Restriction in IgM expression. IV. Affinity analysis of monoclonal anti-phosphorylcholine antibodies

The genetic basis of affinity restriction in IgM antibody has been further studied with monoclonal antibodies directed to the phenylphosphorylcholine moiety. Association constants for the binding of N-(2,4-dinitrophenyl)-p-aminophenylphosphorylcholine to IgM and IgG monoclonal antibodies (anti-PC) have been determined by the method of fluorescence quenching by using the dinitrophenyl group as a sensor of the bound state. On the assumption that the IgM antibodies expressed germ-line V(H) and V(L) genes, whereas the IgG expressed somatic variants of these genes, the differences in binding properties of the isotypes were correlated with their difference in potential diversity. In contrast to the previous findings with pbosphorylcholine as the complete ligand, the inclusion of the phenyl group in the ligand used for affinity determinations yielded results consistent with earlier comparisons of IgG and IgM antibodies in other systems. From these results emerged several significant conclusions. (1) The IgG group of antibodies expresses a maximum affinity not attained by the IgM group. (2) In five of six IgG antibodies the phenyl group enhances the binding constants by factors ranging from sixfold to 82-fold, whereas no more than a twofold increase is observed with IgM. (3) The IgM combining sites are probably complementary only to the phosphorylcholine portion of the determinant. (4) The IgG antibodies do not show an affinity significantly less than the maximum for IgM. It is inferred, therefore, that the IgG variants arise by an antigen-driven selection of B cell clones exhibiting receptors with equivalent or improved affinity for the ligand compared with the germ line-restricted affinity yielding antibodies of refined specificity. This process may be coupled to escape from suppression of germ-line idiotopes. Finally, attention is called to the possibility of a parallel germline restriction in the V(H) gene segments presumably expressed by T cell receptors.