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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1990 Jun;87(12):4814–4817. doi: 10.1073/pnas.87.12.4814

Structural correlates of high antibody affinity: three engineered amino acid substitutions can increase the affinity of an anti-p-azophenylarsonate antibody 200-fold.

J Sharon 1
PMCID: PMC54208  PMID: 2352950

Abstract

The basis for the 200-fold difference in affinity between two hybridoma antibodies specific for the hapten p-azophenylarsonate (Ars) that have diversified by somatic hypermutation was examined. Oligonucleotide-directed mutagenesis was used to sequentially convert the nucleotide sequence of the lower-affinity antibody into that of the higher-affinity one, and the mutant antibodies generated by transfection of hybridoma cells were analyzed for affinity to Ars-tyrosine. The data showed that out of the 19 amino acid differences between the two hybridoma antibodies, the affinity increase could be reproduced by three heavy-chain substitutions that are present in the high-affinity antibody. The combined effect on affinity of amino acid substitutions was generally found to reflect their individual effects. Although the light chain of the high-affinity antibody did not seem to play a major role in the affinity increase, its contribution varied with the kind and number of heavy-chain substitutions. The results hold promise for antibody engineering and are consistent with a stepwise acquisition of somatic hypermutations in which the existing structural context of an antibody most likely influences the affinity-based selection of later substitutions. They further suggest that many substitutions may be tolerated in vivo during the antigen-driven selection process, even though they confer on the antibody no affinity increase.

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Selected References

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