Open AccessSelection of an Anti-CD20, Single-Chain Antibody by Phage ELISA on Fixed Cells
Author(s) -
Stefanie Schmidt,
Michael Braunagel,
Timo Kürschner,
Melvyn Little
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99264st06
Subject(s) - phagemid , microbiology and biotechnology , antigen , antibody , biology , phage display , recombinant dna , immunoglobulin light chain , complementary dna , monoclonal antibody , single chain variable fragment , virology , gene , escherichia coli , bacteriophage , biochemistry , genetics
Cloning the correct genes that code for antibody-variable domains from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from a myeloma cell line. For the rapid functional evaluation of recombinant antibody fragments against cell-surface antigens, we established an efficient expression and screening system using phagemid antibodies and fixed cells. V L and V H -polymerase chain reaction (PCR) products, amplified from hybridoma cDNA, were cloned into the phagemid vector pSEX81. After transduction into E. coli and phage rescue, clones were tested for antigen binding using a phage-enzyme-linked immunosorbent assay (ELISA) procedure with whole cells fixed to ELISA wells. This procedure facilitated the successful cloning of a functional anti-CD20, single-chain antibody from hybridoma cDNA. The CD20 B-lymphocyte surface antigen expressed by B-cell lymphomas is an attractive target for cancer treatment using immunoconjugates or bi-specific antibodies.