Probing the Changes in Antibody Flexibility During Affinity-Maturation Using Single Molecule Spectroscopy

Abstract

Affinity maturation of an immune response is characterized by the expression of antibody proteins with increasing specificity and selectivity over the course of an organism's response to a pathogen. Immature germ line antibodies contain sites that can bind to a host of related small molecules or small regions of larger molecules with low affinity. Over time, new antibodies with increasing affinity and selectivity are expressed. My experiments will test the hypothesis that affinity maturation is a direct result of the loss of flexibility characteristic of the immature germ line antibody binding site. Binding sites from more mature antibodies are pre-conformed to the small molecule epitopes on the pathogen and therefore not flexible, thus reducing the entropic cost of binding, leading to higher affinity.Both intact and Fab fragments of antibodies at various stages of the immune response will be analyzed using fluorescence lifetime imaging microscopy (FLIM) at the single molecule level. This new technique is made possible through the construction of a single molecule fluorescence microscope. Lifetimes and lifetime distributions of donor and acceptor fluorescent labeling dyes tagged to the antibody will be measured for antibodies raised against two classes of small molecule haptens: diketone haptens and PLP-lysine derived haptens. Monoclonal antibodies producing cells have been derived from various points in the affinity maturation process. Lifetime images will be collected with and without hapten.