TY - JOUR

T1 - 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

AU - Stausbøl-Grøn, B

AU - Jensen, K B

AU - Jensen, Kristian Hobolt

AU - Jensen , M Ø

AU - Clark, B F

PY - 2001/5

Y1 - 2001/5

N2 - The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific 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. A 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 non-differentially expressed antigens. A 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 of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

AB - The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte-specific 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. A 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 non-differentially expressed antigens. A 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 of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody-antigen pairs that characterize a specific cell type.

KW - Blotting, Western

KW - Dose-Response Relationship, Drug

KW - Electrophoresis, Polyacrylamide Gel

KW - Enzyme-Linked Immunosorbent Assay

KW - Epidermis/metabolism

KW - Fluorescent Antibody Technique, Indirect

KW - Glutathione Transferase/metabolism

KW - Humans

KW - Immunoblotting

KW - Immunoglobulin Fragments/metabolism

KW - Immunohistochemistry

KW - Keratin-14

KW - Keratinocytes/immunology

KW - Keratins/chemistry

KW - Peptide Library

KW - Polymerase Chain Reaction

KW - Recombinant Fusion Proteins/metabolism

KW - Tissue Distribution

M3 - Journal article

C2 - 11358530

VL - 268

SP - 3099

EP - 3107

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 10

ER -