Figure 2

Phage antibody specificity. In the panel A, the monoclonal phage antibodies MA3.G3 and MA3.B4 are analyzed by ELISA for rHaPrP and brain homogenates recognition. As controls the irrelevant phage antibody anti glucose oxidase and anti tetanus toxoid are also shown. Experiments were repeated at least twice and mean ± SD from representative experiments (triplicate samples) is shown. The cut-off value separating positive from negative sample was calculated as 3 standard deviation above the mean of the value obtained from irrelevant phage antibodies (0,086 OD for rHaPrp, 0,052 OD for brain homogenate). In panel B, the monoclonal phage antibodies and the rodent mAb 3F4 were tested in flow-cytometry on human HL-60 cells. The surface PrPc expression was down-regulated by ATRA inducing granulocytic differentiation. HL-60 cells were cultured in the presence of 1 μM ATRA for 1 and 3 days, then stained for flow cytometry analysis with scFvs MA3.B4, MA3.G3 and mAb 3F4. Basal level of PrPc staining in undifferentiated cells (solid line) is shown relative to an irrelevant phage antibody, specific for glucose oxidase, or an IgG2b isotype control (in gray). The progressive decrease of PrPc staining during differentiation is shown in black.