title: Mutational analysis of arsonate binding by a CRIA+ antibody. VH and VL junctional diversity are essential for binding activity.
abstract: To assess the impact of various heavy and light chain mutations on p-azophenylarsonate binding, murine antibodies have been produced in insect cells (SF9) utilizing a baculovirus expression system. When expressed in this system, an antibody composed of a canonical CRIA+ heavy and light chain can bind antigen and express idiotype indistinguishably from analogous hybridoma-derived antibodies. Antibodies comprised of either light chains mutant at the V-J junction or heavy chains mutant at the V-D junction were found to be incapable of binding arsonate. In addition, substitutions in the first and second complementarity determining regions of the heavy chain were shown to play a role in arsonate binding, most likely related to affinity maturation targeted at the carrier protein. These results confirm the obligatory role that junctional diversity plays in the generation of arsonate-specific antibodies, as well as extend our understanding of the role of other variable region amino acids in arsonate binding.
authors.affiliation: Department of Microbiology, University of Texas Southwestern Medical Center, Dallas 75235.
authors.forename: C A
authors.initials: CA
authors.lastname: Hasemann
authors.forename: J D
authors.initials: JD
authors.lastname: Capra
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D006031:Glycosylation; D006825:Hybridomas; D007074:Immunoglobulin G / Q000302:isolation & purification; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D007313:Insecta; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D014162:Transfection; D010132:p-Azobenzenearsonate / Q000378:metabolism*
chemical_list: D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D010132:p-Azobenzenearsonate