Principle of the Assay
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Adiponectin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Adiponectin present is bound by the immobilized antibody. Following incubation unbound samples are removed during a wash step, and then a detection antibody specific for Adiponectin is added to the wells and binds to the combination of capture antibody- Adiponectin in sample. Following a wash to remove any unbound combination, and enzyme conjugate is added to the wells. Following incubation and wash steps a substrate is added. A coloured product is formed in proportion to the amount of Adiponectin present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450nm. A standard curve is prepared from seven Adiponectin standard dilutions and Adiponectin sample concentration determined.
Adiponectin, alternately named Adipocyte complement-related protein of 30 kDa (Acrp30), adipoQ, adipose most abundant gene transcript 1 (apM1), and gelatin-binding protein of 28 kDa (GBP28), is an adipocyte-specific, secreted protein with potential roles in glucose and lipid homeostasis. Circulating Adiponectin levels are high, accounting for approximately 0.01% of total plasma protein. Adiponectin contains a modular structure that includes an N-terminal collagen-like domain followed by a C-terminal globular domain with significant sequence and structural resemblance to the complement factor C1q. Although they share little sequence identity, similar three-dimensional structure and certain conserved amino acid residues suggest an evolutionary link between the C1q-like domain of Adiponectin and members of the TNF superfamily. Adiponectin assembles into different complexes including trimers (low molecular weight), hexamers (middle molecular weight), and higher order oligomeric structures (high molecular weight) that may affect biological activity. Adiponectin is induced during adipocyte differentiation and its secretion is stimulated by insulin. Two receptors for Adiponectin, termed AdipoR1 and AdipoR2, have been cloned. Although functionally distinct from G-protein-coupled receptors, the genes encode predicted proteins containing 7 transmembrane domains. AdipoR1 is highly expressed in skeletal muscle, while AdipoR2 is primarily found in hepatic tissues.
Injection of Adiponectin into non-obese diabetic mice leads to an insulin-independent decrease in glucose levels. This is likely due to insulin-sensitizing effects involving Adiponectin regulation of triglyceride metabolism. A truncated form of Adiponectin (gAdiponectin) containing only the C-terminal globular domain has been identified in the blood, and recombinant gAdiponectin has been shown to regulate weight reduction as well as free fatty acid oxidation in mouse muscle and liver. The full-length recombinant Adiponectin protein is apparently less potent at mediating these effects. The mechanism underlying the role of Adiponectin in lipid oxidation may involve the regulation of expression or activity of proteins associated with triglyceride metabolism including CD36, acyl CoA oxidase, AMPK, and PPARγ.
Although Adiponectin-regulation of glucose and lipid metabolism in humans is less clear, similar mechanisms may also be in place. A negative correlation between obesity and circulating Adiponectin has been well established, and Adiponectin levels increase concomitantly with weight loss. Decreased Adiponectin levels are associated with insulin resistance and hyperinsulinemia, and patients with type-2 diabetes are reported to exhibit decreased circulating Adiponectin. Thiazolidinediones, a class of insulin-sensitizing, anti-diabetic drugs, elevate Adiponectin in insulin-resistant patients. In addition, high Adiponectin levels are associated with a reduced risk of type-2 diabetes. Using magnetic resonance spectroscopy it has been demonstrated that intracellular lipid content in human muscle negatively correlates with Adiponectin levels, potentially due to Adiponectin-induced fatty acid oxidation.
Adiponectin may also play anti-atherogenic and anti-inflammatory roles. Adiponectin plasma levels are decreased in patients with coronary artery disease. Furthermore, neointimal thickening of damaged arteries is exacerbated in Adiponectin-deficient mice and is inhibited by exogenous Adiponectin. Adiponectin inhibits endothelial cell expression of adhesion molecules in vitro, suppressing the attachment of monocytes. In addition, Adiponectin negatively regulates myelomonocytic progenitor cell growth and TNF-α production in macrophages.
|SYNONYMS||ACDC, ACRP30, ADIPQTL1, ADPN, APM-1, APM1, GBP28|
|Kit Components||96 Wells Quantity/Size|
|Aluminium pouches with a Microwell Plate coated with monoclonal antibody to human Adiponectin (8﹡12)||1 plate|
|Human Adiponectin Standard lyophilized, 2000 pg/ml upon reconstitution||2 vials|
|Concentrated Biotin-Conjugate anti-human Adiponectin monoclonal antibody||2 vials|
|Streptavidin-HRP solution||2 vials|
|Standard /sample Diluent||4 bottles|
|Biotin-Conjugate antibody Diluent||1 bottle|
|Streptavidin-HRP Diluent||1 bottle|
|Wash Buffer Concentrate 20x (PBS with 1% Tween-20)||1 bottle|
|Substrate Solution||1 vial|
|Stop Solution||1 vial|
|Adhesive Films||4 pieces|
|Product data sheet||1 copy|
|Storage||Store at 2 - 8°C|
|REPEATABILITY||The coefficient of variation of both intra-assay and inter-assay were less than 10%.|
|SENSITIVITY||The minimum detectable dose was 15pg/mL.|
|ASSAY RANGE||62.5 - 4000 pg/mL|
|SPECIFICITY||This assay recognizes both natural and recombinant human Adiponectin. The factors listed below were prepared at 50ng/ml in Standard /sample Diluent and assayed for cross-reactivity and no significant cross-reactivity or interference was observed.|
Factors assayed for cross-reactivity
|Recombinant human||4-1BB, APRIL, BAFF/BLyS, CD27, CD30 Ligand, CD40 Ligand, Fas Ligand, GITR Ligand, LIGHT, OX40 Ligand, TNF-α, TNF-β, TRAIL, TRANCE, TWEAK, VEGI, LT-α1/β2, LT-α2/β1|
|Recombinant mouse||Adiponectin, CD27 Ligand, Cd30 Ligand, Fas Ligand, LT-α1/β2, LT-α2/β1, OX40 Ligand, TNF-α, TNF-α(truncated), TRANCE|
|Other proteins||Porcine TNF-α, Rat TNF-α|
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