An analysis of idiotype expression in a high-affinity, somatically mutated variant of a germline-encoded anti-p-azobenzenearsonate antibody.
Using two polyclonal (rabbit) and two monoclonal anti-idiotype (anti-Id) reagents, we investigated structural correlates of the Id of mAb 36-71, a somatically mutated member of the CRIA Id family that has an exceptionally high affinity for the p-azobenzenearsonate (Ars) hapten. The two monoclonal anti-Ids reacted principally with the L chain of 36-71. The polyclonal anti-Ids interacted with both the H and L chain. The amino acid sequences of the VH and VL regions of 36-71 differ in eight and 11 positions respectively from those of the anti-Ars mAb 36-65, an unmutated prototype of the CRIA family. In the presence of 36-71L only three substitutions in 36-65 VH, introduced by mutagenesis, sufficed to restore full expression of the 36-71 Id. The same three substitutions had previously been shown to increase the affinity of 36-65 by a factor of 200, to a level equivalent to that of 36-71. X-ray crystallography had indicated that two of these substitutions introduce conformational changes consistent with the increase in affinity. We propose that these conformational changes may also account for the critical role of the three amino acids in Id expression. We also found that 36-65 is a very poor inhibitor of the interaction of 36-71 with its polyclonal anti-Ids, despite identity of the hapten-contacting residues in the two mAbs and evidence (from hapten inhibition) that the hapten-binding region is part of an important Id. Again, a difference in conformation at the binding site of the two mAbs could account for these observations.
Nisonoff
A
A
Department of Biology, Brandeis University, Waltham, MA 02254.
Oliveira
T M
TM
Sharon
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology; D007130:Immunoglobulin Idiotypes / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D051379:Mice; D008805:Mice, Inbred A; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D010132:p-Azobenzenearsonate / Q000276:immunology*
D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D010132:p-Azobenzenearsonate