Fluorescence lifetime energy-transfer immunoassay quantified by phase-modulation fluorometry

✍ Scribed by Joseph R. Lakowicz; Badri Maliwal; Alvydas Ozinskas; Richard B. Thompson

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 553 KB
• Volume: 12
• Category: Article
• ISSN: 0925-4005
• DOI: 10.1016/0925-4005(93)85016-4

✦ Synopsis

We describe monitoring of an antigen-antibody interaction by phase-modulation lifetime measurements of the donor-labeled antibody upon binding to acceptor-labeled antigen. Two donor-acceptor pairs are used, each based on a donor-labeled IgG directed against acceptor-labeled IgG from another species. The frequency responses demonstrate the expected decrease in the donor lifetime upon antigen-antibody binding. The phase-modulation immunoassay is shown to work in a competitive format by an increase in lifetime upon addition of unlabeled antigen. Lifetime measurements are desirable for use with scattering and/or optically dense samples ,because they are mostly independent of the total signal intensity. The use of energy transfer as the transducing mechanism to detect binding allows this method to be extended for use with longer-wavelength laser sources and, if desired, longer-lived donors to further simplify the instrumentation and reduce the contribution of autofluorescence to the signal.