Fig. 2.

Characterization of total human alpha-synuclein AlphaLISA. The hook-point analysis was evaluated for serial dilutions of biotinylated 4B12 in combination with Europium Acceptor-beads coupled to either LB509 (LB509-Eu) or syn211 (syn211-Eu) using a 19 ng/ml of recombinant GST-tagged h-asyn protein diluted in 10 μg/ml of Tris wild-type rat brain lysate (a). Arrow indicates the highest signal obtained and therefore optimal 4B12-biotin concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled LB509 by serial dilution of either GST-tagged h-asyn or mouse asyn (m-asyn) protein spiked in Tris brain lysate obtained from a naïve rat (10 μg/ml) (b). The lower detection limit (LDL) was 3.7 pg/ml and is indicated by a dashed line. Intra assay variation was calculated based on two standard curves which were measured on the same day but on separate plates (c). Inter assay variation data was calculated based on two pairs of standard curves performed on separate days (d). AU arbitrary units