An improved Western blot assay to assess the clearance of prion protein from plasma-derived therapeutic proteins

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.