title: 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.
authors: a Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University , Sapporo , Japan.
authors: Zhifu
authors: Z
authors: Shan
authors: a Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University , Sapporo , Japan.
authors: Takeshi
authors: T
authors: Yamasaki
authors: a Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University , Sapporo , Japan.
authors: Akio
authors: A
authors: Suzuki
authors: a Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University , Sapporo , Japan.
authors: Rie
authors: R
authors: Hasebe
authors: a Laboratory of Veterinary Hygiene, Graduate School of Veterinary Medicine, Hokkaido University , Sapporo , Japan.
authors: Motohiro
authors: M
authors: Horiuchi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004351:Drug Resistance; D019286:Endopeptidase K / Q000494:pharmacology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D051379:Mice; D018620:PrPSc Proteins / Q000032:analysis*; D017096:Prion Diseases / Q000175:diagnosis* / Q000276:immunology / Q000378:metabolism; D000072002:Prion Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D020033:Protein Isoforms
chemical_list: D000911:Antibodies, Monoclonal; D018620:PrPSc Proteins; D000072002:Prion Proteins; C512657:Prnp protein, mouse; D020033:Protein Isoforms; D019286:Endopeptidase K
keywords: PK-treatment; cell-based ELISA; monoclonal antibody; prion; screening