A fluorescent microplate assay for diarrheic shellfish toxins.
A fluorescent enzyme inhibition assay for okadaic acid using 4-methylumbelliferyl phosphate and fluorescein diphosphate as substrates for the enzyme phosphatase 2A was developed. In the inhibition assay, performed in a microtiter plate, the PP2A was inhibited by adding okadaic acid and the resulting fluorescence enhancement derived from enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The measurable range of okadaic acid was 3.2 to 3200 pg/ml with an IC50 = 0.1 nM. The detection limit of okadaic acid was 2.56 pg/well in buffer solutions and 12.8 ng/g hepatopancreas in shellfish extracts. The coefficient of variation (CV, n = 22) for each point ranged from 18.80 to 37.90% (mean 28.35%). The proposed method is very convenient, rapid, and sensitive by using the enzyme inhibition assay system and fluorescent reaction as a detection system. This work demonstrates that the fluorescent assay can be used to quantify the amount of okadaic acid in shellfish samples and also is valid for very dilute samples, such as phytoplankton samples.
Vieytes
M R
MR
Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
Fontal
O I
OI
Leira
F
F
Baptista de Sousa
J M
JM
Botana
L M
LM
D000818:Animals; D003967:Diarrhea / Q000209:etiology*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods* / Q000706:statistics & numerical data; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D008387:Marine Toxins / Q000032:analysis*; D019319:Okadaic Acid / Q000032:analysis; D009943:Organophosphorus Compounds; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D054648:Protein Phosphatase 2; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012758:Shellfish / Q000032:analysis; D057096:Shellfish Poisoning; D013379:Substrate Specificity
D005452:Fluoresceins; D005456:Fluorescent Dyes; D008387:Marine Toxins; D009943:Organophosphorus Compounds; C074818:3,6-fluorescein diphosphate; C075487:AttoPhos; D019319:Okadaic Acid; C005359:4-methylumbelliferyl phosphate; D006923:Hymecromone; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2