Core Laboratory
7. Human Leptin ELISA
Principles of Assay:
This assay is a direct sandwich ELISA based, sequentially, on: 1- capture of human leptin molecules from samples to the wells of a microtiter plate coated by pre-titered amount of polyclonal rabbit anti-human leptin antibodies, 2- wash away of unbound materials from samples, 3- binding of a biotinylated monoclonal antibody to the captured human leptin, 4- conjugation of alkaline phophatase to biotinylated antibodies, 5- wash away of free antibody-enzyme conjugates, 6- quantification of immobilized antibody-enzyme conjugates by monitoring alkaline phophatase activities in the presence of the substrate p-nitrophenyl phophate. The enzyme activity is measured spectrophotometrically by the increased absorbency at 405 nm due to production of the yellow colored product p-nitrophenol. Since the increase in absorbency is directly proportional to the amount of captured human leptin in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of human leptin.
Sample Collection and Storage:
Serum or plasma can be used. For serum, let blood clot at room temperature for 30 minutes, spin at 2000 to 3000 x.g. for 15 minutes at 4+2oC then transfer and store samples at –20oC for later use. Avoid multiple freeze/thaw cycles. For plasma samples use EDTA tubes and centrifuge immediately, transfer and freeze samples.
Expected Ranges:
Normal fasting range: Leptin levels are directly correlated with degree of adiposity. Mean serum leptin levels from normal lean individuals (BMI ranges 18-25):
Lean Men 3.8 + 1.8 mg/L
Lean Women 7.4 + 3.7 mg/L
Levels rise approximately 2.5 times faster in women per unit BMI as compared to men.
Sensitivity:
The lowest detection range for this assay is 0.5 ng/ml (50 ml sample size).
Cross Reactivity:
There has been no cross reaction detected between Human Leptin and Chicken/Mouse/Ovine/Porcine or Rat leptin. Also no cross reaction found between this assay and Human/Bocine or Porcine Proinsulin, or Human insulin, or Human IGF-I/IGF-II or Glucagon.
Precision:
This information provided by the kit company: %CV within assay from 1.09 to 4.98 , and between the assays reported as 3.89 to 5.33 .
For more information, please contact the Core Laboratory manager or director.