Nori Human Osteoprotegerin (OPG) ELISA Kit Summary

Alternative names for osteoprotegerin: OPG, osteoclastogenesis inhibitory factor, OCIF, tumor necrosis factor receptor superfamily member 11B, TNFRSF11B,

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Osteoprotegerin (OPG), also known as osteoclastogenesis inhibitory factor (OCIF), or tumor necrosis factor receptor superfamily member 11B (TNFRSF11B), is a protein that is encoded by the TNFRSF11B gene.^[1] Osteoprotegerin is a decoy receptor for the receptor activator of nuclear factor kappa B ligand (RANKL). By binding RANKL, OPG prevents RANK-mediated nuclear kappa B (NF-κB) activation which is a central and rapid acting transcription factor for immune-related genes, and a key regulator of inflammation, innate immunity, and cell survival and differentiation.^[2] Osteoprotegerin levels are influenced by voltage-dependent calcium channels Cav1.2. OPG can reduce the production of osteoclasts by inhibiting the differentiation of osteoclast precursors into osteoclasts and also regulates the resorption of osteoclasts in vitro and in vivo. OPG binding to RANKL on osteoblast/stromal cells, blocks the RANKL-RANK interaction between osteoblast/stromal cells and osteoclast precursors. This has the effect of inhibiting the differentiation of the osteoclast precursor into a mature osteoclast. Elevated OPG levels has been reported in heart diseases,^[3] in placebo effect serum responses in IBS patients and in severe mental disorders.^[4]Mutations in TNFRSF11B are associated with osteoarthritis.^[5] OPG is expressed on mesenchymal stem cells, which mediates their suppressive effect on osteoclastogenesis.^[6] In recent years, it has been hypothesized that osteoprotegerin may be a link between bone and cardiovascular disease.^[7] It has been particularly related to the increase in cardiovascular risk in patients suffering from diabetes ^[8]

References

1. Simonet WS, et al. (1997). Cell 89 (2): 309–19.
2. Krakauer T (2008). Int. Rev. Immunol. 27 (5): 286–92.
3. Venuraju SM, et al. (2010). J. Am. Coll. Cardiol. 55 (19): 2049–61.
4. Hope S, et al. (2010). J Psychiatry Neurosci 35 (5): 304–10.
5. Ramos, Y. F (2014). Annals of the Rheumatic Diseases. doi:1136.
6. Takano T (2014). Lab Invest. 94 (3): 288–298.
7. Blázquez-Medela, Ana M (2012). European J Clinical investigation 42 (5): 548–56.
8. Blazquez-Medela, et al. (2012). European J Clinical Investigation 42 (5): 548–56.