In the first step, samples are incubated in the antibody-coated microtiter plate. During the second step, the plate is washed and incubated with the POD-labeled Gla-OC antibody. A substrate is added, and the reaction between POD and the substrate (H2O2, TMBZ) results in a color development. The amount of sample Gla-OC is determined by measuring absorbance using an EIA plate reader. Accurate Gla-OC sample concentrations can be determined by comparing their specific absorbances with the absorbance obtained for the Standard plotted on a standard curve.
Osteocalcin (OC), also known as gamma-carboxyglutamic acid protein, is a small (5.9 kDa), vitamin K-dependent, hydroxyapatite (Ca2+)-binding protein synthesized exclusively by osteoblasts and odontoblasts. This tissue-specific expression of OC makes it an excellent indicator of overall cell activity for bone formation. Osteocalcin has also been shown to be a hormone that controls the regulation of glucose and fat deposition and plays a role in male fertility. Three gamma-carboxyglutamic acid (Gla) residues at positions 17, 21, and 24 of the protein are responsible for binding calcium. Calcium-binding is, in turn, required for such biological activities as activation of the blood coagulation cascade. Thus, Gla-OC likely represents the active form of the protein, while Glu-OC, which has weak binding affinity to hydroxyapatite, represents the inactive form. Serum OC levels correlate well with bone formation rates and, thus, enzyme immunoassay (EIA) measurements of carboxylated OC (Gla-OC) vs. decarboxylated OC (Glu-OC) can be used for clinical evaluation studies. EIA systems may provide better clinical leads than conventional assays which cannot differentiate between active and inactive forms.
