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De novo identification of cellâtype specific antibodyâantigen pairs by phage display subtraction: Isolation of a human single chain antibody fragment against human keratin 14
- StausbÃ¸lâGrÃ¸n, Brian, Jensen, Kim Bak, Jensen, Kristian Hobolt, Jensen, Morten Ãstergaard, Clark, Brian F. C.
- European journal of biochemistry 2001 v.268 no.10 pp. 3099-3107
- DNA libraries, antibodies, antigens, bacteriophages, databases, enzyme-linked immunosorbent assay, fluorescent antibody technique, humans, hybridomas, immunization, immunoblotting, keratin, keratinocytes, polyacrylamide gel electrophoresis
- The aim of this study was to identify novel antibodies directed against cytosolic keratinocyteâspecific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology.âA novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly nondifferentially expressed antigens.âA monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer ofÂ human epidermis by indirect immunofluorescence analysis.âStarting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibodyâantigen pairs that characterize a specific cell type.