Antibody engineering for the analysis of affinity maturation of an anti-hapten response.
The influence of structural variation, previously observed in a panel of V186.2 VH/V lambda 1-expressing anti-NP antibodies from the secondary response, on the affinity of these antibodies was examined by site-specific mutagenesis and recombinant antibody construction. A tryptophan----leucine exchange at position 33 in the VH segment of all but one of the high-affinity antibodies is the most frequently observed somatic mutation and by itself leads to a 10-fold higher affinity; all other somatic exchanges are irrelevant for affinity selection. In the single case of a high-affinity antibody without this common exchange, high affinity is mediated by a combination of mutations (including a one-codon deletion) in VH and the particular D-JH rearrangement carried by this antibody. The data indicate that the pattern of somatic diversification through hypermutation is shaped by affinity selection, but that only a single point mutation is available in the VH and the VL gene of lambda 1 chain-bearing anti-NP antibodies which by itself leads to an increase of hapten-binding affinity. Based on the analysis of two secondary response antibodies from which somatic mutations in VH and VL have been eliminated, it is also concluded that the recruitment of B cell clones into the pathway of hypermutation involves a mechanism which is not based upon affinity differences towards the antigen.
D000595:Amino Acid Sequence; D001483:Base Sequence; D005803:Genes, Immunoglobulin; D014644:Genetic Variation; D005822:Genetic Vectors; D006241:Haptens; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000235:genetics* / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000235:genetics* / Q000276:immunology; D007146:Immunoglobulin lambda-Chains / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids
D006241:Haptens; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007146:Immunoglobulin lambda-Chains