Selection of Antibody Fragments by Means of the Filter-Sandwich Colony Screening Assay

While the selection of antibody fragments from large V-gene libraries via phage display and panning permits the efficient enrichment of pools with antigen-binding activity, the successful identification of individual clones with desired binding specificities constitutes another crucial step. The sandwich-filter colony screening assay has proven as a facile and rapid method in particular for the isolation of recombinant Fab fragments with new or improved antigen-binding activities among 103-105 different clones. In this assay, bacterial colonies harbouring plasmids for the periplasmic expression of antibody fragments are grown on a hydrophilic filter membrane on an agar plate. Upon induction, the antibody fragments are functionally secreted into the periplasm and become partially released from the bacterial colonies. After diffusion across the hydrophilic membrane, the recombinant proteins are finally immobilised on a second hydrophobic membrane that is placed underneath and coated with a capturing reagent. Thus, the antibody fragments become functionally immobilised and, subsequently, they can be probed for binding of a labelled antigen. Antigen-binding activity is directly visualised on this second membrane, for example by using an enzyme conjugate followed by a chromogenic reaction. Colonies that give rise to a strong signal are then identified on the first membrane and propagated for further analysis, preparative expression, and/or subcloning of the V-genes. Apart from Fab fragments, this assay can be applied to many other types of bacterially produced antibody fragments as well as to non-immunoglobulin scaffolds with engineered binding properties.