Schematic representation of the antibody-binding sites for PrP detection in the homogeneous TR-FRET assay. The assay is based on the in-solution binding of two fluorophore-labeled POM antibodies to identical or unique epitopes on PrP. POM antibodies were either labeled with europium chelate (Eu) donor fluorophore or APC-labeled acceptor. The antibody pairs POM2-Eu/POM2-APC (upper), POM2-Eu/3F4-APC (middle), and POM2-Eu/POM1-APC (lower) were used in the assays. POM1 binds to α-helix 1 in the C-terminal globular domain, POM2 binds to the octapeptide repeats (residues 51–91, mouse sequence) in the unfolded N-terminal part of PrP, and 3F4 binds to the charged cluster (CC). The close proximity of the donor and acceptor antibodies upon binding to PrP leads to a fluorescent TR-FRET signal at 665 nm after excitation at 320 nm, which is proportional to the PrP concentration. HC, hydrophobic core region.