title: An improved Western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins.
abstract: Specific detection of the pathogenic prion protein, PrP(Sc), is essential for determining the prion clearance capacity of purification processes for therapeutic proteins. Use of a previously described indirect (two-antibody) Western blot assay sometimes resulted in the appearance of non-specific protein bands that interfered with the detection of small amounts of PrP(Sc)-specific signal, limiting the amount of clearance that could be determined for steps so affected. It is shown that these non-specific signals are due to the interaction between immunoglobulin fragments in the sample and the secondary antibody used in the assay. To circumvent this problem, a direct Western blot assay using a prion-specific primary antibody conjugated to the reporter enzyme alkaline phosphatase was developed. Application of the direct Western blot assay resulted in a significant reduction of non-specific signal while retaining the detection sensitivity for PrP(Sc)-specific signal. Therefore, the direct Western blot assay format is an improved tool for determining prion clearance capacity, particularly for immunoglobulin-rich samples.
authors.affiliation: Department of Pre-Clinical Research and Pathogen Safety, Bayer HealthCare, Division of Biological Products, 85 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Randal C
authors.initials: RC
authors.lastname: Hartwell
authors.forename: Mark S
authors.initials: MS
authors.lastname: Nelson
authors.forename: Michèle M
authors.initials: MM
authors.lastname: Kislan
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Stenland
authors.forename: Jeanette L C
authors.initials: JL
authors.lastname: Miller
authors.forename: Dominique Y
authors.initials: DY
authors.lastname: Pifat
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Petteway
authors.forename: Kang
authors.initials: K
authors.lastname: Cai
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001798:Blood Proteins / Q000009:adverse effects* / Q000302:isolation & purification; D015153:Blotting, Western / Q000379:methods*; D006224:Cricetinae; D018620:PrPSc Proteins / Q000097:blood / Q000276:immunology / Q000493:pharmacokinetics*; D017096:Prion Diseases / Q000378:metabolism* / Q000635:transmission
chemical_list: D001798:Blood Proteins; D018620:PrPSc Proteins; D000469:Alkaline Phosphatase