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Triazophos biomimetic ELISA adsorption detection kit and detection method

A triazophos biomimetic enzyme and detection kit technology, which is applied in the field of chemical detection, can solve the problems of difficult preparation of specific antibodies, long detection time, expensive instruments, etc., and achieve high sensitivity, high sensitivity, and high selectivity.

Inactive Publication Date: 2018-08-10
INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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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 overcome the shortcomings of the traditional triazophos detection method, such as long detection time, expensive equipment, and difficult preparation of specific antibodies, to provide a rapid detection kit and method for triazophos

Method used

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Examples

Experimental program
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Effect test

preparation example Construction

[0038] Preferably, in the kit as described above, the preparation method of the triazophos enzyme-labeled antigen comprises:

[0039] Synthesize the coated antigen from the triazophos hapten by the mixed acid anhydride method, and then couple the coated antigen to the enzyme protein at a molar ratio of 18 to 22:1, more preferably at a molar ratio of 20:1;

[0040] Preferably, the enzyme protein is horseradish peroxidase.

[0041] Preferably, in the above-mentioned kit, the step of synthesizing the coated antigen with the triazophos hapten by the mixed acid anhydride method specifically includes: mixing the triazophos hapten with N,N-dimethylformamide at a concentration of 0.2 mmol ~0.3mmol: 1ml was mixed to obtain a mixed solution, and then n-tributylamine and ethyl chloroformate were added to the mixed solution to react for 50min~70min, and then the obtained reaction solution was mixed with CBS buffer solution in which OVA was dissolved, and after dialysis instant;

[0042]...

Embodiment 1

[0070] This embodiment provides a biomimetic immunoassay method for triazophosphatase labeling, comprising the following steps:

[0071] 1. Synthesis of hapten:

[0072] (1) Synthesis of O-ethylthiophosphoryl dichloride (TZM-1)

[0073] Weigh the phosphorus trichloride (PSCl 3 ) 68g (about 0.4mol) was placed in a three-necked flask with a low-temperature thermometer, cooled to -10~-5°C with an ice-salt water bath, and 55g (about 1.2mol) of absolute ethanol was added dropwise under vigorous stirring, strictly Control the rate of addition so that the temperature of the reaction solution is always not greater than 0°C. After the dropwise addition, the reaction was continued at 10°C for 2h. After the reaction, the reaction solution (100ml×2) was washed with (0±5)°C distilled water, the oil layer was separated and washed with anhydrous Na 2 SO 4 Dry it, then distill it under reduced pressure with a water pump, and collect fractions at 65°C to 75°C to obtain 51.8g of a colorles...

Embodiment 2

[0099] This embodiment provides a biomimetic immunoassay method for triazophosphatase labeling, comprising the following steps:

[0100] Step 1 is the same as in Example 1.

[0101] 2. Preparation of enzyme-labeled antigen

[0102] Take 0.3 mmol of the hapten, dissolve it in 1 ml of DMF, add 60 μl of n-tributylamine and 30 μl of ethyl chloroformate under stirring, and stir at room temperature for 1 h. Take 300μl of the reaction solution and slowly add it dropwise to 6ml of CBS buffer solution (0.01mol / L) dissolved with 120mgOVA. After stirring the reaction at room temperature, put it into a dialysis bag, first dialyze with distilled water for 3 times, and then dialyze with 0.01mol / L of PBS 3d, change the dialysate 3-4 times a day. Take out aliquots and store in a -20°C refrigerator.

[0103] After the antigen is activated, it is coupled to the enzyme protein, and the concentration of the enzyme protein in the reaction solution is 10 mg / ml (the molar ratio of antigen to enzyme...

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Abstract

The invention relates to the field of chemical detection, specifically to a triazophos biomimetic ELISA adsorption detection kit and detection method. The detection kit includes a molecularly imprinted polymer membrane ELISA plate and a triazophos ELISA antigen and is made by taking triadimefon as a template molecule, performing prepolymerization with a functional monomer, a cross-linking agent and an initiator in an inert solvent to obtain a prepolymerized solution, adding the prepolymerized solution to the ELISA plate, performing vacuum drying polymerization, and washing the template molecule off. The provided triazophos adsorption functional material is good in stability, long in service life and great in adverse environment resistance, and the defects that a traditional biological antibody is long is preparation period, is prone to be inactive, and is high in cost are overcome.

Description

technical field [0001] The invention relates to the field of chemical detection, in particular to a triazophos bionic ELISA detection kit and a detection method. Background technique [0002] Triazophos is a moderately poisonous, broad-spectrum organophosphorus insecticide, which has strong contact and stomach poisoning effects. After entering the human body with food, it will cause a large amount of acetylcholine to accumulate at the junction of neuroeffectors, and mushrooms will occur. Alkali-like, nicotine-like and central nervous system symptoms, since December 31, 2016, the use of triazophos on vegetables is prohibited. [0003] At present, the routine detection methods of triazophos include gas chromatography, ultra-high performance liquid chromatography-tandem mass spectrometry, etc., but these methods are complicated in pretreatment, high in cost and slow in detection speed, and are not suitable for on-site real-time detection. Immunoassay has high specificity, high...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/58G01N33/543
CPCG01N33/54353G01N33/581G01N2430/10
Inventor 佘永新
Owner INST OF QUALITY STANDARD & TESTING TECH FOR AGRO PROD OF CAAS
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