Simple, rapid enzyme-linked immunosorbent assay (ELISA) for the determination of rat osteocalcin

Determination of the concentration of osteocalcin in rat serum is frequently performed using a commercially available radioimmunoassay (RIA). However, this assay takes 3 days to complete, uses radioactive material, and has a narrow linear range. The limited range of the RIA makes it necessary to test multiple dilutions of the sample which frequently results in values that differ, depending on the dilution. In order to overcome these limitations, we have developed an ELISA that utilizes the same standards and anti-rat osteocalcin antiserum, as is used in the RIA. The principle of the ELISA is that the osteocalcin in the sample competes with osteocalcin previously immobilized on a microtiter plate to bind to the available anti-rat osteocalcin antibodies. The amount of antibody bound to the immobilized osteocalcin is determined colorimetrically using a secondary antibody coupled to alkaline phosphatase. This ELISA has a three-log linear response with a sensitivity of 0.1-0.15 ng/ml and intra- and interassay coefficient of variance (CV) values of less than 10%. Most importantly, the assay is rapid and only requires a 2-hour incubation of the sample with the antiserum. The incubation time is important since we and others have observed a significant decrease in the osteocalcin level from serum samples incubated for long periods of time with the antiserum, presumably due to degradation of the osteocalcin. In general, the commercially available RIA gives osteocalcin values that are one-half to one-fourth that of the ELISA because the RIA requires a 48-hour incubation time.