2-SITE ENZYME-IMMUNOASSAY FOR MEASURING INTACT HUMAN OSTEOCALCIN IN SERUM

We developed a sensitive two-site sandwich ELISA for quantitative anal ysis of human osteocalcin in serum or plasma. Our method is based on t wo different highly specific antibodies recognizing epitopes at differ ent ends of the protein so that only intact osteocalcin is detected. T he method is fast (total analysis time less than 6 h/96 wells), precis e (intraassay variation less than 2.3% at four different levels; n = 1 0, and interassay variation less than 2.5%, n = 5, respectively), and accurate, with a mean recovery of 105%. The detection limit in serum i s approximately 0.1 mu g/liter. The mean concentration of osteocalcin in normal serum with this assay is 3.3 mu g/liter (SD 3.7 mu g/liter; range 0.1-13.1 mu g/liter; n = 41), and the reference range is 0.28-10 .1 mu g/liter (10 and 90% confidence limits). The method shows a reaso nable positive linear correlation with other osteocalcin assays (Incst ar, r = 0.55, p < 0.05, n = 13; Henning Oscatest, r = 0.52, p < 0.005, n = 34). A good correlation (r = 0.70, p < 0.001) between individual osteocalcin and bone-specific alkaline phosphatase serum concentration s was observed in normal subjects. We found a low or undetectable conc entration of intact osteocalcin in serum of all four of our patients w ith acute primary hyperparathyroidism, and in all five patients with h ypocalcemic secondary hyperparathyroidism, which suggests that PTH eff ectively inhibited the synthesis of osteocalcin in osteoblasts. The se rum concentration of intact osteocalcin was elevated in two of three p atients with chronic primary hyperparathyroidism. In five patients wit h normocalcemic secondary hyperparathyroidism, the serum concentration of intact osteocalcin was low: three had a normal and two had an elev ated value. An age-related decrease in intact osteocalcin in serum of females younger than 50 years and an increase in older women were obse rved.