Affiliations: German Cancer Research Center, Recombinant Antibody Research Unit, 1m Neuenheimer Feld 506, 69120 Heidelberg, Germany
Correspondence:
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Correspondence and reprint requests to: Stefan Dübel, German Cancer Research Center, Recombinant Antibody Research Unit, Im Neuenheimer Feld 506, 69130 Heidelberg, Germany.
Abstract: To increase the avidity of single-chain antibodies (scFv) for their antigen, we have fused them to core-streptavidin. The chimeric protein, expressed by the vector pSTE (plasmid for streptavidin-tagged expression) from Escherichia coli, can form tetrameric complexes, binds its antigen and contains four biotin binding sites per tetrameric complex. An additional cysteine inserted near the carboxy terminus further stabillsed the complex. The scFv fusion protein tetramers could be enriched by affinity chromatography using the biotin analog 2-iminobiotin from periplasmic inclusion bodies after refolding. We have also shown that the scFv fusion protein could be used for direct detection of its antigen in ELISA when stained with biotinylated horseradish peroxidase. The affinity of the scFv-antibody complex was substantially increased by avidity effects due to the tetrameric structure. The biotin binding sites may be used for coupling other antibodies and molecules to form bispecific and bifunctional reagents.
Keywords: Streptavidin, scFv, single chain antibody, bifunctional, bispecific
