Fig. 3

Characterization of S129 phosphorylated human alpha-synuclein specific AlphaLISA. The hook-point analysis was evaluated for biotinylated 4B12 and Europium Acceptor-bead conjugated 11A5 (a). Pair was tested using 14 ng/ml of pS129 h-asyn protein diluted in 10 μg/ml of Tris brain lysate obtained from an asyn knockout mouse. Arrow indicates the optimal biotinylated antibody concentration to use. Standard curve was established for biotinylated 4B12 and Europium Acceptor-bead coupled 11A5 by serial dilution of either pS129 h-asyn or S129A h-asyn protein spiked in wild-type rodent brain lysate (10 μg/ml) (b). The lower detection limit (LDL) was 1.1 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) while the inter assay variation was calculated based on two pairs of standard curves performed on separate days (d). AU arbitrary units