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

利用NAD^+/NADH酶循环反应和lucigenin化学发光反应，建立了碱性磷酸酶（ALP）的高灵敏度化学发光检测方法。这些方法已应用于以ALP为标记酶的酶免疫测定。(1)利用NAD^+/NADH酶循环法测定ALP的化学发光：利用NAD^+/NADH酶循环反应增强NADH的化学发光测定。NADH的检出限提高了10^3倍。本方法以NADP^+为底物测定ALP。ALP的检出限为4 × 10^<-20>mol/次。建立了17-羟基孕激素和hCG的化学发光酶免疫测定法。(2)基于lucigenin与还原性化合物化学发光反应的ALP化学发光测定：在所检测的还原性化合物中，二羟丙酮与lucigenin的化学发光最强烈。以NADP^+为底物，建立了新的ALP化学发光测定方法。ALP生成的NAD^+与甘油/甘油脱氢酶反应生成二羟基丙酮，用lucigenin检测。该方法的检出限为1.25 × 10^<-19> mol/次，低于酶循环法的检出限，但检测过程简单、快速。建立了CCK-8和hCG的化学发光酶免疫测定方法。

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.