AbstractNeurodegenerative tauopathies are caused by transition of tau protein from a monomer to a toxic aggregate. They include Alzheimer disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick disease (PiD). We have previously proposed that tau monomer exists in two conformational ensembles: an inert form (Mi), which does not self-assemble; and seed-competent form (Ms), which self-assembles and templates ordered assembly growth. We proposed that cis/trans isomerization of tau at P301, the site of dominant disease-associated S/L mis-sense mutations, might underlie the transition of wild-type tau to a seed-competent state. Consequently, we created monoclonal antibodies using non-natural antigens consisting of fluorinated proline (P*) at the analogous P270 in R1, biased towards the trans-configuration at either the R1/R2 (TENLKHQP*GGGKVQIINKK) or R1/R3 (TENLKHQP*GGGKVQIVYK) interfaces. Two antibodies, MD2.2 and MD3.1 efficiently immunoprecipitated soluble seeds from AD and PSP, but not CBD or PiD. They stained brain samples of AD, PSP, and PiD, but not CBD. They did not immunoprecipitate, or immunostain tau from control brain. Creation of potent anti-seed antibodies based on the trans-proline epitope implicates local unfolding around P301 in pathogenesis. MD2.2 and MD3.1 may also be useful for therapy and diagnosis.SummaryTau protein undergoes conformational change to self-assemble and trigger neurodegeneration. We have proposed local unfolding events centered on P301 cis/trans isomerization, which expose amyloidogenic sequences. We used a non-natural peptide with a trans-proline to generate monoclonal antibodies that distinguish tau seeds from native tau in human brain. In addition to being important therapeutic and diagnostic leads, the activity of these antibodies supports structural studies implicating local conformational change in tau that underlies disease initiation.