Production of a paraquat-specific murine single chain Fv fragment.
Producing an effective antidote against poisoning by the herbicide paraquat (PQ) has proven to be an elusive goal. One approach that holds some promise is immunotherapy with antibody fragments. In this study we detail the production of a single chain Fv fragment (scFv) specific for paraquat by linking cloned heavy (Vh) and light chain (VL) variable region genes via the peptide spacer (Gly4-Ser)3. These genes were obtained from hybridoma cells secreting a PQ-specific murine monoclonal antibody. The scFv (28 kDa) was expressed at 4% of the total cell protein by the Escherichia coli vector, pPOW, but was associated with the membranes. After solubilization and reduction, the scFv was renatured by rapid dilution. Western blotting confirmed that the refolded scFv had similar structural properties to the parental mAb. The isoelectric point of the scFv (7.0) is equal to the value calculated from the deduced amino acid sequence. Surface plasmon resonance was used to demonstrate specific PQ binding by the refolded scFv(Ka=1.24x10(6)M(-1)) which is similar to that determined for the parent Fab fragment (4.6x10(6)(-1)).
Devlin
C M
CM
University of Queensland, Department of Medicine, Princess Alexandra Hospital, Brisbane, Australia.
Bowles
M R
MR
Gordon
R B
RB
Pond
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005784:Gene Amplification; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D006540:Herbicides / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D010269:Paraquat / Q000276:immunology* / Q000378:metabolism / Q000633:toxicity; D016133:Polymerase Chain Reaction; D011817:Rabbits
D018076:DNA, Complementary; D006540:Herbicides; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D010269:Paraquat