{"doi":"10.1101/2025.09.17.676860","title":"Novel monoclonal antibodies against the C-terminal HEAT domain of Huntingtin","abstract":"Abstract 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":575216,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9567,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1483188,"name":"Rebeka Fanti","orcid":"0000-0001-9995-5875","position":1,"is_corresponding":false},{"id":1483189,"name":"Samira Sadeghi","orcid":"0000-0002-3777-6836","position":2,"is_corresponding":false},{"id":998320,"name":"Renu Chandrasekaran","orcid":"0000-0001-7593-2476","position":3,"is_corresponding":false},{"id":36724,"name":"Aled Edwards","orcid":"0000-0002-4782-6016","position":4,"is_corresponding":false},{"id":36735,"name":"Rachel J. Harding","orcid":"0000-0002-1134-391X","position":5,"is_corresponding":false},{"id":880766,"name":"Douglas W. Houston","orcid":"0000-0002-7438-4655","position":6,"is_corresponding":false},{"id":1483533,"name":"Yang‐Nim Park","orcid":null,"position":0,"is_corresponding":true}],"reference_count":22,"raw_metadata":null,"created_at":"2026-07-19T02:57:48.486077Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}