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 p185erbB-2, a transmembrane glycoprotein, from a library of human Fab genes expressed on the surface of bacteriophage. Magnetic beads coated with the rat antibody ICR55 have been used to capture erbB-2 antigen from Triton X-100 extracts of SKOV3 cells. The antigen-coated beads have then been used to select bacteriophage displaying human Fab with affinity for p185erbB-2. After 4 rounds of selection, 65 phage clones were isolated which bound specifically to p185erbB-2 in a capture assay. Nine of the clones which gave the strongest 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. Supernatants from two of the 9 clones contained human Fab and one of these bound specifically to erbB-2 in a capture assay, stained the membranes of the erbB-2 overexpressing cell lines BT474 and SKBR3 and immunoprecipitated a protein of molecular weight 185 000 kDa from SKOV3 cells. We conclude that a membrane antigen captured by specific monoclonal antibody can be used successfully to select phage displaying human antibodies specific for the antigen.
Sawyer
C
C
Section of Immunology, McElwain Laboratories, Institute of Cancer Research, Belmont, Sutton, Surrey, UK.
Embleton
J
J
Dean
C
C
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001435:Bacteriophages / Q000235:genetics*; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D015723:Gene Library; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D008565:Membrane Proteins / Q000276:immunology*; D051381:Rats; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D008565:Membrane Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins