Leptin (or obese, OB) is a circulating hormone that is expressed abundantly and specifically in the adipose tissue.1 Human ob protein is a 166-amino acid polypeptide with a putative signal sequence and is 84 and 83% homologous to the mouse and rat ob proteins, respectively.2 Northern blot analysis reveals that OB RNA is present at a high level in adipose tissue but at much lower levels in placenta and heart.3 Indeed, Leptin is first described as an adipocyte-derived signalling factor, which, after interaction with its receptors, induces a complex response including control of bodyweight and energy expenditure. Leptin seems in addition to its role in metabolic control to have important roles in reproduction and neuroendocrine signalling.4 Leptin is a powerful inhibitor of bone formation in vivo.5 Comparative mapping of mouse and human DNA indicates that the ob gene is located within a region of mouse chromosome 6 that is homologous to a portion of human chromosome 7q.3 The standard product used in this kit is recombinant human Leptin. Principle of the Assay: This Human Leptin ELISA Kit is a sandwich enzyme-linked immunosorbent assay for the in-vitro quantitative determination of leptin in human plasma, serum and cell culture supernatants. A mouse monoclonal antibody specific for human Leptin has been pre-coated onto the wells of the strip plate. Standards (recombinant human Leptin from expressed in E.coli, V22-C167) and test samples are added to the wells and biotinylated polyclonal goat detection antibody specific for human Leptin is subsequently added, followed by washing with PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with PBS or TBS buffer. TMB substrate is used to visualize the HRP-mediated enzymatic reaction. TMB, in a reaction catalyzed by HRP, produces a blue colored product that changes to yellow upon addition of an acidic stop solution. The intensity of the yellow color is proportional to the concentration of human Leptin in the sample. Assay Range: 62.5pg/ml-4000pg/ml Sensitivity: < 10 pg/ml Crossreactivity: There is no detectable cross-reactivity with other relevant proteins. Uniprot ID: A4D0Y8 Entrez Gene ID: 3952 Kit Components: 143948A: Microtiter Plate, 1x96 wells (12 x 8 strips). Pre-coated with anti-human Leptin antibody. 143948B: Leptin Standard, 2x10ng. Lyophilized recombinant human Leptin. 143948C: Biotinylated anti-Leptin Antibody, 1x130ul 143948D: ABC Complex (Avidin-Biotin-Peroxidase Complex), 1x130ul 143948E: Sample Diluent Buffer, 1x30ml 143948F: Antibody Diluent Buffer, 1x12ml 143948G: ABC Diluent Buffer, 1x12ml 143948H: TMB Substrate, 1x10ml 143948I: TMB Stop Solution, 1x10ml Storage and Stability: Store at 4C for 6 months, at -20C for 12 months. Avoid multiple freeze-thaw cycles. Summary of the Assay: 1. Add samples and standards and incubate the plate at 37C for 90 min. Do not wash. 2. Add biotinylated antibody and incubate the plate at 37C for 60 min. Wash plate 3 times with 0.01M TBS. 3. Add ABC working solution and incubate the plate at 37C for 30min. Wash plate 5 times with 0.01M TBS. 4. Add TMB color developing agent and incubate the plate at 37C in dark for 25-30 min. 5. Add TMB stop solution and read.
