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Single-chain antibody and application thereof of single-chain antibody for detecting phosphorothioate pesticide residues

A single-chain antibody and antibody heavy chain technology, applied in anti-animal/human immunoglobulin, application, fungi, etc., can solve multi-component parallel detection with single detection spectrum, high cost, and inability to realize pesticide residue analysis and other issues, to achieve the effects of cost reduction, high sensitivity, and good cross-reactivity rate

Inactive Publication Date: 2015-06-17
PESTICIDE INST XIBEI AGRI & FORESTRY TECHUNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the antibodies in the organophosphorus pesticide enzyme-linked immunoassay kits developed at home and abroad are mostly prepared by polyclonal antibody or monoclonal antibody technology. This preparation method is expensive and has low yield, which greatly limits the wider application of immunoassay methods. use
At the same time, most of the kits developed have a single detection spectrum, which can only specifically detect a certain compound or pesticide, and cannot achieve multi-component parallel detection of pesticide residue analysis.

Method used

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  • Single-chain antibody and application thereof of single-chain antibody for detecting phosphorothioate pesticide residues
  • Single-chain antibody and application thereof of single-chain antibody for detecting phosphorothioate pesticide residues
  • Single-chain antibody and application thereof of single-chain antibody for detecting phosphorothioate pesticide residues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1, the preparation of recombinant single-chain antibody

[0028] 1. Synthesis of universal haptens of phosphorothioate compounds and preparation of artificial antigens

[0029] 1. Dissolve chloroacetic acid (945mg, 10mmol) with 10mL deionized water, add equimolar amount of sodium hydroxide to obtain sodium chloroacetic acid saline solution; Dissolve salt (1.75g, 10mmol) in a 50mL round-bottomed reaction flask. After heating up to 80°C, add sodium chloroacetate solution dropwise for about 30 minutes, stir magnetically, and terminate the reaction after about 6 hours; Cool to room temperature, acidify with an equimolar amount of hydrochloric acid, extract three times with dichloromethane, combine the organic phases, dry over anhydrous sodium sulfate, and obtain a pale yellow oily liquid by column chromatography. The universal hapten of the phosphorothioate compounds obtained, abbreviated as H2, is shown in formula (I).

[0030]

[0031] 2. The hapten synthe...

Embodiment 2

[0057] Embodiment 2, the preparation of soluble antibody

[0058] 1. Transformation of expression strains

[0059] 1. Streak the expression strain HB2151 on 2×YT solid medium and cultivate overnight at 37°C. Pick a single clone and culture it in 5mL 2×YT liquid medium overnight at 37°C with shaking at 180rpm. Then take 100 μL of the bacterial solution and inoculate it into 10 mL of 2×YT liquid medium, and culture it at 37°C with shaking at 180 rpm until OD 600nm ≈0.3~0.5, the HB2151 strain of the right period was prepared.

[0060] 2. Spread the positive strains on the SOBAG plate and culture overnight at 37°C. The next day, submerge the plate with 3mL 2×YT-AG liquid medium, use a spreader to scrape off the colony, add it to 50mL 2×YT-AG liquid medium, and shake it in a water bath at 37°C until OD 600nm ≈0.5. Take out 5mL and inoculate it into fresh 50mL 2×YT-AG medium, then add 5mL with a titer of about 10 12 The pfu helper phage M13KO7 was incubated at 37°C for 45 minu...

Embodiment 3

[0074] Example 3, Functional Detection of Antibodies

[0075] 1. Detect the binding ability of antibody and antigen

[0076] 1. Using indirect uncompetitive (indirect uncompetitive) ELISA method to determine the optimal working concentration of the obtained scFv single-chain antibody and coated antigen through square matrix titration test. Determine the optimum working concentration of antibody and antigen by square array test method, OD 450nm The value is about 1.0, and the concentration combination with less amount of antibody and antigen is the optimal working concentration of antibody-antigen. The coating antigen prepared in Example 1 was used as the coating antigen. The results showed that the optimal working concentration of the antibody was 1:6,400 dilution concentration, and the optimal working concentration of the antigen was 10 μg / mL.

[0077] The basic operation of the ELISA method is as follows:

[0078] 1) Coating: Coat the enzyme-linked reaction plate with se...

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Abstract

The invention relates to a single-chain antibody and an application thereof of the single-chain antibody for detecting phosphorothioate pesticide residues. The prepared single-chain antibody is polypeptide composed of a light-chain variable range, a heavy-chain variable range of a mice source and connecting peptide between the light-chain variable range and the heavy-chain variable range; the antibody and the heavy-chain variable range are shown in 1ST-119TH amino acid sequences from the N terminal in a sequence 1; the light-chain variable range is shown in 135th-241th amino acid sequences from the N terminal in a sequence 1; the connecting peptide is positioned between the heavy-chain variable range and the light-chain variable range, and the connecting peptide is shown in 120th-134th amino acid sequences from the N terminal in a sequence 1. The experiment result confirms that the prepared single-chain antibody has good cross reaction rate and high affinity to a plurality of phosphorothioate pesticides, is suitable for rapid immunodetection of a plurality of phosphorothioate pesticides, and has wide application prospect in the pesticide residual detection field.

Description

Technical field: [0001] The invention relates to a single-chain antibody and its application in detecting phosphorothioate pesticide residues. Background technique: [0002] Phosphorothioate pesticides belong to the category of organophosphorus pesticides, and their structure is characterized by phosphorothioate-based skeletons. Most of these compounds are in the form of oil or crystals, and the industrial products are light yellow to brown, and most of them have garlic odor. Generally insoluble in water, easily soluble in organic solvents such as benzene, acetone, ether, triazine and oils, relatively stable to light, heat and oxygen, easy to decompose and destroy when encountering alkali. [0003] Phosphorothioate pesticides have the characteristics of high efficiency against pests, broad control spectrum, various modes of action, various varieties, and large differences in toxicity between different varieties, and are widely used in the fields of agricultural production a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/63C12N1/15C12N1/19C12N1/21G01N33/53
Inventor 祁志军王东方刘爽寇立泉赵吉健吴文君
Owner PESTICIDE INST XIBEI AGRI & FORESTRY TECHUNIV