Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

摘要

ABSTRACT Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrPSc). PrPSc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrPC) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrPSc (PrPSc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrPSc (PrPSc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrPSc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119–127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrPSc-sen and PrPSc-res even if all PrPSc molecules were not detected. The analytical dynamic range for PrPSc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%–11% and 2.5–3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrPSc detection did not affect the following PrPSc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrPSc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.