PRODUCTION OF A PARAQUAT-SPECIFIC MURINE SINGLE-CHAIN FV FRAGMENT

Producing an effective antidote against poisoning by the herbicide par aquat (PQ) has proven to be an elusive goal, One approach that holds s ome promise is immunotherapy with antibody fragments, in this study we detail the production of a single chain Fv fragment (scFv) specific f or paraquat by linking cloned heavy (V-H) and light chain (V-L) variab le region genes via the peptide spacer (Gly(4)-Ser)(3). These genes we re obtained from hybridoma cells secreting a PQ-specific murine monocl onal antibody, The scFv (28 kDa) was expressed at 4% of the total cell protein by the Escherichia coil vector, pPOW, but was associated with the membranes, After solubilisation and reduction, the scFv was renat ured by rapid dilution, Western blotting confirmed that the refolded s cFv had similar structural properties to the parental mAb, The isoelec tric point of the scFv (7.0) is equal to the value calculated fi om th e deduced amino acid sequence, Surface plasmon resonance was used to d emonstrate specific PQ binding by the refolded scFv (K-a = 1.24 X 10(6 ) M(-1)) which is similar to that determined for the parent Fab fragme nt (4.6 X 10(6) M(-1)).