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Human Leptin ELISA KIT


product-insert-callout.png Human Leptin ELISA KIT

Leptin ELISA Assay Kit: 
For Research Use Only
Size:  1x96 wells
Sensitivity:  15 pg/mL
Dynamic Range:  31.25 - 1000 pg/ml
Incubation Time:  3.5 hours
Sample Type:  Serum, Plasma, Cell Culture   
Sample Size: 100 µL

Product Developed and Manufactured in the USA
Intended Use
The Eagle Biosciences Human Leptin ELISA Assay Kit (enzyme-linked immunoassay kit) is intended for the quantitative determination of human Leptin concentrations in cell culture supernates, serum, and plasma. The Eagle Biosciences Human Leptin ELISA Assay Kit is for research use only and not to be used in diagnostic procedures.

Assay Principle
The Eagle Biosciences Human Leptin ELISA Assay Kit employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Leptin Α 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 colored 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.

  1. Prepare all reagents and working standards as directed in the previous sections.                            
  2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
  3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
  4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
  5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
  6. Repeat the aspiration/wash.
  7. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C Avoid placing the plate in direct light.
  8. Repeat the aspiration/wash.
  9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
  10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
  11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable) 

Typical Standard Curve:


Assay Background
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) (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).

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7. Ogawa, Y. et al. (1995) J. Clin. Invest. 96:1647.
8.  Verploegen, S.A.B.W. et al. (1997) FEBS Lett. 405:237.
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15. Bacart, J. et al. (2010) FEBS Lett. 584:2213.
16. Tu, H. et al. (2007) J. Cell. Physiol.