Chemiluminescent Assay of NAD (P)H and its Application to Clinical Chemistry

建立了1-甲氧基-5-甲基吩嗪甲基硫酸盐(1-MPMS)/异鲁米诺(IL)/微过氧化物酶(m-POD)化学发光体系测定NAD(P)H的方法。化学发光强度与NAD(P)H浓度在10~(-9)~10~(-12)~10~(-12)~10~(-12)~10~(-12)~10~(-12)~(-12)~(-12)~(-12)~(-12)~(-10)~(-12)~(-12)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1)~(-1G6PDH、β-D-Gal和ALP的检出限分别为10^~(-18)、10~(-20)和10~(-18)。这些酶的化学发光测定被应用于17α-羟孕酮的化学发光酶免疫测定和DNA杂交分析，为了提高该方法的灵敏度，将酶循环系统与NADH的化学发光测定相结合。NADH的标准曲线范围为3×10~(-14)~5×10~(-12)mol/L，检出限为30fmol/次，与细菌荧光素酶生物发光法相当。利用三磷酸腺苷与己糖激酶/丙酮酸激酶的酶循环反应，建立了三磷酸腺苷的增强化学发光分析方法。该方法的灵敏度是前一种方法的1000倍。对三磷酸腺苷的检出限为10fmoL/份。

We have developed a chemiluminescent method for the assay of NAD (P)H using the 1-methoxy- 5-methylphenazinium methylsulphate (1-MPMS) / isoluminol (IL) / microperoxidase (m-POD) system. A linear relationship between chemiluminescence intensity and NAD (P)H concentration (log/log) was obtained ranged from 10^<-9> mol/l to 10^<-12> mol/l. This chemiluminescent assay has been coupled to the assay of glucose-6-phosphate dehydrogenase (G6PDH), beta-D-glactosidase (beta-Gal) and alkaline phosphatase (ALP). The detection limits of G6PDH, beta-D-Gal and ALP were 10^<-18>, 10^<-20> and 10^<-18> molper assay, respectively. The chemiluminescent assay of these enzymes were appplied to chemiluminescent enzyme immunoassay for 17alpha-hydroxyprogesterone and DNA hybridization assay.In order to increase the sensitivity of this method, enzyme cycling system was coupled to the chemiluminescent assay of NADH. The standard curve was obtained in the range from 3 x 10^<-14> to 5 x 10^<-12> mol/l. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase.The chemiluminescent assay of ATP was also developed using hexokinase/G6PDH and i-MPMS/ IL/m-POD system. The enhanced chemiluminescent assay of ATP was developed by using enzyme cycling reaction of ATP with hexokinase/pyruvate kinase. This method is 1000 fold more sensitive than the former method. The detection limit of ATP was 10 fmol/assay.