Open AccessHomogeneous Noncompetitive Immunoassay Based on the Energy Transfer Between Fluorolabeled Antibody Variable Domains (Open Sandwich Fluoroimmunoassay)
Author(s) -
Hiroshi Ueda,
Kazuishi Kubota,
Y Wang,
Kouhei Tsumoto,
Walt Mahoney,
Izumi Kumagai,
Teruyuki Nagamune
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 - 10.2144/99274st04
Subject(s) - förster resonance energy transfer , cyanine , immunoassay , chemistry , fluorescence , cuvette , rhodamine , chromatography , lysozyme , antibody , immunoglobulin light chain , fluorescein , antigen , succinimide , microbiology and biotechnology , biochemistry , biology , physics , genetics , quantum mechanics , immunology
The antigen-dependent stabilization of an anti-hen egg lysozyme (HEL) antibody HyHEL-10 variable region was monitored with fluorescence resonance energy transfer (FRET) between fluorolabeled heavy chain (V H ) and light chain (V L ) fragments. The V H and V L fragments labeled with succinimide esters of fluorescein and rhodamine-X, respectively, were mixed in a cooled cuvette, and the change in fluorescence spectra upon antigen addition was monitored. When excited at 490 nm, significant decrease in the fluorescence at 520 nm and its increase at 605 nm were observed when an increasing amount of HEL was added to the mixture in the concentration range of 1–100 μg/mL. The assay, named open sandwich fluoroimmunoassay (FIA), is noncompetitive and homogeneous and can be conducted with one clone of antibody. With the use of appropriate antibodies, it is thought to be a quick and inexpensive alternative to the conventional laborious and/or expensive immunoassays.