TECO® Adiponectin High Sensitive ELISA. Adiponectin is a 30kDa protein and its percentage in serum proteins is 0.01%. In vivo, it appears with different oligomers and it is mainly synthesized by adipocytes. Until now, IGF-1 is the only known natural inductor of synthesis.
Adiponectin can be use in the research of insulin resistance and metabolic syndrome as well research in increased risk of type 2 diabetes mellitus and cardiovascular disease.
Today, Adiponectin is thought to act as an endogenic insulin sensitizer by decreasing excessive glucose levels without increasing insulin concentrations and by stimulating the burning of adipose tissue in muscle and liver.
Adiponectin is associated with glucose and lipid metabolism and is assumed to have direct antiatherogenic characteristics.
Research significance:
• Obesity
• Atherosclerosis
• Energy metabolism
• Coronary artery disease
• Metabolic syndrome
• Polycystic ovary syndrome (PCOS)
Reagents Supplied
| Symbol | Description | Format |
| 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 2 ng/ml – lyophilized |
1 x 0,75 ml |
| B | Standard B 10 ng/ml – lyophilized |
1 x 0,75 ml |
| C | Standard C 30 ng/ml – lyophilized |
1 x 0,75 ml |
| D | Standard D 70 ng/ml – lyophilized |
1 x 0,75 ml |
| E | Standard E 100 ng/ml – lyophilized |
1 x 0,75 ml |
| L | Control 1 ng/ml – lyophilized, Range as indicated on data sheet |
1 x 0,5 ml |
| H | Control 2 ng/ml – lyophilized, Range as indicated on data sheet |
1 x 0,5 ml |
| 2 | Dilution Buffer Ready for use |
1 x 125 ml |
| 3 | Antibody HRP-Conjugate Ready for use |
1 x 12 ml |
| 4 | TMB Substrate Ready for use |
1 x 12 ml |
| 4 | 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, adhesive | 2 pieces |
| I | Kit instruction | 1 x |
Materials required but not supplied
- Pipettes capable of dispensing 5 µl, 40 µl, 80 µL, 100 µl, 320 µl, 350 µl, 500 µl, 560 µl and 750 µl
- Graduated cylinders for reconstituting or diluting reagents
- Manual Aspiration System and multi-channel pipette or automatic washer
- Aqua dest
- Vortex mixer
- ELISA plate reader suitable for 96 well formats and capable of measuring at 450 and 405 nm (Reference: 590–650 nm)
- ELISA plate shaker (≥ 400 rpm) (orbital shaker)
- Software package for data generation and analysis
Assay Principle
The TECO® assay kit for Adiponectin is a so-called Sandwich-Assay using two specific and high affinity monoclonal antibodies. The Adiponectin in the samples binds to the first antibody coated on the microtiter plate. In the following step the second specific antiAdiponectin-antibody binds in turn to the immobilized Adiponectin. 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 Adiponectin-level of the samples.
Assay Principle
All determinations (standards, diluted serum controls and diluted samples) should be assayed in duplicate. When performing the assay, the standards, control sera and 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 all reagents to stand at room temperature (20–25 °C) for at least 30 minutes. After reconstitution, keep the reagents at room temperature for 15 minutes and then mix gently before use.
- Prepare the frame and the required number of coated strips 1 .
- Allocate the wells of the microtiter plate for standards, controls and samples.Dilute samples (see kit instructions page 11) of and controls (1:300).
- Pipette 100 µl dilution buffer 2 in duplicate into wells (blank).
- Pipette 100 µl of each standards ( A till E ), diluted Control sera ( L and H ) and samples (see dilution protocol ) into the corresponding wells.
- Cover the wells with sealing tape and incubate the plate for 1 hour at room temperature (20–25 °C) on a plate shaker (≥400–500 rpm).
- After incubation, aspirate the wells by using a plate washer or manually decant by inverting the plate. Wash the wells 3x with 350 ml 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.
- Pipette 100 µl of the antibody HRP conjugate 3 in each well.
- Cover the wells with sealing tape and incubate the plate for 30 minutes at room temperature (20–25 °C) on a plate shaker (≥400–500 rpm).
- Wash the wells 3 times with washing buffer as described in step 6.
- Pipette 100 µl of the TMB substrate solution 4 in each well.
- Incubate the plate for 15 minutes, in the dark, at room temperature (20–25 °C).
- Add 100 µl of stop solution E in each well.
- 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 native Adiponectin
Sensitivity
< 0.6 ng/ml
Sample volume
5 µl (dilute >1:300 serum and plasma). For other biological fluids see protocol.
Sample type
Serum, heparin plasma, breast milk, urine, saliva, CSF, cell culture
Sample preparation
- Blood collection – fasting is recommended.
- Samples are stable for maximum 2 days at room temperature. Long-term storage stable for maximum 2 years at -20 °C.
- Max. 5 freeze and thaw cycles.
Incubation time
2 hours
Species
Human
| mg/l | |
| Men Adult | 8-10 |
| Female Adult | 10-12 |
| cut-off | 10 |
Comprehensive reference data related to age and gender are available for this test.
Adiponectin is a 30kDa protein, presenting 0,01 % of serum proteins. It is mainly synthesized by adipocytes, but muscle cells and hepatocytes have also the ability to synthesize Adiponectin. Until now, IGF-I is the only known natural inductor of the synthesis. It consists of a Collagenlike N-terminal and a globular C-terminal domain. In vivo Adiponectin appears with different oligomers. Beside the trimer and ditrimer, high molecular multimers also exist. Up to now two different receptors are known, both receptors are ubiquitary expressed, though the distribution in the tissues varies. The Adiponectin Receptor 1 (AdipoR1) is synthesized especially in muscle- and AdipoR2 in liver tissue.
A special significance for high molecular multimers, as described repeatedly, could not be proven in a comparative study of three test systems.
