ENZYME-LINKED IMMUNOASSAYS USING NANOSECOND FLUOROMETRIC DETECTION

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 beta-naphthyl phosphate as the substrate. Enzymatic activity produces the highly fluorescent tag beta-naphthol. The 8.9-ns lifetime of the tag allows temporal discrimination against the assay blank, providing a 64 x improvement in the detection limit as compared to a steady-state measurement, and a approximately 4100 x improvement over a standard ELISA method incorporating the chromogenic substrate p-nitrophenyl phosphate.