Description
Introduction: Angiopoietin-2 (Ang-2) is one of the critical regulators of tumor angiogenesis. Human and mouse Ang-2 share 85% amino acid identity and Ang-2 is approximately 60% identical to Ang-1. Ang-2 contains an N-terminal coiled-coil domain and a C-terminal fibrinogen-like domain. Located at the N-terminus are stretches of hydrophobic residues typical of secretory signal sequences. Although likely playing different functional roles, both Ang-1 and Ang-2 are ligands for the endothelial cell receptor tyrosine kinase Tie-2. The regulation of Tie-2 activity by Ang-2 is complex, leading to inhibition in certain cell types or conditions, and activation in others. In vitro studies demonstrated that Ang-1, but not Ang-2, could stimulate Tie-2 tyrosine phosphorylation and suggested that Ang-2 could act as a competitive inhibitor of Ang-1 signaling. Consistent with its role as an Ang-1/Tie-2 inhibitor, transgenic overexpression of Ang-2 leads to disruption of embryonic blood vessel formation, a phenotype similar to that of Tie-2 knockouts. In contrast, altering incubation times or elevating Ang-2 concentrations, can under some circumstances, result in Tie-2 activation in HUVECs. It has also been proposed that Ang-2 may play a pro-angiogenic role by mediating destabilizing interactions between endothelial and perivascular cells, enhancing the effects of pro-angiogenic proteins including vascular endothelial growth factor (VEGF). Alone, Ang-2 promotes endothelial cell death and vessel regression, but can act in synergy with VEGF to promote new vessel formation. Ang-2 has been implicated in cancer development due to its role in angiogenesis. Ang-2-mediated destabilization of mature vessel structures accompanied by the presence of VEGF, may contribute to the angiogenesis associated with tumor tissues. Several studies have examined Ang-2 expression in an assortment of human cancers. Ang-2 is elevated in colon carcinoma, gastric carcinoma, glioblastoma, glioma, hepatocellular carcinoma, neuroblastoma and non-small cell lung cancer. Furthermore, Ang-2 levels appear to correlate with advancing tumor stage progression and poor prognosis.
Principle of the Assay: The microtiter plate provided in this kit has been pre-coated with an antibody specific to ANG-2. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody preparation specific for ANG-2 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3',5,5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain ANG-2, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm +/- 2 nm. The concentration of ANG-2 in the samples is then determined by comparing the O.D. of the samples to the standard curve