Intact Human Osteocalcin EIA Kit

About The Assay

Osteocalcin (5800 daltons) is a specific product of the osteoblast.  It is primarily deposited into the ECM of bone and only a small amount of newly synthesized osteocalcin is released directly into the circulation.  Studies suggest that there are various forms of osteocalcin in the circulation and that different antibodies detect both intact and fragments of osteocalcin.  The physiological significance of osteocalcin fragments is unclear but they may be derived from resorption, osteoblastic synthesis, systemic catabolism or all of these.  Since serum proteases may also degrade the intact osteocalcin, it is recommended that blood samples be processed quickly in the presence of protease inhibitors.

The BTI Intact Osteocalcin EIA measures only intact osteocalcin which is synthesized de novo by the osteoblast and it eliminates any interference by circulating fragments.  The assay is a sandwich EIA which employs two monoclonal antibodies directed towards the amino-and the carboxy-terminal regions of the human osteocalcin.  It recognizes only intact osteocalcin, requiring the full 49 residue protein for detection. This assay reacts with human, bovine, monkey and dog osteocalcin.

The assay is highly sensitive (Range: 1-50ng/ml)  and requires only a 25ul sample.  All the necessary reagents, a 96-well strip plate, and a complete 3½ hour protocol are included with the kit.

Procedural Outline

1. Add 25ul standard/sample.  Add antibody and gently swirl.  Incubate 2½ hours at 37^oC.

2. Aspirate and wash 3 times with wash buffer.

3. Add Strep-Av-HRP, swirl, incubate ½ hour at room temperature.

4. Repeat Step 2. Then add 100ul of substrate, swirl, develop 10 minutes in the dark.
    Add 100ul stop solution, swirl, measure absorbance at  450nm.

References

1.   Gundberg C.M., Weinstein R.S. Multiple immunoreactive forms of osteocalcin in uremic serum. J. Clin Invest 77: 1762-67, 1986.

2.   Power M.J., Gosling J.P., Fotrell P.F. Radioimmunoassay of osteocalcin with polyclonal and monoclonal antibodies. Clin Chem 35: 1408-15, 1989.

3.   Calvo M.S., Eyre D.R., and Gundberg C.M. Molecular Basis and Clinical Application of Biological Markers of Bone  Turnover. Endocrine Reviews 17 (4): 333-364, 1996.

 4.   Bodine, P.V.N., Trailsmith, M. and B.S. Komm. Development and Characterization of a Conditionally Transformed Adult Human Osteoblastic Cell Line.  J. of Bone & Miner. Res. 11: 806-819, 1996.
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 5.   Nutall, M.E., Patton, A. J., Olivera, D.L. Nadeau, D.P., &  M.  Gowen.  Human Trabecular Bone Cells are able to Express Both Osteoblastic and Adipocytic Phenotype:  Implications for Osteopenic Disorders. J. of Bone & Miner. Res. 13: 371-382,  1998.

BTI Intact Osteocalcin EIA Kit
Catalog No:  BT-460
(sufficient reagents and precoated 96-well strips)
Quantity:  1 kit 
 

Specifications:
Sample Size:  25ul
Assay Time:  3.5 hours
Sensitivity:  0.5 ng/ml
Range: 1.0- 50 ng/ml
Intraassay Variation:  7%
Interassay Variation:  10.5%

FOR RESEARCH USE ONLY. NOT FOR USE IN HUMANS OR AS AN IN-VITRO DIAGNOSTIC.