基于固定化酶的蛋白质磷酸酶抑制率法测定大田软海绵酸

Research on detection of okadaic acid by using immobilized protein phosphatase inhibition assay

目的建立和优化用于检测大田软海绵酸(OA)的固定化蛋白质磷酸酶2A(PP2A)抑制率法。方法以分离提取的PP2A为材料,根据OA抑制PP2A酶活性的原理,建立用于检测OA的固定化PP2A酶抑制率法,并对最佳酶用量和反应体系参数等进行优化。结果最佳固定化PP2A酶蛋白量2μg、固定化配比为琼脂糖与PP2A酶蛋白量为1:1、p H值为8.4;采取4参数Logistic曲线法进行标准曲线拟合,曲线R2值达0.994,检测限为3.45μg/L,实际样品测定时具有良好的准确性和重复性。结论建立的固定化PP2A酶抑制率法有望作为低成本、操作简单的OA快速筛检方法推广应用。

Objective To develop and optimize an immobilized enzyme inhibition assay for okadaic acid (OA).Methods Based the principle which OA could inhibit the activity of protein phosphatase 2A (PP2A), the assay was established by using the extracted PP2A in this paper. The parameters such as the amount of enzyme and reaction condition were optimized.Results The optimal PP2A immobilized enzyme protein was 2μg, the optimal ratio of agarose and PP2A enzyme protein was 1:1, and the optimal pH value was 8.4. The standard curve fitting was made by the 4-parameter Logistic curve method.R2value of curve fitting was 0.994, which suggested standard curve fitting has good fit. The detection limit of OA was 3.45μg/L. This assay for detecting the actual sample had a good accuracy and repeatability.Conclusion These results suggested that the immobilized enzyme inhibition assay may be used as a low-cost and easy operation method for the rapid detection of OA.