Intended Use
The Human sRAGE ELISA is a sandwich enzyme immunoassay for the quantitative measurement of human sRAGE (soluble receptor for advanced glycation end products).
Contents of Kit
Biotin Labelled Antibody Conc. (100×), concentrated, 0.13 ml
Streptavidin-HRP Conjugate, ready to use, 13 ml
Master Standard, lyophilized, 1 vial
Quality Control HIGH, lyophilized, 2 vials
Quality Control LOW, lyophilized, 2 vials
Dilution Buffer, ready to use, 20 ml
Biotin-Ab Diluent, ready to use, 13 ml
Wash Solution Conc. (10×), concentrated, 100 ml
Substrate Solution, ready to use, 13 ml
Stop Solution, ready to use, 13 ml
Storage
Store the complete kit at 2-8°C. Under these conditions, the kit is stable until the expiration date (see label on the box).
Performance Characteristics
It is intended for research use only.
The total assay time is less than 3.5 hours.
The kit measures sRAGE in serum and plasma (EDTA, citrate, heparin).
Assay format is 96 wells.
Standard is recombinant protein based
Quality Controls are human serum based. No animal sera are used.
Components of the kit are provided ready to use, concentrated or lyophilized.
Detection Range
50-3200 pg/ml
Detection Limit
19.2 pg/ml
Sensitivity
Limit of detection (LOD) (defined as concentration of analyte giving absorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank: Ablank + 3×SDblank) is calculated from the real human sRAGE values in wells and is: 19.2 pg/ml.
Note: Dilution Buffer is pipetted into Blank wells.
General Description
The receptor for advanced glycation end products (RAGE) belongs to the immunoglobulin superfamily and consists of the 319 amino acids-long extracellular region, hydrophobic transmembrane domain and 43-amino acid, highly charged, cytoplasmic tail. The extracellular region of the receptor is composed from three parts: one Ig-like V-type domain and two Ig-like C-type domains. It was shown that the aminoterminal part of the molecule is responsible for binding of the ligands. On the other hand, the carboxyterminal part is responsible for mediating of the signalling actions.
RAGE expression decreases throughout development in several types of cells, including neurons, endothelium, pericytes, smooth muscle cells, mononuclear phagocytes, cardiac myocytes, hepatocytes, renal glomerular epithelial cells or podocytes and Muller and bipolar ganglion cells of the retina. Interestingly, the expression of the RAGE remains at high levels in the lung alveolar epithelial cells in adult tissues.
RAGE is connected to several diseases and pathological states (e.g., diabetic complication, vascular and inflammatory diseases). It was characterized as receptor for advanced glycation end products (AGEs), but this receptor can also bind other ligands such as proinflammatory S100 proteins/calgranulins, amphoterin or amyloid beta-peptide. AGEs are groups of the substances formed via nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids and nucleic acids. AGEs are formed in the body constantly throughout life and accumulate over time. It is natural that in diabetes, when glucose is more available, the formation of AGEs is increased. Their effect in the body can be divided into the crosslinking of the extracellular matrix proteins and thus altering the cellular structure and interaction of AGEs with their receptor on the cell surfaces and thus altering cellular function via signalling. The binding of various proinflammatory molecules to the cell-surface RAGE triggers an intracellular signalling pathway. The target is nuclear factor-κB (NF-κB) which promotes enhanced transcription of proinflammatory genes and also up-regulates the transcription of RAGE.
In addition to full-length RAGE, several truncated isoforms of this receptor have been found. First, there is a splice variant encoding molecule that is lacking N-terminal region and, second, is the variant missing the C-terminus. The RAGE splice variant lacking C-terminal part is missing the transmembrane region, and after secretion to the extracellular environment, it circulates in blood as endogenous secretory RAGE (esRAGE). esRAGE successfully binds to AGEs and, due to this ability, it can act as an antagonist by preventing the activation of cell-surface full-length RAGE. In sera, esRAGE as well as another soluble version of RAGE possibly created by proteolytical cleavage by matrix metalloproteases can be detected. This potential mechanism was initially described in mouse, but it could be present in humans, too. Because of the possible neutralization effect of soluble RAGE, studies have examined the significance of sRAGE serum concentration in patients with various pathological conditions. Decreased level of sRAGE is a biomarker for deficient and/or altered inflammatory control in humans. It was shown that reduced level of sRAGE is associated with higher risk of coronary disease. In Alzheimer disease there is a decrease in serum sRAGE in comparison with patients with vascular dementia and controls. In essential hypertensia, it has been shown that sRAGE concentration in serum was inversely associated with pulse pressure. On the other hand, an increased level of serum sRAGE was found in patients with end-stage renal disease and acute lung injury.
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