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Protein Engineering, Vol. 13, No. 5, 369-376, May 2000
© 2000 Oxford University Press

Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay

Ryoichi Arai1, Hiroshi Ueda1,2,5, Kouhei Tsumoto3, Walt C. Mahoney4, Izumi Kumagai3 and Teruyuki Nagamune1

1 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 2 Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK, 3 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan, and 4 Roche Diagnostics, Chief Technology Office, 2929 7th Street, Suite 100, Berkeley, CA 94710, USA

A site-specific and efficient fluorolabeling of antibody variable regions with green fluorescent protein (GFP) variants and its application to an energy transfer-based homogeneous fluoroimmunoassay (open sandwich FIA) were attempted. Two chimeric proteins, Trx–VH–EBFP and Trx–VL–EGFP, consisting of VH and VL fragments of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and two GFP color variants, EBFP and EGFP, respectively, were designed to be expressed in cytoplasm of trxB – mutant Escherichia coli as fusions with thioredoxin from E.coli The mixture of two proteins could be purified with HEL-affinity chromatography, retaining sufficient intrinsic fluorescence and binding activity to HEL. A significant increase in fluorescence resonance energy transfer (FRET) dependent on HEL concentration was observed, indicating the reassociation of the VH and VL domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1–100 µg/ml could be non-competitively determined. The assay could be performed in a microplate format and took only a few minutes to obtain a sufficient signal after simple mixing of the chimeric proteins with samples. This represents the first demonstration that the FRET between GFP variants is applicable to homogeneous immunoassay.


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