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

ABSTRACT

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP^Sc). PrP^Sc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP^C) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP^Sc (PrP^Sc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP^Sc (PrP^Sc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP^Sc 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 PrP^Sc-sen and PrP^Sc-res even if all PrP^Sc molecules were not detected. The analytical dynamic range for PrP^Sc 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 PrP^Sc detection did not affect the following PrP^Sc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP^Sc 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.

KEYWORDS:

• cell-based ELISA
• monoclonal antibody
• prion
• PK-treatment
• screening

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

The authors have no conflict of interest to declare. The authors also declare no competing financial interests.

Funding

This work was supported by a Grant-in-Aid for Science Research (A) (grant no. 15H02475), a grant from the Program for Leading Graduate Schools (F01), from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This work was also supported by grants for TSE research (H26-Shokuhin-Ippan-004) and Research on Measures for Intractable Diseases from the Ministry of Health, Labour and Welfare of Japan. This work was also supported by the Global Institution for Collaborative Research and Education (GI-CoRE). We thank Zensho Co., Ltd, for the BSL3 facility.