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

骨桥蛋白（Osteopontin，以下简称OPN）是一种钙结合磷酸化蛋白，存在于人乳和血清中，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。这迫使我们研究血清OPN是否可以作为一些骨骼疾病（如骨软化症）的标志物，以及乳汁OPN是否参与婴儿的成骨。我们从人乳中分离到分子量为78kD的OPN，其中n端有7个氨基酸与OPN DNA序列一致，制备了兔多克隆抗OPN抗体。利用该抗体，建立了检测血清中OPN的夹心型酶联免疫吸附试验。然而，虽然ELISA可以测定牛奶中OPN的水平，但在该系统中无法检测到血清OPN。结果表明，该抗体在琼脂糖凝胶中与人血清不发生沉淀反应，但经Western blot检测可与分子量为55和32kD的血清组分发生反应。此外，血清成分对琼脂糖凝胶和ELISA系统中抗体与牛奶OPN的反应具有抑制活性。因此，血清中分子量为55和32kD的成分都被定义为OPN同源物，可能32kD是55kD的片段。在对血清和乳汁中的OPN进行抗原分析时，发现OPN特别是血清中的OPN非常脆弱，容易降解为小分子量组分。这可能是血清OPN在琼脂糖凝胶中不发生沉淀反应，在ELISA系统中不发生结合反应的原因。有必要分离血清中的OPN，并与牛奶中的OPN进行蛋白质性质分析。

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.