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Enzyme-Linked Immunoassays Using Nanosecond Fluorometric Detection

Volume 46, Number 6 (June 1992) Page 994-998

Azimi, Nooshin T.; Wen, Fujiang; Lister, Richard M.; Chen, Dennis A.; Lytle, Fred E.


Nanosecond temporal resolution is combined with an enzyme-linked immunosorbent assay (ELISA) to improve the lower limit of detection for a plant virus, brome mosaic virus. The method uses alkaline phosphatase as the enzyme link and β-naphthyl phosphate as the substrate. Enzymatic activity produces the highly fluorescent tag β-naphthol. The 8.9-ns lifetime of the tag allows temporal discrimination against the assay blank, providing a 64 × improvement in the detection limit as compared to a steady-state measurement, and a ∼4100 × improvement over a standard ELISA method incorporating the chromogenic substrate p-nitrophenyl phosphate.