Affinity Measurements of Antibody Fragments on Phage by Quartz Crystal Microbalance (QCM)

Abstract

U The screening of large combinatorial phage display gene libraries usually leads to a set of diverse recombinant antibody fragment clones specifically binding to the antigen which have to be characterised further and ranked for their usefulness. Beside specificity, the key parameter to be determined is the affinity to the antigen. The dissociation rate is of particular significance for diagnostic and therapeutic applications. For the affinity determination, the most convenient method would be one which directly utilises phage bound antibody fragments from the culture supernatants of isolated E. coli clones. However, affinity determinations of phage particles carrying recombinant antibodies are not easy with established methods like equilibrium ELISA or surface plasmon resonance due to a lack of sensitivity and unspecific effects caused by the large size and filamentous shape of the phage particles. QCM (Quartz Crystal Microbalance), however, provides a method to directly monitor association and dissociation of phage antibodies to their antigen, thus allowing to determine an apparent binding constant. The QCM is an acoustic sensor based on a piezoelectric crystal which can be used for the measurement of specific interactions between immobilized molecules and analytes in solution. Binding of a soluble analyte causes a frequency shift in the resonance frequency wich can be recorded by a frequency counter with high resolution.