Screening and characterization of human phage antibody to permethrin
10.3760/cma.j.issn.1008-1372.2010.11.001
- VernacularTitle:抗氯菊酯人源scFv抗体筛选与鉴定
- Author:
Weina MA
;
Xuelin LIU
;
Hongbin SONG
;
Jianliang SHEN
;
Youzhang HUANG
;
Lizhong GONG
;
Dan XIANG
;
Limei ZANG
- Publication Type:Journal Article
- Keywords:
Pyrethrins/IP;
Antibodies/IP
- From:
Journal of Chinese Physician
2010;12(11):1441-1444
- CountryChina
- Language:Chinese
-
Abstract:
Objective To screen permethrin human single-chain variable region (scFv) antibody for aims of developing rapid detection kit. Methods Phage display technology was used in this study. Permethrin was solid phase coated on Nunc plate as antigen. Semi-synthetic single-chain variable region of human antibody library technology was applied, and single chain variable region was screened from phage antibody library after 3 rounds "adsorption - elution - amplification" of the selection process. 100 clones were random selected as resistance to permethrin clones , enzyme-linked immunosorbent assay (ELISA), crossreactivity and competitive inhibition experiments were used to validate permethrin binding activity with strong scFv clones from the selected phage antibody clones plasmid. The plasmid was digested with restriction enzyme Sfi Ⅰ / Not Ⅰ and subcloned into pCANTAB5E vector. After transformed into E. coli XL1BIue, the plasmid was identified by restriction enzyme analysis. Results After screening in 100 clones, 18 clones had high ELISA absorbance values ( A value) at 490nm wavelength ( A490nm), then bovine serum albumin (BSA) cross-reactions identified five weak cross-reaction. Combined with the triplicate ELISA and competitive inhibition experiment results, one positive clone was acquired at last. And this clone was subcloned into pCANTAB5E vector and transformed into competent cells XL1-Blue. Conclusion Plasmid fragment was consistent with the purpose, which provided the foundation for further study of its specific affinity.
