一种使用固定化酶检测12-α-羟基胆汁酸的生物发光测定法。

A bioluminescent assay for 12-alpha-hydroxy bile acids using immobilized enzymes.

作者信息

Schoelmerich J, Hinkley J E, Macdonald I A, Hofmann A F, DeLuca M

出版信息

Anal Biochem. 1983 Aug;133(1):244-50. doi: 10.1016/0003-2697(83)90250-6.

Abstract

A bioluminescent assay for 12-alpha-hydroxy bile acids was developed using enzymes coimmobilized onto Sepharose 4B. The immobilized enzymes used were a bacterial 12-alpha-hydroxysteroid dehydrogenase, bacterial luciferase, and NADPH:FMN oxidoreductase or bacterial diaphorase. The assay was specific for 12-alpha-hydroxy bile acids and the lower limit of detection was 4 pmol/0.5 ml assay volume with a linear range of 4 to 2000 pmol. Intraassay precision was from 7.8 to 8.2%. Values obtained with this assay showed good agreement with those obtained by gas-liquid chromatography. The system using diaphorase was not stable at 4 degrees C in the absence of added thiol compounds, but could be stabilized by the addition of glutathione (0.5 mM). The assay is a convenient, a rapid, and an extremely sensitive method for the measurement of 12-alpha-hydroxy bile acid concentrations in the serum of patients or experimental animals.

摘要

利用共固定在琼脂糖4B上的酶开发了一种用于检测12-α-羟基胆汁酸的生物发光测定法。所使用的固定化酶为细菌12-α-羟基类固醇脱氢酶、细菌荧光素酶以及NADPH：FMN氧化还原酶或细菌黄递酶。该测定法对12-α-羟基胆汁酸具有特异性，检测下限为4 pmol/0.5 ml测定体积，线性范围为4至2000 pmol。批内精密度为7.8%至8.2%。该测定法所得值与气液色谱法所得值显示出良好的一致性。在没有添加硫醇化合物的情况下，使用黄递酶的系统在4℃不稳定，但通过添加谷胱甘肽（0.5 mM）可使其稳定。该测定法是一种方便、快速且极其灵敏的方法，用于测量患者或实验动物血清中12-α-羟基胆汁酸的浓度。