Panning of a phage VH library using nitrocellulose membranes: Application to selection of a human VH library

We have established a method for selecting binding phages from a phage immu noglobulin heavy chain variable region (VH) Library by panning with nitroce llulose membranes (membrane panning). To evaluate the concentrating ability of membrane panning for binding phages, a phage VH library containing clon es that bind to hen egg white lysozyme (HEL) was used for panning against H EL. The efficiency of our method was as high as that of panning with magnet ic beads. In addition, we performed membrane panning against target protein s and isolated the binding phages. The human VH genes of these phages were cloned and expressed as VH-bacterial alkaline phosphatase (PhoA) conjugates (VH-PhoA) in Escherichia coli. The dose-dependent binding of VH-PhoA to ta rget proteins was confirmed by dot blotting. When applied to disease-associ ated antibodies, these methods will likely benefit clinical research. In ad dition, these techniques may be applicable to systematic analysis in proteo me studies.