Adiponectin HMW Mouse/Rat ELISA
Adiponectin is one of cytokines secreted from adipocyte (adipocytokines, adipokines), and controls lipid metabolism and insulin sensitivity. Adiponectin is an important substance showing anti-diabetic, anti-atherosclerotic, and antiinflammatory actions. In blood, adiponectin monomer molecules are associated to form trimer, hexamer or 12-18mers. The monomer form of adiponectin is found only in adipocytes, and homomultimer, i.e. trimer, hexamer and 12-18-mers formed from adiponectin monomer are found in plasma. Trimer (low molecular weight complexes/LMW) is formed by interaction of noncovalent bonds of triple helix area and by hydrophobic interaction between globular Cq1 domain, and hexamer (middle molecular weight complexes/MMW) or larger complexes (higher molecular weight/HMW) are formed through disulfide bonds between cysteines at 39 of LMW-complexes. Adiponectin is believed to influence on cell growth, angiogenesis, and tissue remodeling by isolating various growth factors through binding with distinct affinity. The affinity is dependent upon the type of complexes, LMW, MMW and HMW. The blood levels of HMW were reported to reflect BMI, sex, effect of body weight decrease, glucose tolerance, liver insulin sensitivity, metabolic syndrome or DM2 more than total adiponectin levels. This means that assay for HMW is expected to be more useful for analysis of metabolic syndrome and DM2. Shibayagi’s Mouse/Rat HMW Adiponectin ELISA KIT is specific to HMW.
INTENDED USE
Mouse/Rat HMW Adiponectin ELISA Kit is a sandwich ELISA system for quantitative measurement of mouse or rat High Molecular Weight Adiponectin. This is for research use only.
STORAGE AND EXPIRATION
When the complete kit is stored at 2-8°C, the kit is stable until the expiration date shown on the label on the box. Reagent, once opened, should be used as soon as possible to avoid losing its optimal assay performance caused by storage environment.
ASSAY PRINCIPLE
In Shibayagi’s Mouse/Rat HMW adiponectin ELISA Kit, standards or diluted samples are incubated in monoclonal anti-adiponectin antibody-coated wells to capture HMW adiponectin. After 2 hours incubation and washing, HRP (horse radish peroxidase)-conjugated anti-adiponectin antibody is added, and incubated for 90 minutes. After washing, HRP-complex remaining in wells is reacted with a chromogenic substrate (TMB) for 30 minutes, and reaction is stopped by addition of acidic solution, and absorbance of yellow product is measured spectrophotometrically at 450 nm. The absorbance is nearly proportional to HMW adiponectin concentration. The standard curve is prepared by plotting absorbance against standard HMW adiponectin concentrations. HMW adiponectin concentrations in unknown samples are determined using this standard curve.
PRECAUTIONS
- For professional use only, beginners are advised to use this kit under the guidance of experienced person.
- Wear gloves and laboratory coats when handling assay materials.
- Do not drink, eat or smoke in the areas where assays are carried out.
- In treating assay samples of animal origin, be careful for possible biohazards.
- This kit contains components of animal origin. These materials should be handled as potentially infectious.
- Be careful not to allow the reagent solutions of the kit to touch the skin, eyes and mucus membranes. Especially be careful for the reaction stopper because it is 1 M sulfuric acid. The reaction stopper and the substrate solution may cause skin/eyes irritation. In case of contact with these wash skin/eyes thoroughly with water and seek medical attention, when necessary.
- Avoid contact with the acidic Reaction stopper solution and Chromogenic substrate solution, which contains hydrogen peroxide and tetramethylbenzidine (TMB). Wear gloves and eye and clothing protection when handling these reagents.
- The materials must not be pipetted by mouth.
- Residual samples and used tips should be rinsed in 1% formalin, 2% glutal aldehyde, or more than 0.1% sodium hypochlorite solution for more than 1 hour, or be treated by an autoclave before disposal.
- Dispose consumable materials and unused contents in accordance with applicable regional/national regulatory requirements.
- Use clean laboratory glassware.
- In order to avoid dryness of wells, contamination of foreign substances and evaporation of dispensed reagents, never forget to cover the well plate with a plate seal supplied, during incubation.
ELISA can be easily affected by your laboratory environment. Room temperature should be at 20-25°C strictly. Avoid airstream velocity over 0.4 m/sec.
REAGENTS SUPPLIED
ASSAY PROCEDURE
Remove the cover sheet of the 96 well-plate after bringing up to room temperature.
- Wash the anti-adiponectin coated plate (A) by filling the well with washing buffer and discard 3 times(*②), then strike the plate upside-down onto several layers of paper towels to remove residual buffer in the wells.
- Pipette 50 µl of diluted samples to the designated sample wells.
- Pipette 50 µl of standard solution to the wells designated for standards.
- Shake the plate gently on a plate shaker (*③)
- Stick a plate seal (*④) on the plate and incubate for 2 hours at 20-25°C.
- Discard the reaction mixture and rinse wells as step (1).
- Pipette 50 µl of HRP-conjugated anti-adiponectin antibody solution to all wells, and shake as step (4).
- Stick a plate seal (*④) on the plate and incubate the plate for 90 minutes at 20-25°C.
- Discard the reaction mixture and rinse wells as step (1).
- Pipette 50 µl of chromogenic substrate solution to wells, and shake as step (4).
- Stick a plate seal (*④) on the plate and incubate the plate for 30 minutes at 20-25oC.
- Add 50 µl of the reaction stopper to all wells and shake as step (4).
- Measure the absorbance of each well at 450 nm (reference wavelength, 620*nm) using a plate reader within 30 minutes.
CALCULATIONS
- Prepare a standard curve using semi-logarithmic or two-way logarithmic section paper by plotting absorbance* (Y-axis) against HMW adiponectin concentration (ng/ml) on X-axis. Physiological or pathological situation of animals should be judged comprehensively taking other examination results into consideration.
- Using the standard curve, read the HMW adiponectin concentration of a sample at its absorbance*, and multiply the assay value by dilution factor if the sample has been diluted. Though the assay range is wide enough, in case the absorbance of some samples is higher than that of the highest standard, please repeat the assay after proper dilution of samples with the buffer solution. * We recommend the use of 3rd order regression curve for log-log plot, or 4 parameters method for log-normal plot in computer calculation.
- Assay results from this product and results from other commercially available mouse/rat (total) adiponectin ELISA kits cannot be compared because of the difference of the assay systems.
Assay range
The assay range of the kit is 3.13 ~ 200 ng/ml.
Specificity
The kit uses two monoclonal antibodies specific to mouse/rat HMW adiponectin.
No crossreaction at 1000 ng/ml: Mouse adiponectin (Trimer), Mouse adiponectin (Monomer), Mouse MCH, Mouse TNF-α Mouse INF-γ Mouse Insulin, Mouse Leptin, Rat adiponectin (Monomer), Rat TNF-α Rat INF- γ Rat Insulin, Rat Leptin
Precision of assay Within assay variation (2 samples, 5 replicates assay,) Mean CV was less than 5 %.
Reproducibility
Between assay variation (3 samples, 4 days, duplicate assay) Mean CV was less than 5 %
Recovery test
HMW Adiponectin was added in 3 concentrations to 2 serum samples and was assayed. The recoveries were 94.4 ~ 105%
Dilution test
2 serum samples were serially diluted by 3 steps.
The dilution curves showed excellent linearity. (R2= 0.999)
REFERENCE ASSAY DATA
These data should be considered as guidance only. Each laboratory should establish its own normal and pathological reference ranges for HMW Adiponectin levels independently.
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