Principle of Method
This assay is a sandwich Enzyme Linked-Immunosorbent Assay (ELISA) for quantitative determination of human Nampt in cells. A monoclonal antibody specific for Nampt 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, Nampt is recognized by the addition of a purified polyclonal antibody specific for Nampt (Detection Antibody). After removal of excess polyclonal antibody, HRP conjugated anti-rabbit IgG (HRP) 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 450nm after acidification and is directly proportional to the concentration of Nampt in the samples.
Nampt, nicotinamide phosphoribosyltransferase, is the rate-limiting enzyme of the mammalian NAD, nicotinamide adenine dinucleotide, biosynthesis pathway from nicotinamide to NMN, nicotinamide mononucleotide (1), which is further converted to NAD by nicotinamide/nicotinic acid mononucleotide adenyltransferase abbreviated Nmnat. Nampt was found to have a significant homology to the mammalian pre-B cell colony-enhancing factor (PBEF) (3) or visfatin (4), which is also called extracellular Nampt (eNampt) (5) due to the fact that it is found as a circulating form in human serum and secreted from differentiated adipocytes. Although visfatin was originally reported as an insulin-mimicking hormone by its capability of binding to and activating the insulin receptor, subsequent studies do not support this observation (5, 6). However, Revollo et al. found that deficiency and chemical inhibition of Nampt caused defects in NAD biosynthesis and glucose-stimulated insulin secretion in pancreatic islets in vivo and in vitro. Additionally, plasma visfatin and NMN levels were reduced in Nampt heterozygous females (5). This study proposed a model that NMN exists in high amounts in plasma, presumably derived from nicotinamide with help of eNampt. This circulating NMN and nicotinamide are uptaken by beta cells via unknown transport mechanism (s) are converted to NAD by Nmnat and intracellular Nampt (hereinafter abbreviated iNampt), respectively, concluding that Nampt-mediated systemic NAD biosynthesis is critical for insulin secretion presumably via a NAD-dependent histonedeacetylase, Sirt1. Since it has been shown that NAD(+) levels in mitochondria remain at physiological levels following genotoxic stress and can maintain cell viability even when nuclear and cytoplasmic pools of NAD(+) are depleted (7), the NAD(+) biosynthetic enzyme Nampt plays a critical role in enhancing life span and protecting against oxidative cell damage. Therefore, measurement of iNampt located within different cell compartments may be able to give us some biological clue on the intracellular functions of Nampt.
nicotinamide phosphoribosyltransferase, iNAMPT, PBEF, Visfatin
Weak reactivity to mouse NAMPT (5%) and rat NAMPT (15%).
Cell Lysates : 0.1 - 10 µL (actual amount of sample loaded in well after dilution)
Calibration Range: 0.25 - 16 ng/ml
Limit of Detection
Intra-assay (Within-Run) C.V.
Inter-assay (Run-to-Run) C.V.
References to Summary
- The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells: J.R. Revollo, et al.; J. Biol. 279, 50754 (2004)
- Identification of a plasmid-encoded gene from Haemophilus ducreyi which confers NAD independence: P.R. Martin, et al.; J. 183, 1168 (2001)
- Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor: B. Samal, et al.; Mol. Cell. 14, 1431 (1994)
- Visfatin: a protein secreted by visceral fat that mimics the effects of insulin: A. Fukuhara, et al.; Science 307, 426 (2005)
- Nampt/PBEF/Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme: J.R. Revollo, et al.; Cell. 6, 363 (2007)
- Molecular characteristics of serum visfatin and differential detection by immunoassays: A. Körner, et al.; J. Clin. Endocrinol. 92, 4783 (2007)
- Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival: H. Yang, et al.; Cell 130, 1095 (2007)