Arakawa H, Maeda M, Tsuji A
School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Anal Biochem. 1991 Dec;199(2):238-42. doi: 10.1016/0003-2697(91)90096-c.
Chemiluminescent assays of various enzymes have been developed using indoxyl derivatives as substrates. The principle of the method is as follows: an enzyme causes hydrolysis of an indoxyl derivative to an intermediate indoxyl that is readily oxidized to indigo dye and simultaneously produces hydrogen peroxide (H2O2). Hydrogen peroxide is detected chemiluminescently using isoluminol-microperoxidase. Alkaline phosphatase (ALP), beta-D-galactosidase (beta-gal), and beta-glucosidase were assayed by this method using 5-bromo-4-chloro-3-indolyl phosphate (BCIP), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and 5-bromo-4-chloro-3-indolyl-beta-D-glucoside, respectively, as substrates. Using BCIP and X-Gal substrates, we have been able to detect 10(-19) mol of ALP and beta-gal, respectively. This assay system can be applied to enzyme immunoassay and DNA probe assay.
已经开发出使用吲哚酚衍生物作为底物对各种酶进行化学发光测定的方法。该方法的原理如下：一种酶使吲哚酚衍生物水解为中间吲哚酚，该中间吲哚酚易于氧化成靛蓝染料并同时产生过氧化氢（H2O2）。使用异鲁米诺 - 微过氧化物酶对过氧化氢进行化学发光检测。碱性磷酸酶（ALP）、β - D - 半乳糖苷酶（β - gal）和β - 葡萄糖苷酶分别使用5 - 溴 - 4 - 氯 - 3 - 吲哚基磷酸酯（BCIP）、5 - 溴 - 4 - 氯 - 3 - 吲哚基 - β - D - 吡喃半乳糖苷（X - Gal）和5 - 溴 - 4 - 氯 - 3 - 吲哚基 - β - D - 葡萄糖苷作为底物，通过该方法进行测定。使用BCIP和X - Gal底物，我们分别能够检测到10^(-19)摩尔的ALP和β - gal。该测定系统可应用于酶免疫测定和DNA探针测定。
