Bone Sialoprotein (BSP) ELISA Assay

Additional Information

Scientific Background

Bone sialoprotein (BSP) is a phosphorylated glycoprotein that comprises 12 % of the total non-collagenous proteins in bone and is thought to be involved in bone mineralisation and remodelling. The polypeptide chain of BSP has a molecular weight of 35 kDa. Native BSP has an apparent molecular weight of 70–80 kDa due to complex post translational modifications involving glycosylation. BSP is synthesized mainly by skeletal-associated cell types, including fibroblasts, hypertrophic chondrocytes, osteoblasts, osteocytes, osteoclasts, as well as trophoblasts. BSP is overexpressed and underglycosylated in human primary breast and prostate cancers that metastasize to the bone, whereby BSP expression correlates with the development of microcalcifications. Underglycosylated BSP is also expressed by other tumours, such as lung cancer, thyroid cancer, multiple myeloma and neuroblastoma, that predominantly metastasise to bones.

Storage and Preparation of Reagents
• To run the assay more than once, ensure that reagents are stored at the conditions stated on the label. Prepare only the appropriate amount necessary for each run. The kit can be used up to 4 times within the expiry date stated on the label.
• Reagents with a volume less than 100µl should be centrifuged before use to avoid loss of volume.
• Preparation of the wash buffer: The wash buffer concentrate (WASHBUF) has to be diluted with ultra pure water 1:50 before use (20ml WASHBUF + 980ml ultra pure water), mix well. Crystals could occur due to high salt concentration in the concentrate. Before dilution, the crystals have to be redissolved at room temperature or in a water bath at 37°C. The WASHBUF is stable at 2–8°C until the expiry date stated on the label. Wash buffer (1:50 diluted WASHBUF) can be stored in a closed flask at 2–8°C for 1 month.
• The lyophilised standards (STD) and controls (CTRL) are stable at 2–8°C until the expiry date stated on the label. Before use, the STD and CTRL have to be reconstituted with 500µl of ultra pure water. Allow the vial content to dissolve for 10 minutes and mix thoroughly to ensure complete reconstitution. Standards and controls (reconstituted STD and CTRL) can be stored at -20°C for 3 weeks. Avoid repeated thawing and freezing.
• Preparation of the conjugate: Before use, the conjugate concentrate (CONJ) has to be diluted 1:101 in wash buffer (100µl CONJ + 10ml wash buffer). The CONJ is stable at 2–8°C until the expiry date stated on the label. Conjugate (1:101 diluted CONJ) is not stable and cannot be stored.
• All other test reagents are ready-to-use. Test reagents are stable until the expiry date (see label) when stored at 2–8°C.