Identification of a Conserved Linear Epitope in SARS-CoV-2 Nucleocapsid Protein Recognized by Monoclonal Antibody N179

Abstract

We previously generated mouse monoclonal antibody N179 against the SARS-CoV-2 nucleocapsid (N) protein and developed a colloidal gold-based immunochromatographic test strip with a 2 ng/mL detection limit and 98% concordance with RT-qPCR. However, the precise epitope recognized by N179 remained undefined. Using GST-fused N protein truncation fragments, Western blotting, and ELISA, we mapped the linear antibody-reactive region to the C-terminal tail of the N protein and identified 390QTVTLL395 as the smallest reactive region under the truncation-mapping conditions used. Multiple sequence alignment of 11 representative SARS-CoV-2 N protein sequences showed complete conservation of this motif across the sequences analyzed. EMBOSS WATER analysis further demonstrated a perfect 6/6 match only in SARS-CoV-2; no identical sequence was found in six other human coronaviruses, four influenza viruses, or five bat coronaviruses. Computational structural analyses predicted that this region may be surface accessible, and all six residues exceeded the default BepiPred 3.0 threshold. These findings identify 390QTVTLL395 as the smallest reactive region defined by truncation mapping and show that this sequence is conserved among the representative SARS-CoV-2 lineages analyzed. Together with the sequence comparison and structural predictions, these results provide sequence-level information that may help explain the previously observed recognition profile of N179. This motif may serve as a reference for the selection and evaluation of diagnostic antibodies targeting conserved regions of the SARS-CoV-2 N protein.