Highly Sensitive Chemiluminescent Assays of Biological Substances using Enzyme Cycling and Cascade Reaction

我们建立了用NAD~+/NADH酶循环反应和荧光素化学发光反应增强碱性磷酸酶(ALP)的高灵敏度化学发光分析方法。这些方法已被应用于以碱性磷酸酶为标记酶的酶免疫测定：(1)NAD~+/NADH酶循环化学发光测定：利用NAD~+/NADH的酶循环反应来增强NADH的化学发光测定。NADH的检出限提高了10^3倍。该方法用于以NADP^+为底物的碱性磷酸酶的测定。碱性磷酸酶的检出限为4×10~(-1)~(-20)摩尔/试液。建立了17-羟孕酮和人绒毛膜促性腺激素的化学发光酶免疫分析方法。(2)基于荧光素与还原化合物的化学发光反应，碱性磷酸酶的化学发光测定：在所考察的还原化合物中，二羟丙酮与荧光素的化学发光最强。以NaDP^+为底物，建立了碱性磷酸酶化学发光分析的新方法。碱性磷酸酶产生的NAD+与甘油/甘油脱氢酶反应生成二羟基丙酮，并用光泽素进行检测。该方法的检出限为1.25×10~(-1)~(-19)·m~(-1)·m~(-1)，低于酶循环法，但方法简便、快速。用该方法建立了CCK-8和hCG的化学发光酶免疫分析方法。

We have developed highly sensitive chemiluminescent assays of alkaline phosphatase (ALP) enhanced by using NAD^+/NADH enzyme cycling reaction and lucigenin chemiluminescence reaction. These methods have been applied to enzyme immunoassays using ALP as label enzyme.(1) Chemiluminescent assay of ALP using NAD^+/NADH enzyme cycling : The enzyme cycling reaction of NAD^+/NADH was applied to enhance the chemiluminescent assay of NADH. The detection limit of NADH is increased 10^3 times. This method was used for the assay of ALP using NADP^+ as substrate. The detecion limit of ALP was 4 x 10^<-20>mol/assay. Chemiluminescent enzyme immunoassays of 17-hydroxyprogesteron and hCG were developed.(2) Chemiluminescent assay of ALP based on the chemiluminescent reaction of lucigenin with reducing compounds : Dihydroxyacetone in examined reducing compounds gives the most intensive chemiluminescence with lucigenin. The new chemiluminescent assay of ALP was developed using NADP^+ as substrate. NAD^+ generated by ALP reacted with glycerol/glycerol dehydrogenase to yield dihydroxyacetone which was detected by lucigenin. The detection limit of this method was 1.25 x 10^<-19> mol/assay which was lower than that of the enzyme cycling method but the assay procedure was simple and rapid. Chemiluminescent enzyme immunoassays of CCK-8 and hCG were developed by this method.