Antibody engineering for the analysis of affinity maturation of an anti-hapten response

EMBO J. 1988 Jul;7(7):1995-2001.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Genes, Immunoglobulin*
  • Genetic Variation
  • Genetic Vectors
  • Haptens
  • Humans
  • Hybridomas / immunology
  • Immunoglobulin Heavy Chains / genetics*
  • Immunoglobulin Heavy Chains / immunology
  • Immunoglobulin Variable Region / genetics*
  • Immunoglobulin Variable Region / immunology
  • Immunoglobulin lambda-Chains / genetics*
  • Immunoglobulin lambda-Chains / immunology
  • Molecular Sequence Data
  • Mutation
  • Plasmids

Substances

  • Haptens
  • Immunoglobulin Heavy Chains
  • Immunoglobulin Variable Region
  • Immunoglobulin lambda-Chains