Definition of Monoclonal Antibody-Fluorescent Ligand Interactions
Due to the characteristic complex cellular and genetic constituents of the immune system, humoral antibody responses exhibit heterogeneity and diversity at various levels. First, immune responses induced by complex macromolecules, containing many structurally different epitopes, result in a mixture of antibodies possessing different specificities and affinities. Second, antibody responses to relatively simple and chemically defined epitopes (haptens) are usually diverse as characterized in terms of the observed range in affinities. The magnitude of affinity measured depends on the biochemical nature and immunogenic properties of the epitope and carrier. Finally, a third level of heterogeneity results from the multiple immunoglobulin classes (or subclasses) resulting from the recombination of different constant (C) genes, which dictate isotypy, with the same variable (V) genes. This report will focus on an experimental approach to understand the structure-function basis of antibodies differing in affinities elicited to the fluorescent probe, fluorescein. The experimental premise to employ a fluorescent epitope is attractive due to: 1) the sensitivity of fluoro-metric measurements; 2) the fact that when fluorescence is an intrinsic property of the ligand, it provides a means by which behavior of the hapten can be studied in complex mixtures; 3) the capacity of fluorescence to reflect changes in the micro-environment; thus, monitoring intensity can provide information about the types of bonds involved in the bimolecular complex as well as the native state of the active site; 4) the potential use of fluorescence polarization measurements to indicate molecular size facilitating measurements of ligand-protein interactions; and 5) the availability of fluorescence quenching and solvent perturbation studies to provide useful functional information which can be correlated with resolution of the physical-chemical structure of the antibody active site.
KeywordsFluorescence Quenching Conformational State Fluorescence Enhancement Deuterium Oxide Affinity Maturation
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