Biologically Active Leptin ELISA Kit

Sample

Serum and plasma

Species Reactivity

Human

Detection Method

sELISA

Intended Use

The Biologically Active Leptin ELISA Kit is intended to be used for quantitative measurement of functional, receptor binding Leptin levels in serum or plasma.

Contents of Kit

1. Microtiter plate: ready for use, coated with human Leptin receptor (rec.). Wells are separately breakable. (8x12) wells
2. Standards (A-H): lyophilized, (recombinant Leptin), concentrations are given on vial labels and on quality certificate. 8 x 1 mL
3. Control Serum 1 (KS1): lyophilized, (human serum), concentration is given on quality certificate. 1 x 500 μL
4. Control Serum 2 (KS2): lyophilized, (human serum), concentration is given on quality certificate. 1 x 500 μL
5. Antibody Conjugate (AK): ready for use, contains biotinylated rabbit anti-hLeptin antibody. 1 x 12 mL
6. Enzyme Conjugate (EK): contains Streptavidin-Peroxidase Conjugate. 1 x 12 mL
7. Dilution Buffer (VP): ready for use. Please shake before use! 1 x 60 mL
8. Washing Buffer (WP): 20-fold concentrated solution. 1 x 50 mL
9. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate, stabilised H₂O₂ Tetramethylbencidine. 1 x 12 mL
10. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
11. Sealing Tape: for covering the microtiter plate. 3

Storage

Store the kit at 2-8°C after receipt until its expiry date. The lyophilized reagents should be stored at –20 °C after reconstitution. Avoid repeated thawing and freezing.
The shelf life of the components after initial opening is warranted for 4 weeks, store the unused strips and microtiter wells airtight together with the desiccant at 2-8°C in the clip-lock bag, use in the frame provided. The reconstituted components standards A-H and Control Sera KS1 and KS2 must be stored at –20°C (max. 4 weeks). For further use, thaw quickly but gently (avoid temperature increase above room temperature and avoid excessive vortexing). Up to 3 of the freeze-thaw cycles did not influence the assay. The 1:20 diluted Washing Buffer WP is 4 weeks stable at 2-8°C

Precision

Intra-Assay Variance
Native human serum samples were measured repeatedly at various positions of the microtiter plate. Intra-Assay variability was on average < 5%.

Inter-Assay-Variability
Serum samples were measured in independent tests. The coefficient of variation was 9% on average.

Sensitivity

Sensitivity was assessed by measuring the blank and calculating the theoretical concentration of the blank + 2SD. The analytical sensitivity of the Biologically Active Leptin ELISA Kit is < 0.01 ng/mL. In 3 independent determinations values ranging from 0.0076 to 0.009 ng/mL were found.

General Description

Leptin a hormone of 146 amino acids shows a molecular weight of 16 kDa (P41159). It consists of four anti-parallel α-helices. Leptin is primarily produced by adipocytes and thus provides a signal to the energy state of the organism. By binding to the leptin receptor (P48357) it influences the activity of the JAK -STAT pathway and thereby regulates energy metabolism, especially the food intake. Leptin levels show a circadian variation (+/- 30%) and are dependent on BMI, pubertal status, and gender. In addition to the expression itself, the bioavailability of leptin, is regulated via a binding protein, as with many hormones with type I cytokine receptors. The binding protein is the extracellular domain of the Leptin receptor formed by proteolytic cleavage of the receptor be metalloproteases (ADAM 10/17).
In healthy persons food intake is reduced by increasing concentrations of leptin and by falling SOB-R quantities. In various pathological situations, this regulatory circuit is however interrupted: e.g. the leptin levels in the circulation of obese persons are increased, but resulting in no satiety. This phenomenon, known as leptin resistance, could have its cause in a reduction in the number of leptin receptors or in influencing the intracellular signal transduction by other parameters.
Lately it was shown that a naturally occurring transversion in the leptin gen (c.298G → T) results in an amino acid exchange of asparagine to tyrosin in position 100 (pD100Y). An in vitro cell culture model revealed that this mutated leptin was still secreted but unable to bind to the receptor and therewith unable to exert intracellular signaling. In vivo this mutation results in disturbance of food intake regulation and in consequence in extreme obesity. Under treatment with recombinant human leptin food intake behavior normalized and concomitantly body weight was reduced significantly.
Since the mutant leptin is recognized by the classical immunological leptin test systems, either a genetic test (sequencing of the ob gene), or a functional assay (binding to the receptor) is necessary for the diagnosis.
The Mediagnost bioLEP assay allows the measurement of leptin in human serum by binding it to the soluble receptor (leptin binding protein). Based on the measurement of receptor binding leptin the Mediagnost bioLEP assay provides additional information on the biological reactivity of the circulating leptin molecules.
Mutations resulting in less or no binding leptin can be detected by a significant reduction of the measured bioactive Leptin. Especially in connection with measurement of total Leptin by classical immunoassays (e.g. DEIA-NB24-17) the receptor-binding characteristics of leptin can easily be detected and quantified. This might help to evaluate mutation rates of the leptin gene by an inexpensive, fast method and accelerate obesity research by improved patient stratification in clinical studies.

Standard Curve

The exemplary data and the standard curve in Figure below cannot be used for the calculation of the test results. You have to establish a standard curve for each test you conduct.