STRUCTURE, FUNCTION, AND DIVERSITY OF MONOCLONAL ANTI-FLUORESCYL ANTIBODIES

The structural basis of antibody affinity and the mechanism by which IgG antibodies undergo increases in affinity with time (maturation) during the immune response were examined using a fluorescein hapten system in conjunction with hybridoma methodology. Monoclonal anti-fluorescyl IgG antibodies (nine clones) were produced by fusion of BALB/c splenocytes and Sp 2/O-Ag 14 myeloma cells. Analyses of these clones were performed in order to compare structure/function relationships and to determine their prevalance in heterogeneous BALB/c immune sera.;Monoclonal anti-fluorescyl antibodies exhibited extensive structural and functional (i.e. K(,A) values ranging from 10('6) to 10('10)M('-1)) diversity. Heavy and light chain recombination studies indicated that such diversity existed in the variable region of both chains (IgG, kappa). Moreover, the absolute requirement and specificity of homologous H and L chains (for idiotype expression and binding site formation) set limits to the degree of diversity produced through combinatorial H and L chain association. In addition, data implied it was unlikely that increases in affinity with time (maturation) were the result of association of different H and L chain regions (V, D, or J). However, binding studies of these hybridoma proteins suggested that interaction of one or several key binding site residues with the ligand could contribute significantly to the binding free energy (affinity). Analyses of heterogeneous antibodies using variable region probes (anti-idiotype reagents), indirectly demonstrated that the structural correlate of such affinity increases was more likely somatic mutational events than selective expression of pre-existing variable regions (clones). Hence, the recently observed hypermutation of IgG proteins may serve as a structural basis for the functional process of affinity maturation.