Human RAGE Quantikine ELISA Kit Summary

Product Summary

The Quantikine Human RAGE Immunoassay is a 4.5 hour solid-phase ELISA designed to measure human RAGE (extracellular domain) in cell culture supernates, serum, and plasma. It contains NS0-expressed recombinant human RAGE/Fc Chimera and has been shown to accurately quantitate the recombinant factor. Results obtained using natural human RAGE 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 RAGE.

Preparation and Storage

Background: RAGE/AGER

RAGE (Receptor for Advanced Glycation End product) is a transmembrane glycoprotein that binds advanced glycation end products (AGEs), beta-amyloid peptides, HMGB1/Amphoterin, and several S100 family proteins. AGEs are adducts formed by the non-enzymatic glycation and oxidation of proteins and lipids. A soluble form can also be generated by MMP-mediated shedding. RAGE is expressed in the CNS during development as well as in adult endothelial cells, smooth muscle cells, pericytes, monocytes, and neurons. It is locally upregulated in vascular inflammation (e.g. diabetes, atherosclerosis, vascular injury, Alzheimer’s disease). At these sites, RAGE binding to S100A1, EN-RAGE/S100A12, or S100B induces inflammatory immune cell adhesion and infiltration as well as vascular smooth muscle proliferation, neointimal expansion, atherosclerotic plaque development, and transport of A-beta into the cerebrospinal fluid. In cancer, RAGE binding to HMGB1, S100A8, or S100A9 promotes tumor growth and metastasis in addition to inflammatory cell infiltration.

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.