Determination of phage antibody affinities to antigen by a microbalance sensor system

Over the past decade, phage display has maturated to be a frequently used,m ethod for the generation of monoclonal antibodies of human origin. The esse ntial step of this method is the "biopanning" of phage carrying functional antibody fragments on their surface on an immobilized antigen. The screenin g of large combinatorial gene libraries with this method usually leads to a set of diverse clones specifically binding to the antigen that need to be characterized further. Beside its specificity, the key parameter to be dete rmined is the affinity of the recombinant antibody fragment to its antigen. Here, we present a mass sensitive microsensor method that allows the estim ation of antibody affinity directly from the phage supernatant. Binding of phage antibodies to the antigen immobilized on a quartz crystal microbalanc e (QCM) induced a mass dependent decrease in frequency. This principle was used to determine the apparent affinity of a single-chain (sc)Fv antibody a gainst the RNA polymerase of Drosophila melanogaster presented on the surfa ce of a filamentous phage (M13) from its association and dissociation rates . The apparent affinity obtained is in accordance with the affinity of the scFv fragment as determined by conventional equilibrium enzyme-linked immun osorbent assay (ELISA) and plasmon resonance methods.