METHODOLOGY FOR SELECTION OF HUMAN-ANTIBODIES TO MEMBRANE-PROTEINS FROM A PHAGE-DISPLAY LIBRARY

We describe a simple antigen capture technique for the selection of a specific human antibody to p185(erbB-2) transmembrane glycoprotein, fr om a library of human Fab genes expressed on the surface of bacterioph age, Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. T he antigen-coated beads have then been used to select bacteriophage di splaying human Fab with affinity for p185(erbB-2) After 4 rounds of se lection, 65 phage clones were isolated which bound specifically to p18 5(erbB-2) in a capture assay. Nine of the clones which gave the strong est reaction in an ELISA were selected for further development and the Fab genes were subcloned into the expression vector pUC119his6mycXba and electroporated into E. coli TG1. Colonies were grown, induced and the supernatants tested for the presence of secreted human Fab, Supern atants from two of the 9 clones contained human Fab and one of these b ound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprec ipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. W e conclude that a membrane antigen captured by specific monoclonal ant ibody can be used successfully to select phage displaying human antibo dies specific for the antigen.