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ELISA kit for detecting fipronil residues on basis of nanobody and application of kit

A nanobody and kit technology, applied in the field of phage display technology, ELISA detection, and genetic engineering, can solve the problems of complex pretreatment, difficult on-site detection, and high cost of analysis methods, achieving less time-consuming, low sample detection cost, Simple pre-processing

Active Publication Date: 2018-01-09
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 fipronil residues

Method used

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  • ELISA kit for detecting fipronil residues on basis of nanobody and application of kit
  • ELISA kit for detecting fipronil residues on basis of nanobody and application of kit
  • ELISA kit for detecting fipronil residues on basis of nanobody and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Preparation of Fipronil Coating Antigen

[0025] The hapten and bovine serum protein are used to prepare the coupling complex as the coating antigen. The preparation method is as follows:

[0026] (1) Weigh 8.8mg 6-(3-cyano-5-(2,6-dichloro-4-(trifluoromethyl)phenyl)cyclopenta-1,3-dien-1-yl)amino )-6-oxohexanoic acid (MW=440.15) (0.02mmol), 2.65mg NHS (MW=115) (0.024mmol), 4.8mgDCC (MW=206) (0.023mmol) dissolved in 200μL anhydrous DMF, Stir at room temperature and react overnight. The reaction solution was centrifuged (5000rpm, 10min), the precipitate was discarded, and the supernatant was the active ester.

[0027] (2) Weigh 20mg BSA (MW=67000) dissolved in 2mL carbonate buffer solution (0.05mol / mL, pH9.5), add 150μL of active ester solution drop by drop with stirring, slowly, about 20-30min Finish adding. Then continue to stir the reaction at room temperature for 4-6 hours.

[0028] (3) The reaction solution is put into a dialysis bag and dialyzed with PBS (0.01...

Embodiment 2

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

[0030] The active ester method is used to couple the hapten to keyhole limpet hemocyanin. The specific method is as follows:

[0031] (1) Weigh 66.02mg 6-(3-cyano-5-(2,6-dichloro-4-(trifluoromethyl)phenyl)cyclopenta-1,3-dien-1-yl)amino )-6-oxohexanoic acid (MW=440.15)(0.15mmol), 17.825mg NHS(MW=115)(0.155mmol), 31.518mg DCC(MW=206)(0.153mmol) dissolved in 1500μL anhydrous DMF , The reaction was stirred overnight at room temperature. The reaction solution was centrifuged (5000rpm, 10min), the precipitate was discarded, and the supernatant was the active ester.

[0032] (2) Take 6 mL of KLH solution (6.8 mg / mL), add 1200 μL of active ester solution dropwise with stirring, and slowly add it in about 20-30 minutes. Then continue to stir the reaction at room temperature for 4-6 hours.

[0033] (3) The reaction solution is put into a dialysis bag and dialyzed with PBS (0.01mol / L pH7.4). Change the fluid every 6-8...

Embodiment 3

[0062] Example 3 Screening of Specific Fipronil Phage Display Nanobody

[0063] Coat the coating antigen of Example 2 in the first well of a 96-well microtiter plate at a concentration of 100 μg / mL at 4°C overnight; the next day, pour out the coating solution and wash with PBST for 3 times. Block the first and second wells of the target plate with BSA and incubate at 37°C for 1 hour; pour out the blocking solution and wash with PBST 3 times; add the phage antibody library of Example 2 to the first well and react for 2 hours; pour out the liquid, Pat dry on clean absorbent paper, wash with PBST 5 times; add 100μL fipronil standard to the first well, react for 1h; aspirate the liquid in the first well, add to the second well, react for 1h, Remove the phage bound to BSA; collect the eluate, take 5 μL for titer determination, and use the rest for amplification.

[0064] Add the phage eluate to fresh E. coli ER2738 bacterial solution, and let it stand for 15 min at 37°C; add carbenicil...

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Abstract

The invention discloses an ELISA kit for detecting fipronil residues on basis of a nanobody 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 coated with a fipronil coating antigen, the reagent comprises an anti-fipronil nanobody, a fipronil standard solution, an enzyme-labelled second antibody, a buffer solution PBS, a scrubbing solution PBST, a substrate solution, a developing solution, a reaction stop solution and the like. In the detection process, the coating antigens adsorbed on theporous wall of the ELISA plate and to-be-detected fipronil compete with each other to react with the antibodies, and results are observed through a developing reaction. The invention further providesa PCR primer which is generally used at C terminals and used for establishing a phage display nanobody library for camelidae animals. The kit has the advantages that the fipronil residues in water andvegetables can be accurately and sensitively detected, the sample pretreatment process is simple, little time is consumed, a large quantity of samples can be detected simultaneously, and the sample detection cost is far lower than that with a conventional instrument detection method.

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 detecting fipronil residue based on nano-antibody and its application. Background technique [0002] Fipronil (Fipronil), also known as fipronil, fipronil, and fipronil, is a widely used N-phenylpyrazole insecticide with trifluoromethylsulfonyl group, which is harmful to The pests of major crops such as grain, cotton, oil, fruits and vegetables, tea have good control effect. During the use of pesticides, many pests are resistant to organophosphate pesticides, carbamate pesticides and pyrethroid insecticides. For such resistant insects, fipronil also has a good effect. Fipronil is highly toxic to aquatic products, bees, silkworms, rodents, and birds, and moderately toxic to mammals. Fipronil can damage human β3GABAA receptor subunits and cause human neurodevelopmental disorders, such as autism , Angleman sy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10C07K16/44G01N33/58G01N33/531
Inventor 许艇王楷林优优刘志平李季布鲁斯·杜普里·哈莫克
Owner CHINA AGRI UNIV
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