Figure 1
C5.2 specificity for pathological tau protein and high affinity for pSer396. (a) Brain samples from 24 weeks old rTg4510 and non-transgenic littermate (non-tg) mice (left image) and from 4 AD and age-matched healthy control (HC) donors (right image) were fractioned into soluble (S1) and sarkosyl-insoluble fraction (P3) and analyzed by western blot (upper image) and dot blot (lower image) for phosphorylated tau at pS396 epitope with C.5.2 (1 µg/ml). AD and HC samples were pooled from 2 males and 2 females each. All four donors were Caucasian and age-matched. Individual AD and HC brains were analyzed with similar results. By western blot, phosphorylated and non-phosphorylated human 4R0N tau with the P301L mutation (tau P301L) from the rTg4510 mice is displayed at 55 kDa, hyperphosphorylated 4R0N tau P301L is mobility-shifted and displayed at 64 and 70 kDa. Phosphorylated and non-phosphorylated six human tau isoforms in AD and HC brains are displayed at 45, 55, 60 and 65 kDa, hyperphosphorylated tau isoforms are displayed at 54, 64, 69 and 74 kDa together with the typical AD smear. (b) Representative images of prefrontal cortex from an AD and an age-matched healthy control (HC) donor processed for C5.2 immunohistochemistry. (c) C5.2 IgG ELISA and (d) C5.2 Fab fluorescence polarization binding curves comparing recognition of four differentially phosphorylated peptides (Table 1). C5.2 binds peptides pS396/pS404 (black) and pS396 (blue), with KD of 15.4 nM and 14.9 nM, respectively, but does not recognize peptide pS404 (green) or the non-phosphorylated (light blue) peptide. The dip in fluorescence for the pS404 and non-phosphorylated peptides around 10 nM Fab concentration is due to protein aggregation and fluorophore quenching. Both experiments confirm C5.2’s phospho-specificity for pS396.
