DIRECT MEASUREMENT VIA PHAGE TITER OF THE DISSOCIATION-CONSTANTS IN SOLUTION OF FUSION PHAGE-SUBSTRATE COMPLEXES

Studies of interactions between filamentous fusion phage particles and protein or nucleic acid molecules have gained increasing importance w ith recent successes of screening techniques based upon random phage d isplay libraries (biopanning). Since a number of different phage are u sually obtained by biopanning, it is useful to compare quantitatively the binding affinities of individual phage for the substrate used for selection. A procedure is described for determination of relative diss ociation constants (K-d(Rel)) between filamentous phage carrying pepti de fusions to the coat protein gpIII and substrates in solution. This novel method is based on the measurement of phage titres. Phage select ed from a random fusion phage library for binding to a monoclonal anti body or a viral structural protein exhibited K-d(Rel) values in the na nomolar and micromolar ranges for their respective substrates, thus va lidating the method over a wide range of binding affinities.