Genetic and fluorescence studies of affinity maturation in related antibodies

Affinity maturation, the process by which an organism's response to infection becomes more specific and more effective over time, occurs after somatic hypermutation of antibody genes in B-cells. This increase in affinity might be a result of the evolution of either specific interactions between antigen and antibody over time (enthalpic factors) or antibody binding site rigidification (entropic factors) or both. Here, monoclonal antibodies, derived from antibodies elicited at different points in the murine immune response after inoculation with the same diketone hapten, have been characterized both genetically and functionally. Though this hapten has previously been shown to produce the catalytic aldolase antibody 38C2, antibodies described here are not catalytic and unlike 38C2, form no covalent enzyme-substrate complex. Thus, they provide a system in which to assess contributions to the evolution of binding affinity. The genes for these non-catalytic antibodies have been sequenced and analyzed both with regard to their relationships to germ line genes, to each other, and to two commercially available catalytic aldolase antibodies. Consequences of particular mutations for antigen binding behavior are discussed. The protein products of these genes have been expressed, purified, and binding properties measured by two complementary techniques: the hapten-induced quenching of the native antibody fluorescence and the changes in the anisotropy of Prodan (6-propionyl-2-(dimethylamino)naphthalene), a fluorescent hapten analogue. Differences in binding, affinity are related back to differences in the lengths and amino acid sequences of the complementary determining region 3 (CDR3) binding loop. Taken together with our earlier results on binding site heterogeneity from tryptophan lifetime analysis [Mohan, G.S., Chiu, P.T., Southern, C.A., O'Hara, P.B., 2004. Steady-state and multifrequency phase fluorometry studies of binding site flexibility in related antibodies. J. Phys. Chem. A 108, 7871-7877], affinity appears to be modulated by a combination of entropic and enthalpic factors, and not dominated by one or the other. Because these antibodies are not related to the same germ line gene, however, these results do not provide evidence for the dominance of enthalpy or entropy in evolving binding affinity in this system. (C) 2005 Elsevier Ltd. All rights reserved.