A microtiter-based assay for hyaluronidase activity not requiring specialized reagents
A microtiter-based assay for hyaluronidase activity not requiring specialized reagents
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DOI：
10.1006/abio.1997.2262
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发表时间：
1997-09-05
影响因子：
2.9
通讯作者：
Stern, R
中科院分区：
文献类型：
--
作者：
Frost, GI;Stern, R
本发明描述了一种用于透明质酸酶活性的灵敏、快速的基于微量滴定的测定，其不需要如迄今所需的高度专业化的生物试剂。透明质酸的游离羧基在使用生物素-酰肼的一步反应中被生物素化。然后将该底物共价偶联至96孔微量滴定板。酶反应完成后，用抗生物素蛋白-过氧化物酶反应检测残留底物，该反应可在标准ELISA酶标仪中读取。由于底物与微量滴定板共价结合，因此不会发生生物素化底物的pH依赖性置换等人为现象。灵敏度允许从培养的细胞和生物样品中快速测量透明质酸酶活性，测定间变化小于5%。使用这种新的检测方法，我们测量了正常人血清中血浆透明质酸酶水平的分布概况。1 μ l血浆样品足以进行一式三份测定。还定量了人包皮原代角质形成细胞培养物中的透明质酸酶活性。与低钙（0.05 mM）培养基中的水平相比，在高钙（1.5 mM）诱导分化的角质形成细胞培养物中观察到透明质酸酶活性增加25倍。基于微量滴定的测定可用作常规临床实验室程序。(C)北京：科学出版社.
A sensitive, rapid microtiter-based assay for hyaluronidase activity is described that does not require highly specialized biological reagents, as required heretofore. The free carboxyl groups of hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide. This substrate is then covalently coupled to a 96-well microtiter plate. At the completion of the enzyme reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read in a standard ELISA plate reader. Because the substrate is covalently bound to the microtiter plate, artifacts such as pH-dependent displacement of the biotinylated substrate do not occur. The sensitivity permits rapid measurement of hyaluronidase activity from cultured cells and biological samples with an interassay variation of less than 5%. Using this new assay, we measured the distribution profile of plasma hyaluronidase levels in normal human sera. A 1-mu l sample of plasma was sufficient for assays in triplicate. Hyaluronidase activity in human foreskin primary keratinocyte cultures was also quantitated. A 25-fold increase in hyaluronidase activity was observed in keratinocyte cultures induced to differentiate in high calcium (1.5 mM), compared to levels in low calcium (0.05 mM) media. The microtiter-based assay may be used as a routine clinical laboratory procedure. (C) 1997 Academic Press.