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J.Health Sci., 47(4), 346-352, 2001
Production and Application of Monoclonal Antibodies to Human Selenoprotein P
Yoshiro Saito,a Yasuko Watanabe,a Eiji Saito,b Tsutomu Honjoh,c and Kazuhiko Takahashi*, a
aDepartment of Hygienic Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12, Nishi 6, Kita-ku, Sapporo 060-0812, Japan, bSecond Department of Internal Medicine, Nihon University School of Medicine, 30-1 Ohyaguchi Kamimachi, Itabashi-ku, Tokyo 173-0032, Japan, and cMorinaga Institute of Biological Sciences, Shimosueyosi 2-1-1, Tsurumi-ku, Yokohama 230-8504, Japan
Selenoprotein P is a selenium-rich extracellular protein and is the major selenoprotein in plasma. Although the biological function of selenoprotein P has not been established, we have recently shown that selenoprotein P has phospholipid hydroperoxide glutathione peroxidase-like activity. To study the structure and function of selenoprotein P, we produced monoclonal antibodies against human selenoprotein P. Immunization of rats with purified selenoprotein P was followed by hybridization, cloning, and the establishment of eleven hybridomas producing specific antibodies against human selenoprotein P. With the addition of six kinds of insoluble rat anti-human selenoprotein P monoclonal antibodies to human plasma, the selenium concentration of the supernatant was decreased to 47% of the control. This suggests that selenoprotein P constituted 53% of total plasma selenium. Western blot analysis of the immunoprecipitate from human plasma showed that the antibodies specifically bound to a 69 kDa protein, representing selenoprotein P. Next, we developed an enzyme-linked immunosorbent assay for selenoprotein P using two monoclonal antibodies. The plasma concentration of selenoprotein P in 77 normal individuals was 5.3 ± 1.1 mu g/ml. Because preferential depletion of selenoprotein P by low density lipoprotein (LDL)-apheresis has been reported, we next measured selenoprotein P concentration of plasma samples from patients before and after LDL-apheresis. We confirmed that the plasma concentration of selenoprotein P was decreased from 5.7 to 2.3 mu g/ml by LDL-apheresis. This result shows that this assay provides a reliable means of measuring the content of selenoprotein P in plasma.
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