Novel monoclonal antibodies against the C-terminal HEAT domain of Huntingtin

BACKGROUND Reliable detection of huntingtin (HTT) is essential for understanding Huntington’s disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVE We sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODS We immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTS Three novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONS These C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.