sRANKL ELISA KIT
INTENDED USE
sRANK ELISA Kit is a sandwich ELISA intended for the quantitative determination of mouse/rat sRANKL in serum and cell culture supernatant. It is for research use only.
SUMMARY AND EXPLANATION OF THE TEST
RANKL, receptor activator of nuclear factor (NF)-B ligand (also: osteoprotegerin ligand, OPGL), its cellular receptor, receptor activator of NF- kB (RANK), and the decoy receptor, osteoprotegerin (OPG) have been identified as the key molecular regulation system for bone remodelling. RANKL, a member of the tumor necrosis factor (TNF) family, is the main stimulatory factor for the formation of mature osteoclasts and is essential for their survival. Therefore, an increase in RANKL expression leads to bone resorption and bone loss. RANKL is produced by osteoblastic lineage cells and activated T lymphocytes. It activates its specific receptor RANK which is located on osteoclasts and dendritic cells.
The effects of RANKL are counteracted by OPG which is secreted by various tissues and acts as an endogenous soluble receptor antagonist.
Imbalances of the RANKL/OPG system have been related to the pathogenesis of Paget’s disease, benign and malignant bone tumors, postmenopausal osteoporosis, rheumatoid arthritis, bone metastases and hypercalcemia. It was shown in several studies that in animal models restoring of the RANKL/OPG balance (e.g. by administering OPG) reduces the severity of these disorders.
It has been shown that RANKL is produced as a membrane-bound protein on murine osteoblasts/stromal cells, and cleaved into a soluble form by a metalloprotease. Stimulators of the osteoclastogenesis such as IL-1beta, IL-6, IL-11, IL-17, and TNF-alpha, increase the expression of RANKL and decrease OPG expression in osteoblasts/stromal cells. Cytokines inhibiting the osteoclastogenesis such as IL-13, INF-gamma, and TGF-beta1, suppress the expression of RANKL and stimulated OPG expression.
PRINCIPLE OF THE TEST
sRANK ELISA Kit
sRANK ELISA Kitis an assay for the direct determination of sRANKLin serum, plasma and urine. In this assay two highly specific antibodiesagainst sRANKL are used. The capture antibody is attached to the wells ofthe microtiter plate, the detection antibody is labeled with biotin.
In a first incubation step the samples and the biotinylated antibody against sRANKL react with the coated capture antibody on the microtiter plate. A sandwich-type complex is formed consisting of the binding antibody on the plate, sRANKL and the biotinylated detection antibody. To remove all unspecific bound substances a washing step is carried out.
In a second step streptavidin – peroxidase is added which reacts with the detection antibody. After another washing step, the solid phase is incubated with the substrate, TMB. An acidic stopping solution is subsequently added. The blue color changes to yellow. The intensity of the yellow color is directly proportional to the concentration of sRANKL in the sample.
A dose - response curve of the absorbance units at 450 nm versus concentration is generated. sRANKL in the samples is determined directly from this calibration curve.
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