TECO® Leptin. Leptin, the product of the ob gene, is a recently discovered proteohormone. It is almost exclusively produced by differentiated adipocytes and is thought to play a key role in the regulation of body weight. Leptin has an influence on the central nervous system, mainly on the hypothalamus, by suppressing food ingestion and increasing energy consumption.

Leptin can be used in the research of reproduction and a number of metabolic and endocrine axes.


Due to its pleiotropic effects, leptin is a valuable research tool with regard to:

• Metabolic syndrome studies
• Adiposity research
• Cachexia and other metabolic studies
• Eating disorder research

Reagents Supplied

SymbolDescriptionFormat
 1 Antibody Coated Wells
12 break apart strips of 8 wells (12 x 8 in total), in a frame, Ready to use
1 plate
 A Standard A
1 ng/ml – lyophilized
1 x 0.75 ml
 B Standard B
10 ng/ml – lyophilized
1 x 0.75 ml
 C Standard C 
25 ng/ml – lyophilized
1 x 0.75 ml
 D Standard D 
50 ng/ml – lyophilized
1 x 0.75 ml
 E Standard E
100 ng/ml – lyophilized
1 x 0.75 ml
 L Control 1
lyophilized, Range as indicated on data sheet
1 x 0.5 ml
 H Control 2
lyophilized, Range as indicated on data sheet
1 x 0.5 ml
 2 Dilution Buffer
Ready for use
1 x 25 ml
 3 Antibody-HRP-Conjugate
Ready for use
1 x 12 ml
 4 TMB Substrate
Ready for use
1 x 12 ml
 5 Wash Buffer
20 times concentrated
1 x 50 ml
 6 Stop Solution – 0.2 M H2S04
0.2 M sulfuric acid, ready for use
1 x 12 ml
 7 Cover for Microtiterplate, adhesive2 pieces
 I Kit instruction1 x

Materials Required and not Supplied

  • Pipettes capable of dispensing 20 µl, 100 µl, 350 µl, 500 µl and 750 µl
  • Graduated cylinders for reconstituting or diluting reagents
  • Manual aspiration system and multi-channel pipette or automatic washer for ELISA plates
  • Aqua dest
  • Vortex mixer
  • ELISA plate reader suitable for 96 well formats and capable of measuring at 450 nm (Reference: 590–650 nm).
  • ELISA plate shaker (≥350 rpm) (orbital shaker)
  • Software package for data generation and analysis

Assay Principle

The TECO® assay kit for human leptin is a so-called sandwich-assay using two specific and high affinity monoclonal antibodies. The leptin in the samples binds to the first antibody coated on the microtiter plate. In the following step the second anti-Leptin-Antibody binds in turn to the immobilized leptin. The second antibody is biotinylated and will be incubated in a mixture with a streptavidin-peroxidase-enzyme conjugate. In the closing substrate reaction the turn of the color will be catalyzed quantitatively depending on the leptin-level of the samples.


Assay Procedure

All determinations (standards, control sera and samples) should be assayed in duplicate. When performing the assay, the standards, control sera and the samples should be pipetted as fast as possible (<15 minutes).

To avoid distortions due to differences in incubation times, HRP conjugate, substrate solution and stop solution should be added to the plate in the same order and with the same time interval as the samples.

Allow a all reagents to stand at room temperature (20–25 °C) for at least 30 minutes.

  1. Prepare the frame and the required number of coated strips  .
  2. Allocate the wells of the microtiter plate for standards, controls and samples.
  3. Pipette 100 µl dilution buffer  2  into all wells.
  4. Add 20 µl dilution buffer in duplicate (Blank).
  5. Pipette 20 µl of each standard ( A  till  E ), control sera ( L  and  H ) and samples into the corresponding wells.
  6. Cover the wells with sealing tape and incubate the plate for 1 hour at room temperature on a plate shaker (≥350 rpm).
  7. After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3 x with 350 µl diluted washing buffer (15 seconds incubation per cycle). After the last wash cycle tap the inverted wells gently on a dry absorbent surface to remove excess wash solution.
  8. Pipette 100 µl of the antibody HRP conjugate in each well.
  9. Cover the wells with sealing tape and incubate the plate for 30 minutes at room temperature on a plate shaker (≥350 rpm).
  10. After incubation wash the wells 3 times with washing buffer as described in step 7.
  11. Pipette 100 µl of the TMB substrate solution  4  in each well.
  12. Incubate the plate for 15 minutes, in the dark, at room temperature (20–25 °C).
  13. Add 100 µl of stop solution  6  in each well.
  14. Measure the color reaction within 30 minutes at 450 nm (reference filter between 590–650 nm). With strong color reaction e. g. >3 OD, also measure at 405 nm.

Protocols for the different automatic ELISA systems are available.

Range

1 – 100 ng/ml, recombinant leptin WHO NIBSC 97/594


Sensitivity

LLOD <0.25 ng/ml
LLOQ 1 ng/ml

Incubation time

2 hours


Sample volume

20 µl


Sample type

Serum, heparin and EDTA plasma, urine, saliva, cell culture.


Sample preparation

  • Normal food intake rhythm provided, samples should be collected till 2 p.m. Leptin shows a moderate circadian variation with a peak at 2 a.m., the leptin values at that time are about 30 to 100 % higher.
  • This variation together with the influence of food intake needs to be taken into account when blood samples are collected. Whole blood should be refrigerated as soon as possible following collection.
  • Samples are stable for maximal 2 days at room temperature.
  • Long-term storage stable for maximal 2 years at -20 °C.
  • Max. 5 freeze and thaw cycles.

Species

Human

Leptin levels depend on age and gender and must be referred to the percentage body fat (such as BMI).

Comprehensive clinical reference data are available for this test.

Leptin ELISA Measurement Assay Test KitLeptin, the product of the ob gene is a recently discovered (1994) 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, 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 research within metabolic and endocrine feedback loops.