title: Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11.
abstract: Here, we report the development and further characterisation of a novel PrP-specific monoclonal antibody: 2A11. By Western blot analysis, 2A11 reacts with PrPC from a variety of species including cow, sheep, pig, hamster, rabbit, cat, dog, deer and mouse but fails to react with human, chicken and turtle PrP. Reactivity to PrPC in Western blot was found to be dependent on the redox state of the protein since binding of mAb 2A11 to its epitope was more effective in reducing conditions. 2A11 binding site was mapped within a region comprised by residues 171-179 (six octarepeats bovine PrP notation; 163-171 for the ovine PrP notation). Interestingly, in immunohistochemistry (IHC) analysis, immunoreactivity was greatly enhanced after proteinase K (PK) sample treatment, while little or no reaction was observed in non-PK-treated BSE samples and samples from healthy animals. Quantitative differences in reactivity to BSE prions after PK treatment were also observed, to a lesser extent, by Western blot analysis. Since definitive diagnosis of prion diseases rely on IHC assays of proteinase K-treated samples, the use of mAb 2A11 might contribute to reduce the occurrence of false positive detection due to incomplete proteinase K digestion.
authors.affiliation: Centro de Investigación en Sanidad Animal, INIA, Ctra. Valdeolmos a El Casar sn, Valdeolmos, 28130 Madrid, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Brun
authors.forename: Joaquín
authors.initials: J
authors.lastname: Castilla
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Ramírez
authors.forename: Kai
authors.initials: K
authors.lastname: Prager
authors.forename: Beatriz
authors.initials: B
authors.lastname: Parra
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Salguero
authors.forename: Diane
authors.initials: D
authors.lastname: Shiveral
authors.forename: Carmen
authors.initials: C
authors.lastname: Sánchez
authors.forename: José M
authors.initials: JM
authors.lastname: Sánchez-Vizcaíno
authors.forename: Alastair
authors.initials: A
authors.lastname: Douglas
authors.forename: Juan M
authors.initials: JM
authors.lastname: Torres
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D001921:Brain / Q000378:metabolism / Q000473:pathology; D002417:Cattle; D019286:Endopeptidase K / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D018622:PrP 27-30 Protein / Q000378:metabolism; D018620:PrPSc Proteins / Q000378:metabolism; D011233:Precipitin Tests / Q000379:methods; D011328:Prions / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D000911:Antibodies, Monoclonal; D018620:PrPSc Proteins; D011328:Prions; D011994:Recombinant Proteins; D018622:PrP 27-30 Protein; D019286:Endopeptidase K