title: Effect of transplantation of antibody heavy chain complementarity determining regions on ligand binding.
abstract: Active site structure-function analyses of anti-fluorescein single chain antibody 4-4-20 and anti-single-stranded DNA single chain antibody 04-01 were conducted studying the ligand binding properties of hybrid antibodies resulting from systematic transplantation of heavy chain complementarity regions (HCDRs) from monoclonal antibody 4-4-20 into 04-01. Two prototype monoclonal antibodies were chosen because the primary structures of their respective light chains were nearly identical but the specificities and shape of their active sites were distinctly different. Based on nearly identical light chains, the diverse active site conformations (i.e. 4-4-20 pocket and 04-01 cleft) and subsequent specificities of the two antibodies were likely dictated by heavy chain properties. As a result, specificities of each HCDR transplant were analyzed in terms of binding reactivity with either fluorescein or (dT)8. Results of binding studies, together with idiotypic and secondary structure analyses, were used to determine the relative contribution of each HCDR to the active site conformations and ligand specificities of monoclonal antibodies 4-4-20 and 04-01. Collectively the various analyses led to the conclusion that the intradomain conformational dynamics and cooperativity necessary for the structural integrity of the low affinity cleft-shaped 04-01 anti-single-stranded DNA active site are probably less stringent than those of a high affinity pocket-shaped anti-fluorescein active site.
authors.affiliation: Department of Microbiology, University of Illinois at Urbana-Champaign 61801.
authors.forename: G A
authors.initials: GA
authors.lastname: Gulliver
authors.forename: E W
authors.initials: EW
authors.lastname: Voss
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D001665:Binding Sites; D002942:Circular Dichroism; D004277:DNA, Single-Stranded / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005454:Fluorescence Polarization; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology*; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data
chemical_list: D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D004277:DNA, Single-Stranded; D000939:Epitopes; D005452:Fluoresceins; D007143:Immunoglobulin Heavy Chains; D008024:Ligands; D016650:Fluorescein-5-isothiocyanate