title: Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.
abstract: Determining the risk of transmissible spongiform encephalopathy (TSE) transmission by blood or plasma-derived products requires sensitive and specific assays for the detection of either infectivity or a reliable marker for infectivity. To this end, a Western blot assay that is both sensitive and reproducible for the detection of PrP(RES), a marker for TSE infectivity, was developed. Using the 263K strain of TSE as a model system, the Western blot assay proved to be sensitive, specific and quantitative over a 3-4 log dynamic range. Compared to the rodent bioassay, the assay was shown to detect PrP(RES) down to approximately 10(3.4) IU/ml which is approximately 5-10 pg of PrP or approximately 10-20 ng brain equivalents. The Western blot was applied to monitor the partitioning of spiked PrP(Sc) through three plasma fractionation steps, cryoprecipitation, fraction I and fraction III, that are common to the purification of several human plasma-derived therapeutic products including albumin and immunoglobulins. The results from these studies demonstrated 1 log, 1 log and 4 logs of PrP(Sc) partitioning away from the effluent fraction for the cryoprecipitation, fraction I and fraction III steps, respectively.
authors.affiliation: Department of Pathogen Safety Research/Biological Products, Bayer Corp., Research Triangle Park, NC 27709, USA. doug.lee.b@bayer.com
authors.forename: D C
authors.initials: DC
authors.lastname: Lee
authors.forename: C J
authors.initials: CJ
authors.lastname: Stenland
authors.forename: R C
authors.initials: RC
authors.lastname: Hartwell
authors.forename: E K
authors.initials: EK
authors.lastname: Ford
authors.forename: K
authors.initials: K
authors.lastname: Cai
authors.forename: J L
authors.initials: JL
authors.lastname: Miller
authors.forename: K J
authors.initials: KJ
authors.lastname: Gilligan
authors.forename: R
authors.initials: R
authors.lastname: Rubenstein
authors.forename: M
authors.initials: M
authors.lastname: Fournel
authors.forename: S R
authors.initials: SR
authors.lastname: Petteway
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D015153:Blotting, Western / Q000379:methods* / Q000706:statistics & numerical data; D001923:Brain Chemistry; D011232:Chemical Precipitation; D006224:Cricetinae; D000939:Epitopes / Q000235:genetics; D005615:Freezing; D006801:Humans; D018620:PrPSc Proteins / Q000097:blood / Q000235:genetics / Q000276:immunology; D017096:Prion Diseases / Q000097:blood / Q000635:transmission; D011328:Prions / Q000097:blood* / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000706:statistics & numerical data
chemical_list: D000939:Epitopes; D018620:PrPSc Proteins; D011328:Prions