| Description |
NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector, both protects the cell from carcinogenic oxidative damage and stabilizes the tumor suppressor p53 protein. At high glycolysis levels NQO1 stabilizes and protects p53 from ubiquitin-independent degradation, a process that is NADH dependent, and also elevates NAD+/NADH levels. NAD+ and NADH play a crucial role in cellular energy metabolism, and a dysregulated NAD+/NADH ratio is implicated in metabolic syndrome. In humans, NQO1 is expressed at high levels in adipocytes and its expression levels are positively correlated with adiposity, glucose tolerance, and liver dysfunction. Thus NQO1 may provide the basis for a new therapy for the treatment of metabolic syndrome. This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human NQO1 in cells. A monoclonal antibody specific for NQO1 has been precoated onto the 96-well microtiter plate. Standards and samples are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, NQO1 is recognized by the addition of a purified polyclonal antibody specific for NQO1 (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (Detector) is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of NQO1 in the samples. This ELISA is specific for the measurement of natural and recombinant human NQO1. It does not cross-react with human adiponectin, human RBP4, human Nampt, human vaspin, human progranulin, human resistin, human clusterin, human ANGPTL3, human CTRP5, human IL-33, human leptin, human GPX3, human NMNAT2, human sirtuin 1, human FTO, mouse Nampt, rat Nampt. The assay range is 0.313 – 20 ng NQO1/ml and a detection limit of 100 pg/ml (based on adding two standard deviations to the mean value of the (50) zero standards).
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