Our hypothesis is that the optimum antibody phenotype selected during immune maturation is one in which both the forward and reverse rate constants for antibody-antigen interaction approach specific limiting values. The key to testing this hypothesis is to probe the immune system with an antigen whose physical properties allow accurate determination of kinetic and affinity constants for binding monoclonal antibodies. Trends in these constants over the course of an immune response will show what biophysical properties in antigen receptors are refined during immune maturation. We developed such an antigen, chicken fatty acid binding protein (FABP) coupled through its single cys residue to the hapten 2-phenyl-5-oxazolone (Ox). FABP is an ideal protein carrier because it is monomeric and lacks trp residues; hence the Ox-FABP conjugate has the same advantageous spectroscopic properties as Ox itself. Ox-FABP will be used to generate mouse hybridomas from the primary, hyperimmune, and memory stages of the immune response. To identify the germline genes used and the key somatic mutations of residues involved in antigen interaction, the immunoglobulin variable region genes of hapten-specific hybridomas will be cloned using the polymerase chain reaction, and their DNA sequences determined. Forward and reverse rate constants for reaction of purified antibodies with Ox-FABP will be determined from stopped-flow fluorescence quench measurements. Affinities will be determined from static fluorescence quench measurements of antibody solutions titrated with hapten-FABP. We have isolated our first Ox-FABP-specific hybridomas already and have sequenced their antibody genes. We find that the response is dominated by a canonical heavy and light chain. Our final Specific Aim is to assess whether the pattern of somatic mutation in the canonical anti-Ox-FABP genes, which is an indicator of the """"""""quality"""""""" of an immune response, can be used as an assay for immunomodulators.
