Production and characterization of single chain Fv directed against beta2-agonist clenbuterol.
The single chain Fv (scFv) directed against beta2-agonist clenbuterol (CBL) was produced by using phage display technology. The heavy chain and light chain variable region genes (VH) VL) were amplified by the polymerase chain reaction (PCR) from CBL specific hybridoma cell lines 5D1 and assembled as a single chain Fv (scFv) fragment with linker peptide (Gly4Ser)3. Then the scFv DNA fragment was cloned into M13 phagemid vector pCANTAB5E and the anti-CBL antibody libraries were constructed. Phages displaying scFv were enriched by panning with CBL-ovalbumin (CBL-OVA) conjugate. After only one round of panning, antigen-positive recombinant phage clones were successfully selected by ELISA. The positive phage was used to infect Escherichia coli HB2151, and the expression of soluble scFv was then induced by IPTG. The scFv showed an improved sensitivity (with IC50 of 0.78 +/- 0.005 ng/mL (n = 4)) when compared with the parent monoclonal antibody (MAb) (with IC50 of 1.34 +/- 0.006 ng/mL (n = 4)) in competitive indirect ELISA (CI-ELISA). Cross-reactivity studies showed that the specificity of scFv was similar to that of MAb. The recombinant scFv prepared in this study could be potentially used instead of conventional antisera or MAb for development of a rapid and affordable immunoassay for the detection of residual CBL in biological matrices.
Pan
Ke
K
Food Quality and Safety Research Institute, South China Agriculture University, Guangzhou, Guangdong Province, 510642, P. R. China.
Wang
Hong
H
Zhang
Hong-Bin
HB
Liu
Hong-Wei
HW
Lei
Hong-Tao
HT
Huang
Li
L
Sun
Yuan-Ming
YM
D000318:Adrenergic beta-Agonists / Q000276:immunology*; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002976:Clenbuterol / Q000276:immunology*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region; D010047:Ovalbumin / Q000276:immunology; D019151:Peptide Library; D016133:Polymerase Chain Reaction; D012995:Solubility; D014162:Transfection
D000318:Adrenergic beta-Agonists; D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; C055966:immunoglobulin Fv; D010047:Ovalbumin; D002976:Clenbuterol