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Optica Publishing Group
  • Applied Spectroscopy
  • Vol. 46,
  • Issue 6,
  • pp. 994-998
  • (1992)

Enzyme-Linked Immunoassays Using Nanosecond Fluorometric Detection

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Abstract

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 <i>p</i>-nitrophenyl phosphate.

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