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Pyrethroid pesticide hapten, preparation method and application thereof

A pyrethroid and hapten technology, applied in the field of food safety immunology detection, can solve the problems of unsuitable samples, high technical requirements, high cost, etc., and achieve the effects of good specificity, simple synthesis method and high yield

Active Publication Date: 2017-09-22
BEIJING KWINBON BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The instrumental analysis method has the advantage of high sensitivity, but it is expensive and te

Method used

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  • Pyrethroid pesticide hapten, preparation method and application thereof
  • Pyrethroid pesticide hapten, preparation method and application thereof
  • Pyrethroid pesticide hapten, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Synthesis and identification of pyrethroid pesticide hapten

[0024] The synthetic route of pyrethroid pesticide hapten is attached figure 1 shown.

[0025] Take 1.0 g of [cyano-(3-phenoxy)-methyl] 2-bromoacetate, add N,N-dimethylformamide to dissolve, add 0.72 g of sodium bicarbonate, stir well, add 0.45g of 1-amino-2,2-dimethylcyclopropanecarboxylic acid, stirred at 60°C for 4h; stop the reaction, add water to extract with ethyl acetate, leave to separate layers, dry the organic phase with anhydrous sodium sulfate, evaporate to dryness, and Silica gel column, petroleum ether / ethyl acetate = 1:1 elution and separation, to obtain 1.1 g of cyclopropane carboxymethrin hapten, which is a pyrethroid pesticide hapten, with a yield of 97.35%.

[0026] The above haptens were identified by H NMR spectroscopy, the results are shown in the attached figure 2 . 1 H-NMR (CDCl 3 ,300MHz) δ: 11.00(1H,s,COOH), 7.41(2H,dd,ArH), 7.17(1H,dd,ArH), 7.14(2H,dd,ArH), 7.08(2H,...

Embodiment 2

[0027] Example 2: Synthesis and identification of artificial antigens of pyrethroid pesticides

[0028] The carboxyl group of the pyrethroid pesticide hapten is used as the active site, and the immunogen is prepared by coupling with human serum albumin (HSA) by the mixed anhydride method; by coupling with ovalbumin (OVA) by the carbodiimide method, the preparation The package was originally.

[0029] 1. Preparation of immunogen by mixed anhydride method

[0030] Take 24mg of cyclopropanecarboxythrin hapten, add 0.5mL of dimethyl sulfoxide to dissolve, add 20μL of triethylamine, stir and mix well, add 20μL of isobutyl chloroformate, and stir for 30min to obtain hapten activation solution A; Add 0.1mol / L phosphate buffer solution to dissolve HSA 100mg to obtain solution B; add solution A dropwise to solution B, stir at room temperature for 4 hours, dialyze 0.01mol / L phosphate buffer solution for 3 days, change every day solution 3 times to obtain the immunogen, and store it at...

Embodiment 3

[0036] Example 3: Preparation, purification and identification of monoclonal antibodies

[0037] 1. Animal immunization

[0038]Take 10 healthy 6-8 week female Balb / c mice (divided into two groups, A and B, 5 in each group), and inject them subcutaneously at multiple points on the back of the neck after initial immunization with complete Freund's adjuvant emulsification, each small The mouse immunization dose was 200 μg of immunogen; after that, booster immunization was injected subcutaneously at multiple points on the back of the neck every two weeks, and Freund's incomplete adjuvant was used for emulsification; for the last immunization, physiological saline was used instead of Freund's incomplete adjuvant, and intraperitoneal injection was used. Dosage is the same as previous times. The specific immunization steps are shown in Table 1.

[0039] Table 1 Mouse immunization program

[0040]

[0041] 7 days after the third, fourth and booster immunizations, blood was coll...

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Abstract

The invention relates to a hapten, a preparation method and application thereof, in particular to a pyrethroid pesticide hapten, a preparation method and application thereof. The pyrethroid monoclonal antibody hybridoma cell line F-3-2 screened based on the pyrethroid pesticide hapten is preserved in China General Microbiological Culture Collection Center with a preservation number of CGMCC No.13842. The titer of a pyrethroid monoclonal antibody secreted by the cell line reaches 2*10<5>, the cross reaction rate with deltamethrin is 100%, the cross reaction rate with cyhalothrin is 50.9%, the cross reaction rate with fluvalinate is 103.6%, the cross reaction rate with cyfluthrin rate is 70.7%, the cross reaction rate with fenvalerate is 74.4%, and the cross reaction rate with benzene permethrin is 69.0%. The pyrethroid pesticide hapten provided by the invention provides the condition for immunodetection of pyrethroid pesticide multi-residue, and has practical application value.

Description

technical field [0001] The invention relates to a hapten and its preparation method and application, in particular to a pyrethroid pesticide hapten and its preparation method and application, and belongs to the technical field of food safety immunological detection. Background technique [0002] Pyrethroid pesticides have the characteristics of wide insecticidal spectrum, rapid drug effect, light and heat stability, and long drug effect time. They are widely used in agriculture, forestry, public health and other fields, but they are harmful to ecosystems and human health. Especially highly toxic to bees and aquatic organisms. The World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) have set strict limits for the residues of pyrethroid pesticides on fruits and vegetables, and my country has also set limits for this type of pesticides (GB 2763-2016). [0003] At present, the detection methods of pyrethroid pesticides are mainly...

Claims

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Application Information

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IPC IPC(8): C07C255/39C07K16/44C07K14/765G01N33/531G01N33/577
CPCC07C255/39C07K14/765C07K16/44C07K19/00G01N33/531G01N33/577
Inventor 万宇平吴小胜崔海峰宋灏贾芳芳何方洋顾蓉蓉刘玉梅
Owner BEIJING KWINBON BIOTECH
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