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Screening method for paraquat simulation antibody and application thereof

A technology of mimicking antibodies and paraquat, which is applied in the field of screening of paraquat mimicking antibodies, can solve the problems of cumbersome determination process, high cost, difficulty in antigen synthesis, etc., and achieve the effect of small molecular weight and clear sequence

Inactive Publication Date: 2015-04-15
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Gas chromatography, high performance liquid chromatography and other instrumental analysis methods sample pretreatment and measurement process are cumbersome and expensive, and are not suitable for screening a large number of samples
Although the immunoassay method is fast, accurate and simple, it is limited by its difficulty in antigen synthesis and the need for animal experiments.

Method used

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  • Screening method for paraquat simulation antibody and application thereof
  • Screening method for paraquat simulation antibody and application thereof
  • Screening method for paraquat simulation antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Screening of Paraquat Mimetic Antibody

[0025] 1. In vitro transcription and translation

[0026] (1) E. coli S30 (Escherichia coli S30Extract System) linear template system from Promega Company was used for in vitro transcription and translation. The reaction system is as follows:

[0027]

[0028] (2) Incubate the system at 37°C for 10 min to complete in vitro transcription and translation. The in vitro translation products were immediately added to 4 volumes of WBTH (pre-cooled) containing 0.5% BSA. Then add 2.6μL Mg(Ac)2 solution and place on ice for 1-2min.

[0029] 2. Solid phase affinity screening

[0030] (1) Coating screening wells: Dilute the PQ-BSA coating to 2 μg / mL with sterilized carbonate buffer, add 100 μL to each well of the microplate (treated with DEPC water), and cover The lids treated with DEPC water were coated overnight at 4°C.

[0031] (2) The next day, pour off the coating solution, and wash the closed well with sterilized PB...

Embodiment 2

[0046] Example 2 Functional expression and purification of paraquat mimetic antibody

[0047] 1. Functional expression of paraquat mimic antibody

[0048] 1. Amplification of Lipocalin-PQ gene after screening

[0049] Amplify the screened gene with primers containing EcoRI and Xhol restriction sites redesigned to amplify the Lipocalin-PQ gene with restriction sites. The primer sequence is as follows: Forward primer EXPRESS (introducing EcoRI restriction site dot): 5′CGC GAATT C ATG AAC GTG TAC CAC GAC GGTG3', reverse primer REPRESS (introduce XhoI restriction site): 5'GCC CTC GAG ATT GTT GAC TTT GCA GGC GGCG3'.

[0050] 2. Construction of recombinant Lipocalin-PQ expression plasmid:

[0051] Plasmid pTIG-TRX was digested with EcoRI and XhoI. 1.5% agarose gel electrophoresis, the Lipocalin-PQ fragment purified by EcoRI and XhoI double digestion and the plasmid pTIG-TRX digested by the same digestion were ligated with T4 DNA ligase, and the host strain BL21(DE3) was tran...

Embodiment 3

[0056] Example 3 Functional Identification of Paraquat Mimetic Antibody

[0057] 1. Detection of the binding activity of the mock antibody Lipocalin-PQ

[0058] PQ-BSA coated enzyme-linked wells, and coated with the same concentration of BSA at the same time, diluted the purified mock antibody Lipocalin-PQ four gradients as the primary antibody solution, mouse anti-6×His tag antibody as the secondary antibody, HRP-labeled sheep Anti-mouse was used as the enzyme-labeled secondary antibody, and the binding activity was detected by direct ELISA method. After color development by TMB, the microplate reader measured the absorbance at 450nm and 630nm, and the difference between the two was the absorbance after the reaction. In order to exclude the non-specific adsorption of the simulated antibody to BSA, the value of PQ-BSA minus the value of BSA was used as the absorbance value of the simulated antibody combined with PQ, and the results were as follows image 3 , the binding of Li...

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Abstract

Belonging to the field of biotechnologies, the invention relates to a screening method for a paraquat simulation antibody and application thereof. The simulation antibody is screened out from a ribosome display Lipocalin simulation antibody library and is subjected to soluble functional expression. The invention also relates to a method of screening the paraquat simulation antibody by a ribosome display technology. The paraquat simulation antibody screened out in the invention can be used for paraquat residual monitoring and development of a kit for rapid detection of pesticide paraquat residual.

Description

technical field [0001] The invention relates to a screening method for a paraquat mimic antibody, which belongs to the field of biotechnology and is used for preparing a highly efficient mimic antibody for specific recognition of paraquat and developing an immune rapid test kit for rapidly detecting paraquat residues. Background technique [0002] Paraquat (paraquat, PQ) is also known as paraquat, gram cloxone, praquat, gram methazone, and its chemical name is 1,1′-dimethyl-4,4′-bipyridine cation; 1,1 '-Dimethyl-4,4'-bipyridine cationic dichloride is a contact type and herbicide. Because of its quick-acting, broad-spectrum, and safety characteristics, it is widely used in garden weeding and non-arable land chemical weeding. It has been promoted and applied in more than 130 countries and regions in the world, especially in North and South America and Asia. my country is the largest producer of paraquat. One of the using and producing countries. Paraquat has a strong toxic ef...

Claims

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Application Information

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IPC IPC(8): C07K16/44G01N33/53
Inventor 宁保安高志贤彭媛白家磊赵丽孙思明
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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