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 1 h, 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.