Chemiluminescent assay of co‐factors

Abstract

Bioluminescent methods are widely used for the assay of the co-factors, NADH and ATP. Although the bioluminescent method is highly sensitive, the enzymes used are unstable and expensive. Therefore a chemiluminescent method would be valuable in clinical routine assay. We have developed a chemiluminescent method for the assay of NADH using the 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase(m-POD) system. In order to increase the sensitivity of this method, enzymatic cycling system was coupled to the chemiluminescent assay of NADH. Alcohol dehydrogenase and malate dehydrogenase were used as the cycling enzyme. The standard curve was obtained in the range from 3 X 10(-14) to 5 X 10(-12) mol/assay. The detection limit of NADH was 30 fmol/assay which was comparable to that of the bioluminescent method using bacterial luciferase. Two chemiluminescent methods for the assay of ATP have been developed. Method 1 is the system using hexokinase/G6PDH and 1-PMS/IL/m-POD, and method 2 is the system based on the enzymatic cycling reaction of ATP using hexokinase/pyruvate kinase. Method 2 is 1000/fold more sensitive than the method 1. The detection limit of ATP was 10 fmol/assay.

Differential Levels of Glutamate Dehydrogenase 1 (GLUD1) in Balb/c and C57BL/6 Mice and the Effects of Overexpression of theGlud1Gene on Glutamate Release in Striatum

“…As a control for the effects of mouse inbreeding, we also added an outbred strain, the CD-1 mice. In order to enhance the sensitivity of the GLUD assay procedure, we used a chemiluminescence detection method of coupled reactions as described in the Methods section (Maeda et al, 1989; Tsuji et al, 1989). The limits of detection of NADH formed using the chemiluminescence procedure were approx.…”

“…The enzyme activity of GLUD was measured in the direction of glutamate oxidation and deamination using a coupled reaction scheme similar to that described (Maeda et al, 1989; Tsuji et al, 1989). In this scheme, the NADH formed during varying incubation time periods was allowed to reduce O 2 to O 2 −• and H 2 O 2 in a coupled reaction with the electron mediator 1-MPMS (1-methoxy-5-methylphenazinium methyl-sulfate).…”

“…In this scheme, the NADH formed during varying incubation time periods was allowed to reduce O 2 to O 2 −• and H 2 O 2 in a coupled reaction with the electron mediator 1-MPMS (1-methoxy-5-methylphenazinium methyl-sulfate). The O 2 −• and H 2 O 2 formed were detected as light generated by the chemiluminescence reaction with isoluminol in the presence of m-POD (micro-peroxidase) (Maeda et al, 1989; Tsuji et al, 1989). The GLUD enzyme reactions with brain homogenates were performed in 96-well chemiluminescence microplates and were initiated by the addition of 10 μl of homogenate (50 μg/ml final protein concentration) to 90 μl of reaction buffer (final concentrations in mM: 125.0 ammonium acetate, 1.0 EDTA, 0.9 NAD, 1.0 ADP, 1.0 leucine, 20.0 l -glutamate and 50.0 triethanolamine, pH 7.5).…”

Resident bacteria contribute to opportunistic infections of the respiratory tract

“…To investigate the underlying mechanism, we directly measured the NAD + content in the culture medium using a colorimetric assay [33]. The accumulation of NAD + reached its peak after culturing S. chromogenes for 4 h (Fig 4A).…”

Chemiluminescent and Bioluminescent Assays for Polyadenylic Acid and Applications to DNA Probe Assay and Immunoassay

“…In both methods, NADPH generated by the enzymatic reaction is finally measured by the CL assay using 1-methoxy-5-methylphenazinium methylsulfate (1-MPMS)/ isoluminol(IL)/ microperoxidase (m-POD). 2 We have recently developed a CL assay of poly A based on the enzymatic cycling reaction of ATP and its application to a CL DNA probe assay using biotinylated poly A as label. We also developed a BL immunoassay for human thyroid-stimulating hormone (hTSH) using the biotinylated poly A as label and luciferase reaction as the detection system.…”