We immunized rabbits with an unfolded NTD linear peptide epitope including Trp68 to generate polyclonal and monoclonal antibodies (pAb, mAb). mAb antibody affinity to the immunizing peptide was determined by surface plasmon resonance (SPR).  NTD was expressed in E. coli, and properly folded monomer status was confirmed by size exclusion chromatography, followed by studies with denaturing and native gel electrophoresis and immunoblotting. Antibody specificity was confirmed by immunohistochemistry (IHC) on patient samples, and immunocytochemistry (ICC) of HEK293 cells transfected with TDP-43 triple-tandem mutation of the nuclear localization sequence (ΔNLS).

SPR of mAbs revealed picomolar affinity to the epitope. Recombinant NTD displayed pAb immunoreactivity only under denaturing conditions.  IHC of ALS/FTLD CNS sections, but not normal CNS, was reactive to antibodies. ICC revealed immunoreactivity for mislocalized/aggregated ΔNLS-TDP-43, but not nuclear wild-type TDP-43.  Antibodies also failed to recognize TDP-43 in physiologic stress granules in HEK293 cells. A ΔNLS-TDP-43 construct in which Trp68 was mutated to serine did not display Immunoreactivity in transfected cells, indicating that Trp68 is immunodominant in the immunizing peptide.  

We have developed a family of antibodies sensitive to solvent exposure of NTD Trp68 that are selective for misfolded/aggregated, disease-associated TDP-43 while sparing physiologically important molecular species, which may find utility in biomarker and immunotherapy applications for TDP-43 associated diseases.