Production and Application of Monoclonal Antibodies to Human Selenoprotein P.

Abstract

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 µg/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 µg/ml by LDL-apheresis. This result shows that this assay provides a reliable means of measuring the content of selenoprotein P in plasma.

Cell Death Caused by Selenium Deficiency and Protective Effect of Antioxidants

“…It has been reported that these seleniumcontaining materials are incorporated and can be the cellular source of selenium used for synthesis of selenoprotein (22). SeP, which is a selenium-rich extracellular glycoprotein (11,33,34) that functions as selenium transport protein (22,35,36), was the most effective of the materials tested (Table I).…”

Comparison of different forms of dietary selenium supplementation on gene expression of cytoplasmic thioredoxin reductase, selenoprotein P, and selenoprotein W in broilers

“…1 g of liver and kidney in 9 ml of homogenization buffer (10mM Tris-HCl, pH 7.4, 0.1mM EDTA-2Na, 10M sucrose, and 0.1% peroxide-free Triton X-100) was homogenized on ice with an Ultra-Turrax T8 (IKA-Werke GmbH & Co. KG, Staufen, Germany) at 8000 rpm for 10 s. Homogenates were centrifuged at 3500 g and 4°C for 15 min and the supernatant was used for analysis of SelP concentration. The serum, liver, and kidney SelP concentrations were measured using ELISA, as described previously (Saito et al 2001). The assays were conducted with the commercial kits (Life Sciences Advanced Technologies, Saint Petersburg, USA).…”

Novel compound heterozygous mutations in the SBP2 gene: characteristic clinical manifestations and the implications of GH and triiodothyronine in longitudinal bone growth and maturation

“…Plasma selenoprotein P (SeP) concentrations were determined using a sandwich ELISA with rat antihuman SeP MABs BD1 and AH5, as described earlier (11). The optical density of the plates was measured at 450 nm using an OPTImax tunable microplate reader (Molecular Devices, Sunnyvale, CA, USA).…”