Adiponectin, Mouse BioAssay™ ELISA Kit
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This ELISA is a sandwich enzyme immunoassay for the quantitative measurement of mouse Adiponectin/Acrp30 protein in serum. It is intended for in vitro research use only.
• The total assay time is less than 3 hours.
• The kit measures serum Adiponectin / Acrp30.
• Quality Controls are mouse serum based.
• Components in the kit are provided ready-to-use, concentrated, and lyophilized.
Adiponectin, also referred to as Acrp30, AdipoQ and GBP-28, is a recently discovered 244 aminoacid protein, the product of the apM1 gene, which is physiologically active and specifically and highly expressed in adipose cells (adipokine). The protein belongs to the soluble defence collagen superfamily; it has a collagen-like domain structurally homologous with collagen VIII and X and complement factor C1q-like globular domain. Adiponectin forms homotrimers, which are the building blocks for higher order complexes found circulating in serum. Adiponectin receptors AdipoR1 and AdipoR2 have been recently cloned; AdipoR1 is abundantly expressed in skeletal muscle, whereas AdipoR2 is predominantly expressed in the liver.
Paradoxically, adipose tissue-expressed adiponectin levels are inversely related to the degree of adiposity. A reduction in adiponectin serum levels is accompanied by insulin resistance states, such as obesity and type 2 diabetes mellitus. It is also reported in patients with coronary artery disease. Increased adiponectin levels are associated with type 1 diabetes mellitus, anorexia nervosa and chronic renal failure. Adiponectin concentrations correlate negatively with glucose, insulin, triglyceride concentrations and body mass index and positively with high-density lipoprotein-cholesterol levels and insulin-stimulated glucose disposal.
Adiponectin has been shown to increase insulin sensitivity and decrease plasma glucose by increasing tissue fat oxidation. It inhibits the inflammatory processes of atherosclerosis suppressing the expression of adhesion and cytokine molecules in vascular endothelial cells and macrophages, respectively. This adipokine assumes the role of a scaffold of newly formed collagen in myocardial remodeling after ischaemic injury and also stimulates angiogenesis by promoting cross-talk between AMP-activated protein kinase and Akt signalling in endothelial cells.
The surface of the wells in a microtitration plate is coated with monoclonal anti-mouse Adiponectin specific antibody. The standards, quality controls (QC), and diluted samples are pipetted into the wells. Any mouse Adiponectin/Acrp30 present is captured by immobilized antibody and unbound protein is washed away after the first incubation period. Then, a horseradish peroxidase (HRP) conjugated polyclonal anti-mouse Adiponectin antibody is added to the wells and incubated. Following another washing step, to remove unbound antibody-HRP conjugate, a substrate solution (H2O2 and TMB) is added to the wells. The enzymatic reaction yields a blue product that turns yellow when acidic stop solution is added. The intensity of the color, measured spectrophotochemically at 450nm, is directly proportional to the amount of mouse Adiponectin bound in the initial step. The concentrations of the diluted test samples are then read off the standard curve that is constructed by plotting the absorbance values against each respective mouse Adiponectin standard level using a four-parameter function. The dilution factor needs to be taken into consideration when calculating the actual concentration of Adiponectin/Acrp30 analyte in the test samples.
1. Antibody Coated Microtiter Strips, 96 wells, sealed
2. Conjugate Solution, 13ml
3. Mouse Adiponectin Master Calibrator, 8ng, 2 vial
4. Quality Controls: High and Low, 2 vials each. Refer to the Certificate of Analysis for the actual Quality Control values.
5. Dilution Buffer Concentrate (10x), 22ml
6. Wash Solution Concentrate (5x), 100ml
7. Substrate Solution (TMB), 13ml
8. Stop Solution, H2SO4, 13ml
Storage and Stability:
Store components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
Reagents Required but Not Supplied:
1. Test tubes for diluting samples
2. Precision pipettes to deliver 10-1000ul and disposable tips
3. Multichannel pipette 50-200ul
4. Microplate reader with 450 ± 10nm filter
5. Orbital microplate shaker capable of agitation at approximately 300rpm
6. Software package facilitating data generation and analysis
7. Microplate washer (optional). Manual washing is possible but not preferable.
8. Glassware (graduated cylinder and bottle for Wash Solution)
9. Distilled water
Limits of Assay:
Samples exceeding an Adiponectin level of 8ng/ml should be measured at higher dilution (e.g. 1:20 000) and the new dilution factor needs to be taken into consideration (20 000 in this case).
The sensitivity of the mouse Adiponectin ELISA, defined by the 99% confidence interval, is 0.104ng/ml. Sensitivity or limit of detection (LOD) is determined as a mean absorbance value plus three standard deviations of thirty Blank* readings (ABlank + 3x SDBlank). Then the corresponding concentration is calculated.
*The dilution buffer is pipetted into Blank wells
The antibodies in this kit are highly specific for mouse Adiponectin protein. No cross-reactivity with sera of other animal species (rat, hamster, rabbit, sheep, goat, cattle, swine, horse) has been observed. The assay recognizes natural and recombinant mouse Adiponectin. No cross-reactivity has been observed for mouse cytokines: RELM- , RELM- , Leptin, Leptin-receptor, Resistin; as well as for rat Leptin and human Adiponectin at 100ng/ml.
very little cross-reactivity (0.3%) has been measured for rat recombinant adiponectin at 100ng/ml.
Important Note: This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.