一种用于微囊藻毒素-LR的荧光微孔板检测方法。

A fluorescent microplate assay for microcystin-LR.

作者信息

Fontal O I, Vieytes M R, Baptista de Sousa J M, Louzao M C, Botana L M

机构信息

Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, 27002, Spain.

出版信息

Anal Biochem. 1999 May 1;269(2):289-96. doi: 10.1006/abio.1999.3099.

DOI:10.1006/abio.1999.3099

PMID:10222000

Abstract

A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The concentration of microcystin-LR causing 50% inhibition of PP1 and PP2A activity (IC50) was 0.01 nM for PP1 and 0.08 nM for PP2A. The measurable range of microcystin-LR was 800 to 0.08 pg/well for both enzymes. The described assay is fast and very sensitive for the detection of microcystin-LR. Furthermore, this assay can be successfully applied to the study of toxins that inhibit PP1 or PP2A.

摘要

利用蛋白磷酸酶1（PP1）和2A（PP2A）的新型荧光底物6,8 - 二氟 - 4 - 甲基伞形酮磷酸酯，开发了一种用于微囊藻毒素 - LR的荧光酶抑制测定法。在微量滴定板中进行微囊藻毒素 - LR的PP1和PP2A抑制测定，并在荧光酶标仪中对底物酶促水解产生的荧光进行定量。导致PP1和PP2A活性50%抑制（IC50）的微囊藻毒素 - LR浓度，对于PP1为0.01 nM，对于PP2A为0.08 nM。两种酶对微囊藻毒素 - LR的可测量范围均为800至0.08 pg/孔。所描述的测定法对于微囊藻毒素 - LR的检测快速且非常灵敏。此外，该测定法可成功应用于抑制PP1或PP2A的毒素研究。