De novo identification of cell-type specific antibody-antigen pairs by phage display subtraction - Isolation of a human single chain antibody fragment against human keratin 14

The aim of this study was to identify novel antibodies directed against cyt osolic keratinocyte-specific antigens from a phage display antibody reperto ire by using phage display subtraction. Phage display is a method of displa ying foreign molecules on the surface of filamentous bacteriophage particle s. It allows the interaction between two cognate molecules to be analysed t hrough affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, anti bodies can be obtained in vitro without the need for immunization or the hy bridoma technology. A novel subtractive strategy for selecting antibodies f rom phage libraries was applied. Phage antibodies were selected against imm obilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retri eval of phage antibodies with specificities to commonly nondifferentially e xpressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identifie d as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotti ng based on 2D PAGE and a corresponding 2D PAGE protein database. In accord ance with the expected tissue localization of K14, the identified scFv stai ned the basal layer of human epidermis by indirect immunofluorescence analy sis. Starting with crude cell lysates, phage display subtraction in combina tion with 2D PAGE and 2D PAGE protein databases can be used to identify ant ibody-antigen pairs that characterize a specific cell type.