Cloning, expression and characterization of a single-chain antibody fragment to the herbicide paraquat.
New cost effective methods for the detection and removal of pesticides from water samples are required to meet modern safety standards. The recent development of techniques to produce antibody fragments in bacteria has provided the opportunity to exploit antibodies as specialized chemicals for affinity detection/removal technologies. The variable heavy and light polypeptide chains of the anti-paraquat monoclonal antibody PQXB1/2 have been cloned into the single-chain antibody (ScAb) expression vector pBG1. The construct was expressed in Escherichia coli and 0.4 mg functional antibody produced from 1 dm3 of induced culture. Characterization of ScAb by antigen binding profile and competition ELISA showed it to have a sensitivity one order of magnitude below that of the parent monoclonal. ScAb was purified as a monomer or dimer and analysed by HPLC size exclusion chromatography. When immobilized on polystyrene beads the ScAb could remove 85% of a paraquat-bovine serum albumin conjugate from solution in a single step.
Graham
B M
BM
Department of Molecular and Cell Biology, University of Aberdeen, Marischal College, UK.
Porter
A J
AJ
Harris
W J
WJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000032:analysis; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D007074:Immunoglobulin G / Q000032:analysis; D008969:Molecular Sequence Data; D010269:Paraquat / Q000276:immunology*; D011137:Polystyrenes
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D007074:Immunoglobulin G; D011137:Polystyrenes; D010269:Paraquat