PrPTSE detection

Prion Blood Confirm

Optimisation, evaluation and validation of a Protein Misfolding Cyclic Amplification (PMCA) based confirmatory screening assay to detect prions in human blood. A French and British Collaborative Study

2009: 2 years / Grant: 252 000 euros

Project: Given the risk of prion transmission following blood transfusion, researchers in the UK and France proposed in this project to combine their efforts, expertises and resources for the development, evaluation and validation of a PMCA based confirmatory test to detect vCJD infectivity in blood.

Main results and related published data: A plasma pre-treatment step was first designed to optimize PrPTSE detection and/or to remove PMCA inhibitory factors from plasma. Four plasma pretreatment methods (beads coated with two different PrPTSE ligands, NaCl precipitation and streptomycin precipitation) were shown to be compatible with PMCA and allowed to improve the detection of PrPTSE in plasma spiked with a 108 dilution of a 10% (w/v) vCJD brain homogenate and a 103 dilution of a 10% (w/v) vCJD spleen homogenate. Transmission of PMCA derived products to tg650 transgenic mice was performedin parallel to titer their infectivity.

Prion Blood Confirm Valid

Validation of a PMCA based confirmatory screening assay for the detection of PrPTSE as a marker for vCJD infectivity in human plasma

2012: 1 year / Grant: 30 000 euros

Project: The objective of the “Prion Blood Confirm Valid” project was to pursue the “Prion Blood Confirm” project and to validate (analyticspecificity,sensitivity and diagnosticspecificity) the method according to the European guidelines.

Main results and related published data: Further work was performed to validate the performance of the original assay developed in the “Prion Blood Confirm” project regarding the guidelines issued by the Official Journal of the European Union 22 12 2011 (List A of Annex II to Directive 98/79/EC) for the CE marking of vCJD assays for blood screening. A 3-step method coupling 1) a plasminogen capture 2) a PMCA amplification step and 3) a detection of PK resistant PrPTSE Western blot was shown to allow the detection of a brain homogenate dilution of 10-9 ie analyticalsensitivity and no false positive results in the 80 plasma samples of blood donors used as negative controls indicating a diagnostic specificity of 100% (95% interval confidence: 95.5-100).