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EK000027-EK0482: Human OPN ELISA Kit
Range 156pg/ml-10,000pg/ml
Sensitivity < 10 pg/ml
Specificity: no detectable cross-reactivity with any other cytokine.
Application: for quantitive detection of human OPN in sera, plasma, body fluids, tissue lysates or cell culture supernates.
Expiration: four months at 4°C and eight months at -20°C.
Background
Osteopontin (OPN) also called urinary stone protein, secreted phosphoprotein 1 (SPP1), bone sialoprotein, and early T lymphocyte activation 1 (ETA1). Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extrOPNllular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extrOPNllular matrix, allowing them to perform their respective functions during osteogenesis.1 Osteopontin is presumably involved in stone formation as stone matrix.2 The deduced protein sequence reveals a 317-amino acid protein (34,982 Da) containing a 16-amino acid hydrophobic signal sequence and a 33,352-Da protein destines to undergo extensive post-translational modifications before being secreted from the cell. The gene is located on human chromosome 4.3 The standard product used in this kit is recombinant human OPN, consisting of 17-314 amino acid sequence with the molecular mass of 32.9KDa. As a result of glycosylation, the molecular mass is 60-65KDa.
Principle
The ELISA Kit is based on standard sandwich enzyme-linked immune-sorbent assay technology. The target specific antibodies are precoated onto 96-well plates. The target from the sample is bound to the microwell. The biotinylated target specific detection antibodies are added to the microwells and followed by washing with the PBS or TBS buffer. Avidin-Biotin-Peroxidase Complex is added and unbound conjugates are washed away with the PBS or TBS buffer. TMB, the HRP (horseradish peroxidase) substrate, is used to visualize color change resulting from the HRP enzymatic reaction. TMB is catalyzed by HRP to produce the blue color. The color changes into yellow after the acidic stop solution is added. The density of the yellow color is proportional to the target amount from the sample captured in the microwells.
Reference
1. Beck, G. R., Jr.; Zerler, B.; Moran, E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc. Nat. Acad. Sci. 97: 8352-8357, 2000.
2. Kohri, K.; Suzuki, Y.; Yoshida, K.; Yamamoto, K.; Amasaki, N.; Yamate, T.; Umekawa, T.; Iguchi, M.; Sinohara, H.; Kurita, T. Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin. Biochem. Biophys. Res. Commun. 184: 859-864, 1992.
3. Fisher, L. W.; McBride, O. W.; Termine, J. D.; Young, M. F. Human bone sialoprotein: deduced protein sequence and chromosomal localization. J. Biol. Chem. 265: 2347-2351, 1990.
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