title: Cloning, expression, and identification of anti-carbofuran single chain Fv gene.
abstract: Phage display method was used to clone anti-carbofuran (CBF) single chain Fv (scFv) gene. The heavy chain and light chain variable region genes were amplified by the polymerase chain reaction from the CBF-specific hybridoma cell lines 5D3 and assembled as a scFv DNA fragment with linker peptide (Gly(4)Ser)(3). The scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBF antibody libraries were then constructed. After one round of panning with CBF-ovalbumin (CBF-OVA) as a conjugate, antigen-binding positive recombinant phage clones were successfully selected by enzyme-linked immunosorbent assay (ELISA). The positive phages were used to infect Escherichia coli HB2151 cells and the expression of the soluble scFv antibodies was then induced by IPTG. The scFv antibody was about 31 kDa by SDS-PAGE and showed HRP-anti-E-tag antibody-recognized activity by Western blotting. The indirect competitive ELISA (icELISA) showed that the recombinant scFv antibody could competitively combine with CBF, with the IC(50) value of 1.07 ng/mL. The cross reactivity studies showed that the anti-CBF scFv antibody, similar to the parent monoclonal antibody, poses high specificity to CBF and has little reactivity to the analogs. Taken together, these findings suggest that the recombinant scFv antibody can be used for further developing immunoassay method for CBF.
authors: Key Laboratory of Food Quality and Safety of Guangdong Province, College of Food Science, South China Agricultural University, Guangzhou 510642, PR China.
authors: Hong
authors: H
authors: Wang
authors: Jinyi
authors: J
authors: Yang
authors: Xixia
authors: X
authors: Liu
authors: Yan
authors: Y
authors: Liang
authors: Hongtao
authors: H
authors: Lei
authors: Yudong
authors: Y
authors: Shen
authors: Xiaoyan
authors: X
authors: Xu
authors: Yuanming
authors: Y
authors: Sun
authors: Zhenlin
authors: Z
authors: Xu
authors: Yongsheng
authors: Y
authors: He
mesh_terms: D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D000918:Antibody Specificity; D002235:Carbofuran / Q000032:analysis* / Q000276:immunology; D003001:Cloning, Molecular; D015870:Gene Expression; D007118:Immunoassay; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; D002235:Carbofuran