Human Leptin (gene name OB) is a 16 kDa, 146 amino acid (aa) residue, non-glycosylated polypeptide that regulates adipose tissue mass and energy balance (1 - 6). Mature human Leptin shares 87% and 84% aa identity with mouse and rat Leptin, respectively(1,7). Human Leptin is active in both the mouse and rat systems (8, 9). Leptin is expressed almost exclusively by adipocytes and its production is influenced by hormones, cytokines and nutrients (5, 7, 10) and circulates in the plasma, crosses the blood-brain barrier, and is present in human breast milk (3 - 6, 11). The human Leptin receptor (designated ObR or LEPR) is a 150 kDa, 1144 aa residue, type I transmembrane glycoprotein of the IL-6 receptor family of Class I cytokine receptors (12, 13). The gene for ObR undergoes considerable splicing, forming variants a-d with cytoplasmic domains of variable length, plus the potentially soluble form ObRe (13, 14). The long form, ObRb (formerly OB RL), is expressed mainly in the hypothalamic arcuate nucleus and is essential for signal transduction (6, 15, 16). In a concentration-dependent manner, Leptin signaling can have diverse effects, causing neurons that express pro-opiomelanocortin (POMC) peptides to reduce food intake, and neurons that express neuropeptide Y and agouti-related protein (NpY and AgRP) to increase food intake (4, 6). Leptin is fundamentally a “starvation signal” that, when low, prompts increased appetite and decreased energy expenditure (4, 6, 10). Leptin deficiency influences the immune system, depressing Th1 responses and causing increased frequency of infections (4). Leptin also regulates puberty, blocking the onset of puberty, or of menses if Leptin deficiency exists due to excessive thinness, such as results from starvation, extreme exercise-induced weight loss, anorexia or cancer-induced cachexia (3, 4).

This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Leptin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Leptin present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for Leptin is added to the wells and binds to the combination of capture antibody- Leptin in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of Leptin present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven Leptin standard dilutions and Leptin sample concentration determined.

Figure 1:Schematic diagram of the assay

1. Aluminium pouches with a Microwell Plate coated with antibody to human Leptin (8x12)

2. 2 vials human Leptin Standard lyophilized, 2000 pg/ml upon reconstitution

3. 2 vials concentrated Biotin-Conjugate anti-human Leptin antibody

4. 2 vials Streptavidin-HRP solution

5. 4 bottle Standard /sample Diluent

6. 1 bottle Biotin-Conjugate antibody Diluent

7. 1 bottle Streptavidin-HRP Diluent

8. 1 bottle Wash Buffer Concentrate 20x (PBS with 1% Tween-20)

9. 1 vial Substrate Solution

10. 1 vial Stop Solution

11. 4 pieces Adhesive Films

12. package insert

NOTE: [96 Tests]

Unopened Kit：Store at 2 -8° C. Do not use past kit expiration date. opened/ReconstitutedReagents：Please refer to the datasheets for detail information.

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