Structural characterization of idiotopes by using antibody variants generated by site-directed mutagenesis.
Four anti-idiotopic mAB, 107, MB, AI, and AD8, react with mouse hybridoma protein 36-65 specific for the hapten p-azophenylarsonate. The four antiidiotypic antibodies do not react with hybridoma protein 36-71, a somatically mutated variant of 36-65 whose H and L chain V region sequence differs at 19 amino acid positions. To determine which regions of 36-65 are important for the interaction with each of the four anti-idiotypic antibodies, variants of 36-65 containing one or more of the 36-71 substitutions were generated by oligonucleotide-directed mutagenesis of the rearranged 36-65 H chain V region gene, followed by expression of mutant proteins containing either the 36-65 or the 36-71 L chain in transfected hybridoma cells. Idiotypic characterization of the mutant proteins showed that reactivity correlates with the 36-65 H chain, but some contributions from the 36-65 L chain come into play. In the 36-65 H chain V region, idiotopes were mapped to the first and third complementarity-determining regions for anti-idiotypic antibodies 107, MB, and AI, and to all three complementarity-determining regions for anti-idiotypic antibody AD8. The binding of all four anti-idiotypic antibodies to hybridoma protein 36-65 was hapten inhibitable. However, a comparison between the effect of individual 36-71 substitutions on idiotope expression and their effect on Ag-binding affinity suggests that none of the four anti-idiotypic antibodies bodies mimics the structure of Ag.
Sharon
J
J
Department of Pathology, Hubert H. Humphrey Cancer Research Center, Boston University School of Medicine, MA 02118.
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004252:DNA Mutational Analysis; D006241:Haptens; D007074:Immunoglobulin G / Q000235:genetics / Q000648:ultrastructure; D007130:Immunoglobulin Idiotypes / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides
D000888:Antibodies, Anti-Idiotypic; D006241:Haptens; D007074:Immunoglobulin G; D007130:Immunoglobulin Idiotypes; D009841:Oligonucleotides