title: A solid-phase immunoassay of protease-resistant prion protein with filtration blotting involving sodium dodecyl sulfate.
abstract: The precise diagnosis for bovine spongiform encephalopathy (BSE) is crucial for preventing new transmission to humans. Several testing procedures are reported for determining protease-resistant prion protein in various tissues as a major hallmark of prion diseases such as BSE, scrapie, and Creutzfeldt-Jakob disease. However, contamination of materials from tissues or degradation of the specimens sometimes disturbs the accuracy of the assay. Here, we have developed a novel method for solid-phase immunoassay of the disease-specific conformational isoform, PrP(Sc), using filtration blotting of protein in the presence of sodium dodecyl sulfate (SDS) followed by a filtration-based immunoassay with a single anti-prion protein antibody, together with the improved fractionation procedure involving high concentrations of surfactant/detergent. The SDS/heat treatment renders unfolded PrP(Sc) quantitative retention on a polyvinylidene difluoride filter and allows enhancement of the analyte signal with immunodetection; thus, all of the tested specimens are determined with 100% accuracy. In addition, the immunoassay is completed in approximately 1h, indicating its usefulness not only for the screening of BSE specimens but probably also for the postmortem BSE diagnosis of fallen stock as the antibody recognizes the core part of PrP(Sc). The solid-phase immunoassay method, including the filtration blotting with SDS, would be applicable to determining even more sensitively proteins other than PrP(Sc), especially those having rigid conformations.
authors.affiliation: New Business and Technology Development Operations, Wako Pure Chemical Industries, Chuo-ku, Osaka 540-8605, Japan. kobayashi.yoshiteru@wako-chem.co.jp
authors.forename: Yoshiteru
authors.initials: Y
authors.lastname: Kobayashi
authors.forename: Naoyuki
authors.initials: N
authors.lastname: Kohno
authors.forename: Shoko
authors.initials: S
authors.lastname: Wanibe
authors.forename: Kazunari
authors.initials: K
authors.lastname: Hirayasu
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Uemori
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Tagawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Yokoyama
authors.forename: Morikazu
authors.initials: M
authors.lastname: Shinagawa
mesh_terms: D000818:Animals; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005374:Filtration; D018621:PrPC Proteins / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*
chemical_list: D018621:PrPC Proteins; D012967:Sodium Dodecyl Sulfate