AMPLIFIED ASSAY OF ALKALINE-PHOSPHATASE USING FLAVIN-ADENINE DINUCLEOTIDE PHOSPHATE AS SUBSTRATE
AMPLIFIED ASSAY OF ALKALINE-PHOSPHATASE USING FLAVIN-ADENINE DINUCLEOTIDE PHOSPHATE AS SUBSTRATE
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DOI：
10.1016/s0003-2697(05)80020-x
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发表时间：
1992-10-01
影响因子：
2.9
通讯作者：
RABIN, BR
中科院分区：
文献类型：
--
作者：
HARBRON, S;EGGELTE, HJ;RABIN, BR
描述了一种简单易用、稳健、定量和极灵敏的碱性磷酸酶比色测定法（EC 3.1.3.1），设计用于采用抗体或基因探针的诊断测定中的检测系统。该技术基于假体生成的新原理，根据该原理，用于主要分析物连接的酶标记的目的设计的底物（假体原）被水解以产生用于检测酶系统的假体基团。该方法采用的前体是在核糖环3′-位磷酸化的FAD衍生物（FADP），标记酶是碱性磷酸酶，检测器是D-氨基酸氧化酶-辣根过氧化物酶偶联系统。基本上，碱性磷酸酶的每一个分子的每一次转换都会产生一个D-氨基酸氧化酶分子用于检测。因此，由于FADP的碱性磷酸酶的相对高的周转数，在短时间内实现了初始信号的巨大放大。该系统可以被格式化为一个稳定的，预先形成的，冻干制剂含有所有的分析成分，这是简单地通过添加缓冲溶液重建。该方法可以使用微量滴定板在25°C下在30分钟内定量小于0.1阿莫尔的碱性磷酸酶。
A simple to use, robust, quantitative, and extremelysensitive colorimetric assay for alkaline phosphatase (EC 3.1.3.1), designed to be used as a detection system in diagnostic assays employing antibodies or gene probes, is described. This technology is based on the novel principle of prosthetogenesis, according to which a purpose-designed substrate (a prosthetogen) for a primary analyte-linked enzyme label is hydrolyzed to produce a prosthetic group for a detector enzyme system. The prosthetogen employed here is a derivative of FAD which is phosphorylated at the 3′-position of the ribose ring (FADP), the label enzyme is alkaline phosphatase, and the detector is a D-amino-acid oxidaselhorseradish peroxidase-coupled system. Essentially each turnover of every molecule of alkaline phosphatase produces a molecule of D-amino-acid oxidase for detection. Thus enormous amplification of the initial signal is achieved in short time periods because of the relatively high turnover number of alkaline phosphatase for FADP. The system can be formatted as a stable, preformed, freeze-dried preparation containing all analytical components, which is reconstituted simply by addition of buffer solution. This methodology can quantitate less than 0.1 amol of alkaline phosphatase in 30 min at 25°C using microtiter plates.