Dissecting the human peripheral B-cell compartment with phage display-derived antibodies

Previously we have employed a large semisynthetic phage antibody display library, in combination with subtractive selection by flow cytometry to isolate phage antibodies specific for subpopulations of leucocytes. In this study, human tonsillar B cells were incubated with the phage library and IgD- CD38- memory B lymphocytes and attached phage antibodies were selected by cell sorting. In a panel of 17 monoclonal phage antibodies obtained, five displayed binding to cells of multiple haematopoietic lineages or broadly reacted with B-lineage cells. Immunofluorescent, immunohistochemical and biochemical studies permitted the characterization of the target molecules recognized by these phage antibodies. The remaining 12 antibodies displayed restricted binding to small subpopulations of peripheral human B cells. These results show that subtractive selections with phage antibody display libraries in combination with flow cytometry yield antibodies that bind to differentially expressed molecules on closely related cell populations, and can be used as a tool in a variety of assays.