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A vhh-elisa kit suitable for the analysis of triazophos residues

A kit, the technology of triazophos, applied in the field of ELISA kits, can solve the problems of difficult on-site detection, complicated pretreatment, high cost of analysis methods, etc., and achieve low cost of sample detection, less time-consuming, and simple pretreatment process Effect

Active Publication Date: 2020-07-31
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a method with high specificity, high sensitivity, high accuracy and high precision for the shortcomings of current pesticide residue instrument analysis methods such as high cost, complicated pretreatment, poor specificity, low sensitivity and difficulty in on-site detection in experiments. , The operation method is simple, and can be used for rapid detection of large batches of samples, ELISA detection kit for the analysis of triazophos residues

Method used

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  • A vhh-elisa kit suitable for the analysis of triazophos residues
  • A vhh-elisa kit suitable for the analysis of triazophos residues
  • A vhh-elisa kit suitable for the analysis of triazophos residues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1 3

[0024] Example 1 Preparation of Triazophos-coated Antigen

[0025] A conjugated complex was prepared with hapten and bovine serum albumin as the coating antigen. The preparation method is as follows:

[0026] (1) Weigh 7.4mg O-ethyl-O-3-(1-phenyl-1,2,4-triazolyl)-N-(3-carboxymethyl)phosphorothioate (MW=370 ) (0.02mmol), 2.65mg of NHS (MW=115) (0.024mmol), 4.8mg of DCC (MW=206) (0.023mmol) were dissolved in 200 μL of anhydrous DMF, and stirred overnight at room temperature. Centrifuge the reaction solution (5000rpm, 10min), discard the precipitate, and the supernatant is the active ester.

[0027] (2) Dissolve 20mg of BSA (MW=67000) in 2mL of carbonate buffer solution (0.05mol / mL, pH9.5), add 150μL of active ester solution drop by drop under stirring, slowly, and finish adding in about 20min . Then the stirring reaction was continued at room temperature for 4 h.

[0028] (3) The reaction solution was put into a dialysis bag and dialyzed with PBS (0.01mol / L pH7.4). The liq...

Embodiment 2 3

[0029] Example 2 Construction of Triazophos Phage Display Nanobody Library

[0030] The active ester method was used to couple the hapten to keyhole limpet hemocyanin, and the specific method was as follows:

[0031](1) Weigh 59.7mg O-ethyl-O-3-(1-phenyl-1,2,4-triazolyl)-N-(5-carboxypentyl)phosphorothioate (MW=398 ) (0.15mmol), 17.825mg NHS (MW=115) (0.155mmol), 31.518mg DCC (MW=206) (0.153mmol) were dissolved in 1500 μL of anhydrous DMF, and stirred overnight at room temperature. Centrifuge the reaction solution (5000rpm, 10min), discard the precipitate, and the supernatant is the active ester.

[0032] (2) Take 6mL of KLH solution (6.8mg / mL), add 1200μL of active ester solution drop by drop under stirring, and add slowly in about 20 minutes. Then the stirring reaction was continued at room temperature for 4 h.

[0033] (3) The reaction solution was put into a dialysis bag and dialyzed with PBS (0.01mol / L pH7.4). The liquid was changed every 6 hours, and the liquid was ch...

Embodiment 3

[0060] Example 3 Screening of specific triazophos phage display nanobodies

[0061] Coat the first well of a 96-well ELISA plate with the coated antigen of Example 2, at a coating concentration of 100 μg / mL, overnight at 4°C; the next day, pour out the coating solution, wash 3 times with PBST, and the enzyme The first and second wells of the target plate were blocked with BSA, incubated at 37°C for 1 hour; poured out the blocking solution, and washed 3 times with PBST; added the phage antibody library of Example 2 to the first well, and reacted for 2 hours; poured out the liquid, Pat dry on clean absorbent paper, wash 5 times with PBST; add 100 μL triazophos standard to the first well, react for 1 h; suck out the liquid in the first well, add to the second well, react for 1 h, Phage bound to BSA were removed; the eluate was collected, 5 μL was used for titer determination, and the rest was used for amplification.

[0062] Add the phage eluate to fresh Escherichia coli ER2738 ...

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Abstract

The invention discloses a nanobody-based ELISA kit for detecting triazophos residues and an application of the kit. The kit comprises a kit body, an ELISA plate arranged in the kit body and a reagent, wherein each hole of the ELISA plate is enveloped with a triazophos envelope antigen, and the reagent comprises an anti-triazophos nanobody, a triazophos standard solution, an enzyme-labeled second antibody, a buffer solution PBS (phosphate buffered saline), a scrubbing solution PBST, a substrate solution, a developing solution, a reaction stop solution and the like. During detection, the envelope antibodies adsorbed on the hole walls of the ELISA plate compete with to-be-detected triazophos to react with the antibody, and the result is observed through a chromogenic reaction. The concentration of to-be-detected triazophos can be calculated by detecting triazophos with the known concentration and drawing a standard curve. The kit has the advantages that the triazophos residues in water and vegetables can be detected accurately and sensitively, the sample pre-treatment process is simple and consumes short time, a large quantity of samples can be detected simultaneously, and the sample detection cost is far lower than that of traditional instrument detection methods.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering, phage display technology and ELISA detection, in particular to an ELISA kit for detecting triazophos residues based on a nanobody and an application thereof. Background technique [0002] Triazophos is a widely used organophosphate insecticide, which has good control effects on pests that harm grain, cotton, oil, fruits and vegetables, tea and other major crops. Triazophos is highly toxic to fish, bees, and silkworms, and moderately toxic to mammals. After entering the human body, it will cause a large amount of acetylcholine to accumulate at the junction of neural effectors, causing muscarinic, nicotinic, and central nervous system reactions. symptom. [0003] After the use of highly toxic organophosphorus insecticides such as methamidophos and parathion was restricted or banned, the usage of triazophos has increased rapidly in recent years. Due to the long half-life and easy resid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/44G01N33/543
CPCC07K16/44G01N33/54306G01N2430/10
Inventor 许艇王楷刘志平林优优李季布鲁斯·杜普里·哈莫克
Owner CHINA AGRI UNIV
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