Vieytes M R, Fontal O I, Leira F, Baptista de Sousa J M, Botana L M
Departamento de Fisiología, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain.
Anal Biochem. 1997 Jun 1;248(2):258-64. doi: 10.1006/abio.1997.2127.
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.
开发了一种以4-甲基伞形酮磷酸酯和荧光素二磷酸酯作为磷酸酶2A的底物来检测冈田酸的荧光酶抑制试验。在微孔板中进行的抑制试验中，通过添加冈田酸抑制PP2A，并在荧光酶标仪中对底物酶促水解产生的荧光增强进行定量。冈田酸的可测量范围为3.2至3200 pg/ml，IC50 = 0.1 nM。在缓冲溶液中，冈田酸的检测限为2.56 pg/孔，在贝类提取物中为12.8 ng/g肝胰腺。每个点的变异系数（CV，n = 22）范围为18.80%至37.90%（平均28.35%）。所提出的方法通过使用酶抑制试验系统和荧光反应作为检测系统，非常方便、快速且灵敏。这项工作表明，荧光测定法可用于定量贝类样品中冈田酸的含量，并且对于非常稀释的样品（如浮游植物样品）也是有效的。
