Wu M C, Takagi K, Okuyama S, Ohsawa M, Masahashi T, Narita O, Tomoda Y
J Chromatogr. 1986 Apr 25;377:121-9. doi: 10.1016/s0378-4347(00)80767-7.
We studied the use of an immobilized enzyme, covalently bound to aminopropyl-CPG, in the analysis of individual delta 5-3 beta-hydroxysteroid sulphates. A microcolumn with immobilized 3 beta,17 beta-hydroxysteroid dehydrogenase was prepared and used together with high-performance liquid chromatography (HPLC). The reduced nicotinamide-adenine dinucleotide produced from delta 5-3 beta-hydroxysteroids by this enzyme was fluorimetrically determined. The immobilized enzyme was sufficiently stable for at least one month or for 180 tests when used repeatedly. A clinical trial demonstrated that this HPLC-immobilized enzyme method is superior to the soluble enzyme method, giving reliable and reproducible results at a low cost.
我们研究了一种固定化酶在个体δ5-3β-羟基甾体硫酸盐分析中的应用，该酶通过共价键与氨丙基-CPG结合。制备了固定有3β,17β-羟基甾体脱氢酶的微柱，并与高效液相色谱（HPLC）联用。通过该酶由δ5-3β-羟基甾体产生的还原型烟酰胺腺嘌呤二核苷酸用荧光法测定。该固定化酶具有足够的稳定性，反复使用时至少可稳定一个月或进行180次测试。一项临床试验表明，这种HPLC-固定化酶方法优于可溶性酶方法，能以低成本给出可靠且可重复的结果。
