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

Abstract

Background

Reliable detection of huntingtin (HTT) is essential for understanding Huntington's disease (HD) biology and for 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 in common immuno-applications using parental and HTT-knockout cell lines, and in patient-derived fibroblasts.

Results

Three novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines and in patient-derived cells, 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 in vitro and in cells 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.

Plain Language Abstract

Huntington's disease (HD) happens because of a genetic change that adds too many repeats of a sequence called CAG in the huntingtin (HTT) gene. This creates a longer-than-normal stretch of glutamine amino acids (known as polyQ) at the start of the HTT protein, making the protein unstable and prone to clumping, which can harm brain cells. To track huntingtin in the lab and to see how it changes with disease or treatment, detection tools are needed, including antibodies. Many existing antibodies recognize the beginning part of the huntingtin protein, but since the polyQ can vary from person to person, those tools don’t always give consistent results. On the other end of the protein, far fewer good-quality antibodies exist. In this new study, we developed new antibodies that recognize the other end of HTT. We tested these in cells and found one antibody, called 2F8, that was most reliable. 2F8 worked across several common lab tests including western blotting, immunofluorescence, and ELISA. It detected HTT consistently no matter how long the polyQ stretch was, or whether the protein was bound to one of its key partners, HAP40. These new antibodies give the HD research community better tools to study both the normal job of huntingtin and the role it plays in HD, helping the field move toward more reliable research and, eventually, better treatments.