Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single mo lecule level will provide an improved tool for generating proteins with com plex and distinct properties from large molecular libraries. To establish s uch an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescen tly labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes f or detection of the phage-antigen complex, either with labeled antiphage an tibody or using a labeled antigen. As a model system, we studied a human mo noclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and it s cognate antigen (rE2 or rE1/E2). We could thus assess the specific intera ctions and determine the fraction of specific versus background phage (26% specific phage), Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highl y specific detection system.