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[A fluorimetric microplate assay for detecting diarrheic shellfish poisoning toxins]
Haiyan Huang1, Aijun Huang, Yingbin Fu
1Toxicology Laboratory, Shenzhen Center for Disease Control and Prevention, Shenzhen 518055, China. hhy424@126.com
Objective:
To establish a new assay for detecting the cytotoxicity of diarrheic shellfish poisoning (DSP) toxins. The assay is based on the depolymerization of F-actin induced by okadaic acid (OA), which is one of main components of DSP toxins.
Methods:
HL-7702 Liver cells were stained with Oregon Green-514 phalloidin as a fluorescent marker for F-actin. The change of the fluorescence of F-actin treated with OA was measured with a fluorimetric microplate reader. The detection results of this assay and ELISA were compared to evaluate the reliability of the assay.
Results:
OA caused an increase of F-actin depolymerization in a dose-dependent manner. There was a linear relationship between the concentration of OA and the depolymerization of F-actin in the range of 2.5 - 40 nmol/L of OA in 24 hours (R2 = 0.993). The detection limit of the F-actin fluorescence assay for OA was 2.01 microg/100g muscles in shellfish extracts, and the recoveries were 90% - 100%. The results of the fluorescence assay were consistent with other methods mentioned above (R2 = 0.830).
Conclusion:
F-actin fluorescence assay was a promising method for the detection of OA in shellfish for its convenience, shortcut and sensitivity.
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