Fluorolabeling of antibody variable domains with green fluorescent protein variants: application to an energy transfer-based homogeneous immunoassay.
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-V(H)-EBFP and Trx-V(L)-EGFP, consisting of V(H) and V(L) 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 V(H) and V(L) domains of these chimeric proteins due to co-existing antigen. With this open sandwich FIA, an HEL concentration of 1-100 microg/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.
Arai
R
R
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Ueda
H
H
Tsumoto
K
K
Mahoney
W C
WC
Kumagai
I
I
Nagamune
T
T
D001483:Base Sequence; D017931:DNA Primers; D004735:Energy Transfer; D005455:Fluorescent Antibody Technique; D005461:Fluorine / Q000737:chemistry; D049452:Green Fluorescent Proteins; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry*
D017931:DNA Primers; D007135:Immunoglobulin Variable Region; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D005461:Fluorine