STUDIES ON CALCIUM BINDING PROTEIN,OSTEOPONTIN,ITS DIAGNOSTIC AND THERAPEUTIC APPLICATION FOR BONE DISORDERS.

骨桥蛋白（OPN）是一种存在于人乳和血清中的钙结合磷蛋白，被认为通过与成骨细胞和破骨细胞以及钙离子相互作用参与骨组织的重塑。本研究从人乳中分离出分子量为78kD的OPN，其N端7个氨基酸与OPN的DNA序列一致，并制备了兔抗OPN的多克隆抗体。利用该抗体，我们建立了一种夹心式ELISA检测血清中OPN。然而，尽管ELISA在测定乳OPN水平中起作用，但在该系统中不能检测到血清OPN。结果表明，该抗体在琼脂糖凝胶中不与人血清发生沉淀反应，但经Western blot检测，该抗体可与分子量为55和32kD的血清组分发生沉淀反应。此外，血清组分在琼脂糖凝胶中以及在ELISA系统中显示出对抗体与乳OPN之间的反应的抑制活性。因此，血清中分子量为55和32 kD的组分均为血清中OPN的同源物，其中32 kD可能是55 kD的片段。在对血清和乳汁中OPN进行抗原性分析时发现，OPN尤其是血清中的OPN非常脆弱，容易降解成小分子量组分。这可能是血清OPN在琼脂糖凝胶中不显示沉淀反应以及在ELISA系统中不显示结合反应的原因。因此，有必要分离血清中的OPN，并与牛奶中的OPN进行比较，分析其蛋白质性质。

Osteopontin (OPN hereafter) , a Ca binding phosphoprotein present in human milk as well as in serum is thought to be involved in the remodeling of the bony tissues by interacting with osteoblasts and osteoclasts, as well as with Ca ions. This forced us to investigate whether the serum OPN can be a marker for some bony disorders, such as osteomalacia and whether the milk OPN is involved in the osteogenesis of the infants.We isolated OPN with mol.wt of 78kD from human milk, of which N-terminal seven amino acids were identical to those deduced from consensus DNA sequence of OPN,and prepared polyclonal anti-OPN antibodies in rabbits. Using this antibody, we developed a sandwich type ELISA for measuring OPN in serum. However while the ELISA worked in determining levels of milk OPN,serum OPN could not be detected in this system. It was disclosed that while the antibody did not show precipitation reaction in agarose gel with human serum, it reacted with serum components with mol.wt.of 55 and 32kD by Western blot procedure. Furthermore, the serum components showed an inhibitory activity against reaction between the antibody and milk OPN in agarose gel as well as in the ELISA system. Thus, the serum components with mol.wt.of 55 and 32kD were defined both to be OPN homologue in serum, possibly the 32kD being fragment of the 55kD.During the antigenic analysis of OPN from serum and milk, it was notified that OPN especially that from serum was quite fragile and was easily degraded into small mol.wt.components. This would be a reason why serum OPN did not show precipitin reaction in agarose gel as well as binding reaction in ELISA system. It should be necessary to isolate OPN in serum and analyze its protein nature in comparison with that in milk.