Contents of Kit
1. Microtiter plate: ready for use, coated with mouse-anti-Leptin-antibody. Wells are separately breakable. (8x12) wells
2. Calibrators (CAL A-E): lyophilized, (recombinant human Leptin), concentrations are given on vial labels and on quality certificate. 5 x 750 μL
3. Control 1 (CTR1): lyophilized, (human serum), concentration is given on quality certificate in ng/mL. 1 x 500 μL
4. Control 2 (CTR2): lyophilized, (human serum), concentration is given on quality certificate in ng/mL. 1 x 500 μL
5. Antibody-HRP-Conjugate (DET): ready for use, contains mouse-anti-hLeptin-antibody biotinylated + streptavidin horseradish peroxidase conjugate. 1 x 12 mL
6. Dilution Buffer (DIL): ready for use. Please shake before use! 1 x 25 mL
7. Washing Buffer (WB): 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 (STP): ready for use, 0.2 M sulfuric acid. 1 x 12 mL
10. Sealing Tape: for covering the microtiter plate. 2
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 calibrators A-E and Controls CTR1 and CTR2 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
General Description
Leptin, the product of the ob gene, is a 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. 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 by 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, 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.
For clinical purposes, it is important to note that serum leptin levels show a moderate circadian variation with a peak during the night at about 2 a.m.. The leptin values at this time are about 30 to 100% higher than the levels measured in the morning or early afternoon. This variation together with the influence of food intake needs to be taken into account, when blood samples are collected. Under fairly standardized conditions, i.e. normal eating cycles and blood sampling in the morning or early afternoon, a single leptin measurement is informative.
For the appropriate interpretation of measured leptin levels, reference ranges are required. Because body fat mass is the major confounding variable, these ranges should be referred to measures of the percentage body fat such as body mass index (BMI) or percent body fat determined by, e.g., bioelectric impedance assessment (BIA). Leptin levels are higher in females than in males and an age dependence was shown in children and adolescents. Therefore, reference ranges referring to measures of body fat should be stratified according to gender and pubertal development.
Leptin levels are high in most obese patients suggesting the presence of leptin insensitivity. In a small percentage of patients, however, leptin levels have been found inappropriately low with respect to their fat mass. It remains for future studies to prove that these patients represent a new pathophysiologic entity: leptin deficiency. Since leptin has also been shown to be of great importance for reproductive functions, possible new pathophysiologic mechanisms may be discovered relating infertility to insufficient leptin production.
The discovery of leptin has released an avalanche of research activities seeking to understand the regulation and actions of this hormone. Most importantly, it has provided a key to better understand the physiology of body weight regulation and to unveil possible pathophysiologic mechanisms in both obesity and eating disorders. Further, it may provide new insights into certain causes of infertility.
This enzyme immunoassay kit is suited for measuring human leptin in serum or plasma, and conditioned adipocyte culture media for scientific and diagnostic purposes. Measuring leptin in anorectic or cachectic patients, young children or in specimen other than serum, such as urine, cerebrospinal fluid, and certain cell culture media, is also possible with this kit.
The comparison with BMI-related reference ranges may be useful to detect conditions of relative leptin deficiency as a possible cause of obesity or provide an indication for leptin resistance respectively.
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