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METHOD:
An Enzyme Immunoassay for the Determination of Intact Osteocalcin (Bone GLA Protein) in Human Serum or Plasma. For Research use only ─ not for use in diagnostic procedures
A solid-phase enzyme-immunoassay on coated micro-titre plates for osteocalcin in serum or plasma, using an osteocalcin-horseradish-peroxidase conjugate and a monoclonal antibody raised against bovine osteocalcin.
Areas of application:
Serum osteocalcin measurements are used in various clinical situations, including metabolic bone diseases, renal disorders; hyperthyroidism and diseases related to excess glucocorticoids.
OSTEOCALCIN AS A MARKER
Osteocalcin is the most abundant noncollagenous protein in mature human bone where it constitutes 1% to 2% of the total protein. Synthesized by osteoblasts, it is incorporated into the bone matrix. Human osteocalcin is a 49-residue protein (MW. 5669) with three gamma-carboxyglutamic acid residues, at positions 17, 21 and 24. These three residues confer on it a very strong ability to bind to hydroxyapatite.
Some of the newly synthesized osteocalcin escapes into the blood and its concentration in serum reflects the rate of bone formation rather than the rate of bone resorption. Although its function is not known, it is a more specific biochemical index of bone metabolism than other more established indices such as urinary hydroxyproline and total serum alkaline phosphatase activity.
Assay Procedure
1. Prior to commencing the assay the sample distribution and identification plan should be established.
2. Preparation of reagents:
a) Conjugate: Dilute 1:150 in diluent solution.
b) Wash solution: Dilute 1:20 with distilled water.
c) OPD tablets: Each tablet contains 10mg OPD, dissolve at 2mg/ml.
d) Dispense 1ml of osteocalcin diluent solution into a separate tube.
3. Select the required number of strips and place them in a strip holder.
4. Transfer 10 ul of each standard, in duplicate, to the appropriate wells, followed by 10 ul of
osteocalcin free serum. Transfer 10 ul serum sample to the correct wells followed by 10 ul of
osteocalcin diluent solution. ( Note: Use a positive displacement pipette for these additions).
5. Add 200 ul of diluted conjugate to each well.
6. Incubate overnight at 4 ℃
7. Prepare OPD buffered substrate ( 2mg/ml OPD in buffered substrate)
8. Aspirate the contents of the wells and wash 4 times with diluted wash solution.
9. Add 150 ul OPD buffered substrate to each well. Incubate in the dark for 30 minutes.
10. Stop the reaction with 4M H2SO4. and read absorbance at 492nm (reference 690nm if available).
Kit Components:
Anti Osteocalcin coated strips (12x8 well strips)
Zero Osteocalcin standard (150ul/tube)
Osteocalcin standard (150ul/tube)
Osteocalcin free plasma (1 ml)
Osteocalcin HRP conjugate solution (150 ul)
Osteocalcin diluent solution (22.5 ml)
Buffered substrate solution (20 ml)
Wash concentrate solution (20X, 45 ml)
Stopping solution (7 ml)
OPD tablets (4 tablets)
Adhesive plate sealers (4x) | 
