Human Total Adiponectin/Acrp30 Quantikine ELISA Summary

Product Summary

The Quantikine Human Total Adiponectin Immunoassay is a 4.5 hour solid-phase ELISA designed to measure total (low, middle, and high molecular weight) human Adiponectin in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human Adiponectin and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human Adiponectin showed linear curves that were parallel to the standard curves obtained using the Quantikine kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring Adiponectin.

Preparation and Storage

Background: Adiponectin/Acrp30

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 (1-4). 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 (1, 5, 6). Although they share little sequence identity, similar threedimensional structure and certain conserved amino acid residues suggest an evolutionary link between the C1q-like domain of Adiponectin and members of the TNF superfamily (7). 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 (1, 7, 8). Adiponectin is induced during adipocyte differentiation and its secretion is stimulated by insulin (1, 9). Two receptors for Adiponectin, termed AdipoR1 and AdipoR2, have been cloned (10). 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 (11). This is likely due to insulin-sensitizing effects involving Adiponectin regulation of triglyceride metabolism (11). 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 (2, 12). The full-length recombinant Adiponectin protein is apparently less potent at mediating these effects (2, 12). 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 PPAR gamma (12-14). 

Although Adiponectin-regulation of glucose and lipid metabolism in humans is less clear, similar mechanisms may also be in place (15). A negative correlation between obesity and circulating Adiponectin has been well established (6, 16, 17), and Adiponectin levels increase concomitantly with weight loss (18). Decreased Adiponectin levels are associated with insulin resistance and hyperinsulinemia, and patients with type-2 diabetes are reported to exhibit decreased circulating Adiponectin (19, 20). Thiazolidinediones, a class of insulin-sensitizing, anti-diabetic drugs, elevate Adiponectin in insulin-resistant patients (21). In addition, high Adiponectin levels are associated with a reduced risk of type-2 diabetes (22). 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 (15). 

Adiponectin may also play anti-atherogenic and anti-inflammatory roles. Adiponectin plasma levels are decreased in patients with coronary artery disease (20). Furthermore, neointimal thickening of damaged arteries is exacerbated in Adiponectin-deficient mice and is inhibited by exogenous Adiponectin (23). Adiponectin inhibits endothelial cell expression of adhesion molecules in vitro, suppressing the attachment of monocytes (24). In addition, Adiponectin negatively regulates myelomonocytic progenitor cell growth and TNF-alpha production in macrophages (25, 26).

Assay Procedure

Refer to the product datasheet for the complete assay procedure.

Bring all reagents and samples to room temperature before use. It is recommended that all samples, standards, and controls be assayed in duplicate.

1.     Prepare all reagents, standard dilutions, and samples as directed in the product insert.

2.     Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3.     Add 50 μL of Assay Diluent to each well.

4.     Add 50 μL of Standard, control, or sample to each well. Cover with a plate sealer, and incubate at room temperature for 2 hours.

5.     Aspirate each well and wash, repeating the process 4 times for a total of 5 washes.

6.     Add 100 μL of Conjugate to each well. Cover with a new plate sealer, and incubate at room temperature for 2 hours.

7.     Aspirate and wash 5 times.

8.     Add 100 μL Substrate Solution to each well.

9.     Add 100 μL of Stop Solution to each well. Read at 450 nm within 30 minutes. Set wavelength correction to 540 nm or 570 nm.