Time-Resolved Fluorescence Energy Transfer Immunoassay
Time-resolved methods, including both time- and frequency-domain techniques, are of growing importance in fluorescence immunoassay due to their selectivity and ability to reject background fluorescence (Soini and Hemmila, 1979). Phase methods are especially interesting because the instrumentation is simple, and can potentially be made compact and solid state. We decided to test the utility of variable-frequency phase fluorometry for measurement of an antigen-antibody binding reaction, a reaction which could also be quantitated by a separate method. Thus antibodies and antigens were labeled with fluorescence energy transfer acceptors and donors, respectively. This allowed binding to be measured from changes in the donor’s decay kinetics (quenching), or from changes in the acceptor’s fluorescence excitation spectrum, which are caused by energy transfer.
KeywordsEnergy Transfer Fluorescence Anisotropy Heme Pocket Donor Fluorescence Fluorescence Immunoassay
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