MEASUREMENT OF INTACT RAT OSTEOCALCIN IN OSTEOBLAST (ROS17 2.8) CELLSAND IN OVARIECTOMIZED RATS WITH A SANDWICH ENZYME-IMMUNOASSAY/

We developed a sandwich enzyme immunoassay system for intact rat osteo calcin to improve the region specificity for the detection of this mol ecule. We synthesized two peptides of N-terminal 20 residues and C-ter minal 10 residues of rat osteocalcin. After conjugating these peptides with carrier protein, we obtained anti-N- and anti-C-terminal rat ost eocalcin antibodies in rabbits raised against these two peptides, resp ectively. By using these antibodies, we measured intact rat osteocalci n levels in a two-site immunoassay manner. These antibodies did not sh ow the cross-reactivity to human osteocalcin. The immunoreactive peak corresponding to the intact molecules was detected by our intact osteo calcin method after high-performance liquid chromatographic fractionat ion of osteocalcin fragments in plasma from uremic rats. Furthermore, the intact rat osteocalcin was stable over 8 hours at 25 degrees C. In tact rat osteocalcin levels extracellularly secreted from ROS 17/2.8 c ells were measured by this method, showing time- and dose dependent si gnificant increases when administered 1,25(OH)(2)D-3. The inhibition f or the secretion of intact osteocalcin by actinomycin D was also detec ted quantitatively with this method. In ovariectomized rats, intact os teocalcin levels in plasma were acutely elevated after ovariectomy, an d its elevation was significantly depressed by 17 beta-estradiol admin istration. These data suggest that this sandwich method is able to mea sure the intact form of osteocalcin secreted by osteoblasts. As the an tibodies identify the specific regions of osteocalcin molecule, this m ethod would be useful for sensitive estimation of bone turnover for va rious experimental conditions in rats.