NR ASPG
AU Hartwell,R.C.; Nelson,M.S.; Kislan,M.M.; Stenland,C.J.; Miller,J.L.; Pifat,D.Y.; Petteway,S.R.Jr.; Cai,K.
TI An improved Western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins
QU Journal of Virological Methods 2005 May; 125(2): 187-93
PT journal article
AB Specific detection of the pathogenic prion protein, PrPsc, 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 PrPsc-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 PrPsc-specific signal. Therefore, the direct Western blot assay format is an improved tool for determining prion clearance capacity, particularly for immunoglobulin-rich samples.
MH Alkaline Phosphatase/metabolism; Animals; Biological Assay; Blood Proteins/*adverse effects/isolation & purification; Blotting, Western/*methods; Hamsters; PrPsc Proteins/blood/immunology/*pharmacokinetics; Prion Diseases/*metabolism/transmission
AD 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
SP englisch
PO Niederlande