Osteocalcin, a major non-collagenous bone protein, is synthesized by osteoblasts during bone formation. Most of the osteocalcin is incorporated into the bone matrix and a small amount is secreted into the circulation. Studies have shown that serum osteocalcin levels are directly related to osteoblastic activity and bone formation.
The BTI Mouse Osteocalcin EIA kit is a sandwich ELISA assay. Two mouse osteocalcin antibodies are employed, each directed toward an end (C or N terminal) of the osteocalcin molecule. One mouse osteocalcin antibody is bound to the wells which binds the mouse osteocalcin standard or sample, the other biotinylated antibody completes the sandwich.
This sandwich ELISA kit is specific for intact mouse osteocalcin only. Both carboxylated and decarboxylated mouse osteocalcin are recognized. Mouse Osteocalcin can be measured at a 1:10 to 1:20 dilution from serum or heparinized plasma. Bone extracts and serum-free cell culture supernates can be measured directly. This kit shows good correlation (r=0.90) with the BTI mouse osteocalcin RIA.
The protocol has an overnight incubation and yields a
1-50ng/ml range. As little as 5-10 microliters of mouse serum is required for duplicate determinations. Each kit contains all the necessary reagents, precoated 96-well strips and a complete protocol.
1. Tsuji, H.,Cawthorne,C. and B.Ecarot. Abnomal Modulation of Serum Osteocalcin by Dietary Phosphate and 1,25-Dihydroxyvitamin D3 in the Hypophosphatemic Mouse. Journal of Bone and Mineral Research 11: 1234-1240 (1996).
2. Wenstrup, R. J.,Fowlkes, J. L., Witte, D.P. and J.B. Florer. Discordant Expression of Osteoblast Markers in MC3T3-E1 Cells that Synthesize a High Turnover Matrix. The Journal of Biological Chemistry. 271: 10271-10276 (1996).
3. Kousteni, S. et al. Reversal of Bone Loss in Mice by Nongenotropic Signaling of Sex Steroids. Science. 298: 843-846 (2002).
1. Add 25ul standard/sample/control/blank to individual wells, then add 100ul of biotin-MOC Ab, cover, incubate at 4ºC for 18-24 hours.
2. Aspirate and wash three times.
3. Add 100ul of HRP-Strep A to each well, cover, and incubate in the dark at rm. temp for 30 minutes.
4. Repeat Step 2, then add 100ul of TMB-Perox. to each well, cover, and incubate in the dark at room temperature for 15 minutes.
5. Add 100ul of stop solution, swirl and read at 450nm.