A direct relationship between the partitioning of the pathogenic prion protein and transmissible spongiform encephalopathy infectivity during the purification of plasma proteins.

BACKGROUND: Experimental evidence from rodent models indicates that blood can contain transmissible spongiform encephalopathy (TSE) infectivity, which suggests a potential risk for TSE transmission via proteins isolated from human plasma. Because methods that can reduce TSE infectivity typically are detrimental to protein function, infectivity must be removed to ensure the safety of these therapeutic proteins. Animal bioassays are conventionally used to detect infectivity, but the pathogenic form of the prion protein (PrP(Sc)) can serve as a marker for TSE infectivity. STUDY DESIGN AND METHODS: Seven plasma protein-purification steps were performed after the plasma intermediates were spiked with TSE-infected material. Resulting fractions were analyzed for PrP(Sc) by using a Western blot assay and for TSE infectivity by using an animal bioassay. Western blots were quantitated by an endpoint dilution analysis, and infectivity titers were calculated by the Spearman-Kärber method. RESULTS: PrP(Sc) partitioning paralleled TSE infectivity partitioning, regardless of the nature of the protein-purification step. The detection ranges for PrP(Sc) and infectivity were 0 to 5.3 log and 1.1 to 8.9 log median infectious dose per unit, respectively. Clearance of PrP(Sc) and infectivity ranged from 1.0 to 6.0 log. CONCLUSION: Purification steps for isolating therapeutic proteins from human plasma showed the removal of both PrP(Sc) and TSE infectivity. PrP(Sc) partitioning coincided with infectivity partitioning, which showed a close relationship between PrP(Sc) and TSE infectivity. By exploiting this association, the in vitro Western blot assay for PrP(Sc) was valuable for estimating the partitioning of TSE infectivity during plasma protein purification.

Monitoring plasma processing steps with a sensitive Western blot assay for the detection of the prion protein.

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.

Detailed analysis of a family of RNAs transcribed from varicella-zoster virus open reading frame 21 during replication.

The expression of VZV open reading frame (ORF) 21 during viral replication was examined at the RNA level. Three distinct mRNAs homologous to ORF 21, 3.6, 3.0, and 2.4 kb in size, were detected by Northern blot hybridization. Sequence analysis of eight ORF 21 cDNAs indicated that the corresponding RNAs were 3' coterminal and unspliced. The three largest cDNAs contained the entire ORF 21 coding sequence. Two upstream alternative polyadenylation signals located within ORF 21 were not utilized. Sequence analysis of the site of polyadenylation suggests a virus-specific cis-acting element is present in these transcripts.

Expression of the Epstein-Barr virus BamHI A fragment in nasopharyngeal carcinoma: evidence for a viral protein expressed in vivo.

A family of mRNAs that are transcribed rightward through the BamHI A fragment have been detected in C15, a nasopharyngeal carcinoma (NPC) which has been passaged in nude mice. Northern (RNA) blot hybridizations indicate that these RNAs are also expressed in three other NPCs which have been established in nude mice and in an NPC obtained at biopsy. Moreover, hybridization in situ detected transcription from BamHI A in 12 NPCs and 1 Epstein-Barr virus (EBV)-containing carcinoma of the parotid gland. In each case, transcription was detected in all of the malignant epithelial cells. Transcription was not detected in two cases of EBV-positive lymphoma biopsies by in situ hybridization nor in latently infected EBV-positive lymphoblastoid cell lines by Northern blot hybridization. The consistent transcription of these sequences in latently infected epithelial malignancy but not in lymphoid cells suggests that this viral function is associated with latent EBV infection of epithelial cells. Sequence analysis of a cDNA synthesized from the C15 tumor, representing the 3' end of BamHI A messenger RNA, revealed an open reading frame (ORF). Translation of this ORF in vitro produced several peptides that were immunoprecipitated with antisera from patients with NPC. The detection of antibodies to the protein encoded by the ORF present in the BamHI A cDNA indicates that BamHI A encodes a protein which is expressed in vivo and is antigenic.

Production of rat monoclonal antibody from rat x mouse hybridoma cell lines using microencapsulation technology.

The cell growth and monoclonal antibody production characteristics of two rat x mouse heterohybridoma cell lines, designated 187.1 and M1/9.3, were investigated using a biocompatible microencapsulation technology. Both cell lines, derived from the fusion of immunized rat spleen cells with either the NS1 or X63Ag8.653 myeloma cell lines, were found to reach a maximum intracapsular cell density of 1.3 to 1.5 X 10(7) cells/ml during a 27-d culture period. During this period, rat monoclonal antibody accumulated in the intracapsular space of both cultures to a final concentration of 2.0 to 2.8 mg/ml. Comparison of the concentration of rat monoclonal antibody in the extracapsular vs. the intracapsular space of both cultures indicated that significantly less than 1% of the antibody produced by the encapsulated hybridoma cells was capable of transiting the microcapsule membrane during the culture period. Due to the partition of the rat monoclonal antibody within the intracapsular space, the initial purity of the antibody harvested from 21-d microcapsule cultures of 187.1 and M1/9.3 cells was approximately 48 and 75% by weight, respectively. Analysis of the intracapsular protein by sodium dodecyl sulfoxide gel electrophoresis at different times during the culture period demonstrated that the principal contaminant associated with the unpurified antibody was bovine serum albumin.