All three anti-tau antibodies strongly recognized human tau fibrils on SPR ( Figure S1 available online). Because
the fibrils have multiple identical epitopes, this website we could not directly calculate the association and dissociation rates. We also assessed the antibodies by immunoblotting and immunostaining. On western blots, all three antibodies bound to human tau (Figure 1H). HJ9.3 and HJ9.4 bound to mouse tau, while HJ8.5 did not (Figure 1H). Consistent with our prior findings (Yamada et al., 2011), there appeared to be less reassembly buffer (RAB)-soluble tau in 9-month-old compared to 3-month-old P301S mice. We found that HJ8.5 stained human tau in 3-month-old and 9- to 12-month-old transgenic P301S mouse brains. Tau immunoreactivity was present throughout the cell bodies and processes (Figure S2). In 9- to 12-month-old P301S mice with tau aggregates, HJ8.5 detected tau aggregates in cell bodies (Figure S2A). Other antibodies produced similar results (Figure S2B). All antibodies bound to neurofibrillary tangles and neuropil threads in AD brain (Figure S2). To evaluate seeding
activity present in P301S brain lysates, we adapted a cellular biosensor system that we have previously described (Kfoury et al., 2012). buy Rucaparib This is based on expression of the repeat domain of tau (aa 243–375) containing the ΔK280 mutation fused either to cyan or yellow fluorescent protein (RD(ΔK)-CFP/YFP). Uptake of exogenous aggregates into these cells triggers intracellular aggregation of RD(ΔK)-CFP/YFP that is detected by fluorescence resonance energy transfer (FRET) recorded on a fluorescence plate reader (Kfoury et al., 2012). Clarified brain lysates from 12-month-old P301S mice added to the biosensor cell system induced strong aggregation of the RD(ΔK)-CFP/YFP reporter, indicating the presence of tau seeding activity (Figure 2A). The seeding activity from 12-month-old P301S brain homogenate mice roughly corresponds
to 50 nM (monomer equivalent) of recombinant full-length fibrils (data Levetiracetam not shown). There was little to no aggregation induced by lysates from tau knockout mice, wild-type mice, or 3-month-old P301S mice lacking tau pathology (Figure 2A). We assessed the anti-tau antibodies (HJ8.5, HJ9.3, and HJ9.4) for their ability to block the uptake and seeding activity from the 12-month-old P301S brain lysates. HJ3.4 (mouse monoclonal anti-Aβ antibody) was a negative control. The anti-tau antibodies effectively blocked seeding activity (Figure 2B). To determine their relative efficacy, we titrated the antibodies (0.125, 0.25, 0.5, 1, and 2 μg/ml) against a fixed amount of P301S brain lysate (Figure 2C). The HJ8.5 antibody blocked seeding activity at concentrations as low as 0.25 μg/ml compared to controls. At 0.5 μg/ml, both HJ8.5 and HJ9.3 antibody significantly blocked uptake and seeding activity compared to control. HJ9.4 was least potent in blocking the uptake and seeding activity, consistent with its higher affinity for mouse tau.