Characterization of four nucleic acid-binding single-chain Fv fragments by direct and competitive solid-phase radioimmunoassays.
The heavy- and light-chain variable region genes of four different nucleic acid-binding antibodies (Jel 274 and Jel 72 (specific for duplex DNA), Jel 103 (specific for poly(rI)), and Jel 318 (specific for DNA triplexes)) were cloned. Single-chain Fv fragments (scFv) in which the heavy and light chains were joined by a linker were constructed by polymerase chain reaction. The linker of 21 amino acids also served as a tag since it consisted of a repeating heptapeptide that was recognized by a specific antipeptide antibody. scFv were expressed in the cytoplasm of Escherichia coli as inclusion bodies. After purification and renaturation, a cross-linking assay was used to demonstrate that > 90% of scFv were in the form of monomers. The specificity of scFv was analyzed by both direct and competitive solid-phase radioimmunoassays. scFv.103 retained its specificity for poly(rI), whereas the other three scFv still bound the original antigen, but subtle changes in the overall specificity were noted. Thus, in some cases, the conformation of the binding site may be different in the context of an scFv compared with the original IgG.
Kuderova
A
A
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Tanha
J
J
Lee
J S
JS
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011863:Radioimmunoassay
D007128:Immunoglobulin Fragments; D009838:Oligodeoxyribonucleotides; C055966:immunoglobulin Fv; D004247:DNA