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

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07557061
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 試験
• Research Field: Pediatrics
• Research Institution: HOKKAIDO UNIVERSITY
• Principal Investigator: KOBAYASHI Kunihiko HOKKAIDO UNIVERSITY SCHOOL OF MEDICINE DEPARTMENT OF PEDIATRICS,PROFESSOR, 医学部, 教授 (60091451)
• Co-Investigator(Kenkyū-buntansha): MAFUNE Naoki HOKKAIDO UNIVERSITY SCHOOL OF MEDICINE DEPARTMENT OF LABORATORY MEDICINE,ASSISTA, 医学部, 助手 (70241304) ; CHIBA Hitoshi HOKKAIDO UNIVERSITY HOSPITAL,CLINICAL LABORATORY,LECTURER, 医学部・附属病院, 講師 (70197622)
• Project Period (FY): 1995 – 1996
• Keywords: OSTEOPONTIN / CALCIUM BINDING PROTEIN / HUMAN MILK / OSTEOMALACIA / PHOSPHOPROTEIN

Research Abstract

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.