with Freund's complete adjuvant Mix equal amounts of the same doses, and after complete emulsification, inject 100 μL into the abdomen and back of mice. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations, and immunization was performed every 3 weeks, for a total of 4 booster immunizations. One week after the second booster immunization, blood was taken from the tail vein to test the titer and inhibition. After the fourth booster immunization, mice with higher titer and inhibition rate were selected for cell fusion, and the dose was doubled for booster immunization 3 days before...

Embodiment 4

[0132] Example 4 Establishment and specificity detection of anti-fenitrothion detection method

[0133] 1. Experimental method

[0134] The monoclonal antibody prepared in Example 3 was used to establish a standard curve, the working concentration of the coating source was 1000 ng / mL, and three groups of parallel experiments (n=3) were used.

[0135] Antiserum indirect competition ELISA detection steps are as follows:

[0136] S1. Coating: Dilute the coating source (fenitrothion complete antigen shown in formula (IV-1)) with pH 9.6 carbonic acid buffer to 1 μg / mL, add to the wells of the microtiter plate, 100 μL / well, 37°C overnight in a water bath.

[0137] S2. Washing: Pour off the liquid in the wells, wash the plate twice with a plate washer, add 250 μL of washing solution to each well, and dry the liquid in the wells.

[0138] S3. Blocking: add 120 μL of blocking solution to each well, block at 37°C for 3 hours, shake off the liquid in the well, and place it upside down...

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Abstract

The invention discloses a preparation method and an application of a direct recognition fenitrothion antibody. The structural formula of a hapten of fenitrothion is as shown in a formula (II) or (I) in the specification. The hapten is coupled to a carrier protein, and two artificial antigens are successfully prepared. One artificial antigen is used for immunizing animals to prepare hybridoma cellscapable of secreting the fenitrothion antibody. The other artificial antigen is used as a coating antigen, and a fenitrothion detection method based on indirect competitive enzyme linked immunosorbent assay is further built. The detection method for detecting fenitrothion pesticide residues has the advantages of simplicity, convenience, rapidness, strong specificity and high sensitivity, detectedIC50 value is 7.13ng / mL, detection limit is (IC10) 1.7ng / mL, and linear detection range is 2.9-17.9ng / mL.

Description

technical field [0001] The invention relates to the technical field of immunoassay, more specifically, to a preparation method and application of an antibody that directly recognizes fenitrothion. Background technique [0002] my country is a large agricultural country, and a large amount of pesticides are consumed in agricultural production every year. The residual pesticides are likely to cause serious problems such as environmental pollution and acute food poisoning. Fenitrothion belongs to organophosphorus pesticides and is one of the widely used insecticides. For the detection of fenitrothion, the instrumental analysis method is currently the main analysis method, but the instrumental analysis method requires expensive instruments, dedicated personnel for operation and maintenance, complex pre-treatment, and cannot achieve rapid on-site detection. In order to make up for the shortcomings of instrumental analysis, it is necessary to develop an analytical method that can...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/24C07F9/20C07K14/79C07K14/765C07K14/795C07K14/77C07K1/107C07K16/44G01N33/543
Inventor 徐振林陈子键孙远明傅慧君肖治理沈玉栋杨金易王弘雷红涛
Owner SOUTH CHINA AGRI UNIV
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