An alternating selection strategy for cloning phage display antibodies.
Phage display is a powerful technique that can be used to develop antibodies to target molecules. One approach for antibody phage display is to select phage from a large naive library of antibody immunoglobulin variable region fragments (Fv) expressed on the surface of phage. Phage that display antibody fragments of interest are selected by their ability to bind the target antigen immobilized on a solid support surface. A major difficulty often encountered with this approach is that phage that bind to additional antigens that are present during the phage selection steps are also selected. We have developed an alternating selection approach to minimize selection of unwanted phage. In the alternating selection approach, two selection methods are used. Each selection method contains different contaminating antigens. This approach was used to select phage that bind a phosphoryated form of the E47 transcription factor. Phage were selected based on their ability to bind a phospho-peptide in solution and alternatively a phospho-protein coated on a polyvinyl micortiter plate. This approach proved significantly better than selection with only one method. With one selection technique, 2 of 48 (4%) selected clones bound to the target antigen. With another selection technique, 15 of 48 (31%) selected clones bound to the target antigen. With alternating selection, 71 of 93 (76%) of the clones bound to the target antigen.
Lu
J
J
University of California, Irvine, Pathology Department, Medical Science D470, Irvine, CA, USA.
Sloan
S R
SR
D000906:Antibodies / Q000235:genetics* / Q000378:metabolism; D000941:Antigens / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D066298:In Vitro Techniques; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000906:Antibodies; D000941:Antigens; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins