Structure-Guided Development of Genotype-Specific Recombinant Antibodies for Rapid Norovirus Detection via Dual-Mode Immunoassays

Norovirus (NoV) is a leading cause of acute gastroenteritis. We expressed GI.1 and GII.4 VP1 proteins and generated genotype-specific monoclonal antibodies (1C1 for GI.1; 7B5 and 10B8 for GII.4). Variable heavy chains and variable light chains were obtained, and full-length heavy/light chain vectors were constructed. Recombinant antibodies (rAbs) expressed in Expi293F and ExpiCHO cells showed an affinity comparable to that of ascite-derived antibodies. Using these rAbs, we developed a dual-antibody sandwich ELISA (DAS-ELISA) and a gold immunochromatographic strip (GICS). In oyster samples, DAS-ELISA limits of detection (LOD) were 3.23 ng/g (GI.1) and 2.16 ng/g (GII.4); visual limits of detection of GICS were 10 ng/g and 5 ng/g, respectively. Validation with 60 clinical samples showed good concordance with PCR, confirming the field-screening suitability. Molecular docking and molecular dynamics simulations identified 12 key interacting residues at the antibody-VP1 interface. Site-directed mutagenesis confirmed that mutating these CDR-localized residues reduced binding affinity by 60–90%, whereas mutation of a framework residue (GLN1) had only a minor effect. These findings provide structural insights into future antibody humanization or affinity maturation.