Studies were performed with PMCA to determine whether a cell-free system can recapitulate the effect of Mabs on PrP C conversion observed in infected cells. This system also allowed us to evaluate whether accessibility of Mab to membrane associated PrP C in the living cells influences the PrP C to PrP Sc conversion process. Mabs (12-50 mg/ml final concentration) were added throughout the sPMCA 40 protocol along with the 10% NBH spiked with a 10 24 dilution of 263K infected brain homogenate as described in the Methods section. This dilution of infected brain homogenate does not result in detectable PK resistant PrP Sc immunostaining (Fig. 3A) and, therefore, did not interfere with the detection of newly formed PrP Sc . At the completion of sPMCA 40 , the samples were digested with PK (100 mg/ml) and analyzed on immunoblots using biotinylated Mab 2F11 which reacts equally with both hamster PrP C and PrP Sc . It is interesting to note that although the 263K-infected brain homogenate displayed the 3 band pattern typical for the multiple glycosylated forms of PrP C and PrP Sc (Fig. 3A) , the sPMCA 40 products in the positive controls and Mab-treated reactions consisted of only a single diglycosylated 30 kDa PrP Sc band observed after PK digestion at the higher levels of inhibition, .50%, but had two bands or a smear when there was less inhibition (Fig. 3B) .