Specific determination of the proteinase K-resistant form of the prion protein using two-site immunometric assays. Application to the post-mortem diagnosis of BSE

The aim of this work was to establish an immunological test suitable for sp ecifically detecting PrPres in tissues from animals or humans developing TS Es. We chose to use as detection method a conventional two-site immunometri c assay (sandwich immunoassay) because over the last 20 years this techniqu e has clearly been shown to be more sensitive and specific than other tests . We have established numerous two-site immunometric assays based on the us e of monoclonal antibodies and suitable for measurement of PrPsen in variou s mammalian species (human, bovine, ovine, mouse and hamster). A detection limit below 100 pg/ml was estimated from standard curves established using ovine recombinant PrP. PrPres was selectively detected by processing sample s (currently brain homogenates) to enable specific purification and concent ration of PrPres, which was finally solubilized by a strong denaturing trea tment. This sample-processing procedure can be achieved within 30 minutes. The capacity of this test to detect bovine PrPres was estimated in the fram ework of an evaluation study organized by the Directorate-General XXIV of t he European Commission during May 1999. On this occasion, a blind test on 1 400 brain stem samples taken from either healthy (1000) or BSE-infected (30 0) cows demonstrated 100% sensitivity and specificity. In addition, dilutio n experiments showed that the test can significantly detect PrPres in homog enates diluted 1/300 and was at least as sensitive as a conventional bioass ay performed on mice.