title: Production, purification and characterisation of genetically derived scFv and bifunctional antibody fragments capable of detecting illicit drug residues.
abstract: We have generated a single chain antigen binding protein (scFv) recognising morphine. Variable regions of heavy (V(H)) and light (V(L)) chain antibody genes isolated from a murine immune repertoire were connected via a glycine-serine linker and cloned into the expression vector pAK 400. The scFv was produced in Escherichia coli JM83 yielding a functional protein of approximately M(r) 30000. Immunoaffinity chromatography using M3G-BSA-Sepharose column proved most effective for scFv purification. Purity was monitored by SDS-PAGE and Western blotting and the scFv characterised using ELISA and BIAcore. The scFv was capable of specifically binding free morphine in solution and was applicable to real sample analysis in saliva. In order to express a bivalent "minibody" the scFv gene was recloned into a vector containing a gene encoding a helix for dimerisation. The scFv was expressed as a protein of M(r) 75000 and retained its antibody binding capabilities. Cloning the scFv gene into a vector containing the bacterial alkaline phosphatase gene produced a bifunctional molecule, which retained the binding activity of the parental scFv along with the enzymatic activity of alkaline phosphatase.
authors.affiliation: Applied Biochemistry Group, School of Biotechnology, Dublin City University, Glasnevin, 9, Co. Dublin, Dublin, Ireland. joanne.brennan4@mail.dcu.ie
authors.forename: Joanne
authors.initials: J
authors.lastname: Brennan
authors.forename: Paul
authors.initials: P
authors.lastname: Dillon
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
mesh_terms: D015153:Blotting, Western; D002846:Chromatography, Affinity; D004350:Drug Residues / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D013287:Illicit Drugs / Q000032:analysis*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids
chemical_list: D013287:Illicit Drugs; D007128:Immunoglobulin Fragments