EVOLUTION OF MOLECULAR DIVERSITY &EPITOPE RECOGNITION

Immune maturation is viewed as a Darwinian process that involves
continuous selection of the structure (s) of best fit. The long
term objective of this study is to understand the molecular basis
of antigen recognition and its contribution as a selective force in
shaping the antibody response to phosphocholine-protein (PC-
KLH) conjugates. The evolution of antibodies (in particular Group
II) recognizing different molecular aspects of the PCcarrier
complex will be studied using a combination of molecular genetic
and physical measurements of serum antibodies, hybridomas and
monoclonal antibodies obtained at various times during the
immune response to PC-KLH. The specific aims are 1) Determine
the molecular progression of the antibody response to PC-KLH via
hybridoma analysis. The emergence of Group II antibodies will be
determined and analyzed for isotype and/or clonotype restriction.
The role of somatic mutation will be determined and specific gene
usage will be assessed. 2) Determine the vulnerability of the T15+
clonotype to antigen-binding loss mutations. 3) Analyze the
evolution of epitope recognition with regard to fine specificity,
affinity, and physical interaction between structural features of
phenyl-PC and the active site. 4) Analyze the relative
contributions of PC-phenyl-tyrosine and PC-phenyl-histidine to
evolution of the Group II response. The methods to be used
include mRNA sequencing, in situ hybridoma lysis and mRNA
hybridization to obtain gene usage information. Affinity of
monoclonal antibodies will be measured by fluorescence
spectroscopy. Antibody fine specificity will be assessed using a
panel of hapten analogs. Physical interactions between antibody
combining sites and structural components of the hapten will be
assessed by nuclear magnetic resonance spectroscopy.