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ELISA kit for one-step detection of triazophos residues and use thereof

A kit, the technology of triazophos, is applied in the field of ELISA kits for detecting triazophos residues by one-step method, can solve the problems of complicated pretreatment, long time consumption, many operation steps and the like, and achieves simple pretreatment process and less time consumption Effect

Active Publication Date: 2018-01-19
CHINA AGRI UNIV
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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 aim at the disadvantages of current pesticide residue instrument analysis methods such as high cost, complex pretreatment, poor specificity, low sensitivity and difficulty in on-site detection in experiments; Influenced by the specificity and sensitivity of the secondary antibody, it has disadvantages such as relatively low stability, and provides a high-specificity, high-sensitivity, high-accuracy, high-precision, simple operation method, and can be used for large-scale samples quickly Detectable, ELISA kit for one-step detection of triazophos residues

Method used

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  • ELISA kit for one-step detection of triazophos residues and use thereof
  • ELISA kit for one-step detection of triazophos residues and use thereof
  • ELISA kit for one-step detection of triazophos residues and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Preparation of triazophos-coated antigen

[0020] (1) Weighing 7.4mg O-ethyl-O-3-(1-phenyl-1,2,4-triazolyl)-N-(3-carboxymethyl)thiophosphoramidate, 2.65mg NHS , 4.8 mg of DCC was dissolved in 200 μL of anhydrous DMF, stirred and reacted at room temperature overnight; the reaction solution was centrifuged, the precipitate was discarded, and the supernatant was active ester;

[0021] (2) (2) Dissolve 20 mg of BSA in 2 mL of 0.05 mol / mL pH9.5 carbonate buffer solution, add 150 μL of the above-mentioned active ester dropwise under stirring, and complete the addition in 20 to 30 minutes; then continue to stir at room temperature for reaction 4 ~6h;

[0022] (3) After the reaction, the reaction solution was put into a dialysis bag and dialyzed with PBS (0.01mol / L, pH7.4); the solution was changed every 6 hours for a total of 5-6 times. After dialysis, centrifuge, discard the precipitate, and collect the supernatant as the antigen coating solution.

Embodiment 2

[0023] Example 2 Construction of Triazophos Phage VHH-AP Genetic Engineering Antibody

[0024] In the early stage, the specific triazophos nanobody (SEQ ID NO: 1) was screened by phage display technology, and the preserved specific triazophos nanobody plasmid was extracted, and the VHH gene fragment was cloned, and the sticky end was modified with restriction endonuclease SfiI , the VHH gene fragment was ligated to the vector Pecan 45 by T4 ligase (see Wang J, Majkova Z, Bever C RS, et al. One-Step Immunoassay for Tetrabromobisphenol A Using a Camelid SingleDomain Antibody–Alkaline Phosphatase Fusion Protein[J]. Analytical Chemistry, 2015, 87(9): 4741. and Liu X, Xu Y, Wan D B, et al. Development of a nanobody-alkalinephosphatase fusion protein and its application in a highly sensitive directcompetitive fluorescence enzyme immunoassay for detection of ochratoxin A incereal [J].Analytical Chemistry,2015,87(2):1387. The carrier Pecan 45 was donated by Dr. Jinny L.Liu and Dr. Ell...

Embodiment 3

[0033] Example 3 Expression of Specific Triazophos VHH-AP Genetic Engineering Antibody

[0034] Extract positive monoclonal plasmids, transform them into Escherichia coli TOP10F' competent cells, and spread them on solid medium for overnight culture after thawing. The next day, pick a single clone and culture it in SB-carbenicillin medium (the working concentration of carbenicillin is 50mg / L), and add IPTG to induce overnight expression; Column purification, that is, to separate and purify the VHH-AP genetic engineering antibody by using histidine tag and nickel chloride affinity chromatography in a nickel column to obtain a high-purity anti-triazophos VHH-AP genetic engineering antibody, which is sequenced by amino acid Analysis, the amino acid sequence of the resulting VHH-AP genetically engineered antibody is shown in SEQ ID NO:4.

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Abstract

The invention discloses an ELISA kit for one-step detection of triazophos residues and a use thereof. The kit comprises a box body, an ELISA plate in the box body and reagents. All pores of the ELISAplate are coated with a triazophos coating antigen. The reagents comprise an anti-triazophos VHH-AP genetically engineered antibody, a triazophos standard solution, a buffer PBS, a washing liquid PBST, a developing solution and a reaction termination solution. In detection, the coating antigen adsorbed by the pore walls of the ELISA plate and triazophos to be detected compete with each other to react with the antibodies and through the developing reaction, the result is observed. The invention also provides C-terminal universal PCR primers for constructing a VHH-AP genetically engineered antibody. The method can accurately and sensitively detect triazophos residues in product water, soil and vegetables, has simple sample pretreatment processes and small time consumption and realizes detection of a large amount of samples and a sample detection cost far lower than that of the existing detection method. Compared with the traditional ELISA kit, sample detection time is greatly shortened.

Description

technical field [0001] The invention relates to the fields of genetic engineering, phage display technology and ELISA detection technology, in particular to an ELISA kit for one-step detection of triazophos residue and its application. Background technique [0002] Triazophos is a carbamate pesticide. The routine detection of this kind of pesticide residues mainly uses instrumental analysis methods, including gas chromatography (GC) and high performance liquid chromatography (HPLC). The instruments required for these analytical methods are expensive, the pretreatments such as sample separation, extraction, purification, and derivation are complicated, the analysis speed is slow, and the detection sensitivity is low, which makes it difficult to meet the needs of on-site rapid detection. Many physical and chemical analysis techniques have their own limitations, such as the poor thermal stability of triazophos, which is difficult to analyze by gas chromatography, and liquid chr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/531
Inventor 许艇王楷刘志平李季布鲁斯·杜普里·哈莫克
Owner CHINA AGRI UNIV
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