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Related Experiment Videos

A fluorescent microplate assay for diarrheic shellfish toxins

M R Vieytes1, O I Fontal, F Leira

  • 1Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.

Analytical Biochemistry
|June 1, 1997
PubMed
Summary

A new fluorescent enzyme inhibition assay accurately detects okadaic acid, a marine toxin. This sensitive method quantifies okadaic acid in shellfish and phytoplankton, ensuring food safety.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Marine Biology

Background:

  • Okadaic acid is a potent marine toxin causing diarrhetic shellfish poisoning.
  • Accurate and sensitive detection methods for okadaic acid are crucial for food safety and environmental monitoring.
  • Existing detection methods can be complex and time-consuming.

Purpose of the Study:

  • To develop a rapid, sensitive, and convenient fluorescent enzyme inhibition assay for okadaic acid.
  • To validate the assay's performance in buffer solutions and complex matrices like shellfish extracts.
  • To establish the assay's utility for quantifying okadaic acid in environmental samples such as phytoplankton.

Main Methods:

  • Development of a fluorescent enzyme inhibition assay using phosphatase 2A (PP2A) as the target enzyme.

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  • Utilized 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for PP2A.
  • Quantification of fluorescence enhancement in a microtiter plate format using a fluorescence plate reader following PP2A inhibition by okadaic acid.

    • Main Results:

    • The assay demonstrated a measurable range of 3.2 to 3200 pg/ml for okadaic acid.
    • An IC50 of 0.1 nM was determined for okadaic acid inhibition of PP2A.
    • The detection limit was as low as 2.56 pg/well in buffer and 12.8 ng/g in shellfish hepatopancreas extracts.
    • The coefficient of variation ranged from 18.80% to 37.90% (mean 28.35%).

    Conclusions:

    • The developed fluorescent assay is a convenient, rapid, and sensitive method for okadaic acid detection.
    • The assay is effective for quantifying okadaic acid in shellfish samples.
    • The method is also suitable for analyzing very dilute samples, including phytoplankton, highlighting its broad applicability.