Sensitive chemiluminescence assay of reduced nicotinamide adenine dinucleotide using enzyme cycling and its applications

Abstract

A highly sensitive assay system for NADH using an enzyme cycling method was developed. The proposed method is simpler and more rapid than the previous enzyme cycling method. In this method, alcohol dehydrogenase (ADH) and malate dehydrogenase (MDH) is used as a cycling reaction, and malic enzyme as an indicator reaction enzyme. Alkaline phosphatase (ALP) was determined using this method; it was also applied to the determination of 17α-hydroxyprogesterone (17-OHP) and/or human chorionic gonadotropin (hCG). The procedure was as follows: to an assay tube was added 10 μl of an NAD+ or NADH solution and 50 μl of an enzyme cycling solution containing 4 U/ml ADH, 6 U/ml MDH, 0.6 mM oxalacetate and 0.36 M ethanol; the resulting solution was incubated at room temperature overight. Then, 100 μl of an indicator solution containing 200 μ of NADP+, 1 mM of MgCl2 and 0.625 U/ml of malic enzyme were added to the assay tube, which was subsequently incubated at room temperature for 20 min. The reaction mixture was immediately mixed with 500 μ of 1-methoxy-phenazinium methylsulfate (5×10-6M); after standing for 30 s at room temperature, 500 μl of an isoluminol (2.4× 10-4 M)/microperoxidase (1×10-6M) (1 : 1) solution. © 1991, The Japan Society for Analytical Chemistry. All rights reserved.