Mouse/Rat Leptin ELISA Kit

Sample

Serum and plasma

Species Reactivity

Mouse and rat

Detection Method

sELISA

Intended Use

This enzyme immunoassay kit is suited for measuring Leptin in mouse and rat serum for scientific purposes.

Contents of Kit

1. Microtiter plate: ready for use, coated with goat anti-mouse/rat Leptin-antibody. Wells are separately breakable. (8x12) wells
2. Standards (A-G): lyophilized, (recombinant mouse Leptin), concentrations are given on vial labels and on quality certificate. 7 x 1 mL
3. Control Serum (KS): lyophilized, (mouse serum), concentration is given on quality certificate. 1 x 200 μL
4. Antibody Conjugate (AK): 100-fold concentrated, contains goat biotinylated anti-mouse/rat leptin antibody. 1 x 120 μL
5. Enzyme Conjugate (EK): 100-fold concentrated, contains HRP (Horseradish-Peroxidase)-labelled. 1 x 120 μL
6. Dilution Buffer (VP): ready for use. Please shake before use! 1 x 120 mL
7. Washing Buffer (WP): 20-fold concentrated solution. 1 x 50 mL
8. Substrate (S): ready for use, horseradish-peroxidase-(HRP) substrate, stabilised Tetramethylbencidine. 1 x 12 mL
9. Stopping Solution (SL): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
10. 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. Reconstituted components must be stored at –20°C (or colder). For further use, thaw quickly but gently (avoid temperature increase above room temperature and avoid excessive vortexing). Freezing is only possible once! The 1:20 diluted Washing Buffer WP is 4 weeks stable at 2-8°C.

Precision

Inter-assay and intra-assay variation coefficients were found to be < 10 %.

Sensitivity

The practical sensitivity of the assay is 10 pg/ml, i.e., 1 pg/well (calculated by extrapolation of the standard curve).

General Description

Leptin, the product of the ob gene, is a recently discovered single-chain proteohormone with a molecular weight of 16 kD which is thought to play a key role in the regulation of body weight. Its amino acid sequence exhibits no major homologies with other proteins. Leptin is almost exclusively produced by differentiated adipocytes. It acts on the central nervous system, in particular the hypothalamus, thereby suppressing food intake and stimulating energy expenditure. Leptin receptors - alternatively spliced forms exist that differ in length - belong to the cytokine class I receptor family. They are found ubiquitously in the body indicating a general role of leptin which is currently not fully understood. A circulating form of the leptin receptor exists which acts as one of several leptin binding proteins.
Besides its metabolic effects, leptin was shown to have a strong influence on a number of endocrine axes. In male mice, it blunted the starvation-induced marked decline of LH, testosterone, thyroxine and the increase of ACTH and corticosterone. In female mice, leptin prevented the starvation-induced delay in ovulation. Ob/ob mice, which are leptin deficient due to an ob gene mutation, are infertile. This defect could be corrected by administration of leptin, but not through weight loss due to fasting, suggesting that leptin is pivotal for reproductive functions.
All these actions may, at least in part, be explained by the suppressive effect of leptin on neuropeptide Y (NPY) expression and secretion by neurons in the arcuate nucleus. NPY is a strong stimulator of appetite and is known to be involved in the regulation of various pituitary hormones, e.g. suppression of GH through stimulation of somatostatin, suppression of gonadotropins or stimulation of the pituitary-adrenal axis.
The most important variable that determines circulating leptin levels is body fat mass. Obviously, under conditions of regular eating cycles, leptin reflects the proportion of adipose tissue showing an exponential relationship. This constitutive synthesis of leptin is modulated by a number of non-hormonal and hormonal variables. Stimulators in both rodents and humans are overfeeding, high fat diets, insulin and glucocorticoids. Suppression has been shown for fasting, cAMP and beta--3-adrenoceptor agonists. From these findings it becomes clear that leptin is an integral component of various metabolic and endocrine feedback loops.

Standard Curve

The following data is for demonstration only and cannot be used in place of data generation at the time of assay.

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