A novel cELISA using a high-performance monoclonal antibody for serological detection of porcine rotavirus

Porcine rotavirus (PoRV) is a major cause of severe diarrhea in piglets, highlighting the need for efficient serological tools to support surveillance and vaccine evaluation. While virus neutralization tests (NTs) remain the gold standard for detecting functional antibodies, they are labor-intensive and low-throughput. To address this limitation, we developed a competitive enzyme-linked immunosorbent assay (cELISA) based on a high-affinity monoclonal antibody (mAb16) that targets the VP4 spike protein of PoRV. When validated against a panel of porcine serum samples previously characterized by NT, the cELISA demonstrated excellent diagnostic performance, with an area under the receiver operating characteristic curve (AUC) of 0.995, 96.00% sensitivity, and 98.28% specificity. Notably, the percent inhibition values from the cELISA exhibited a strong positive correlation with neutralizing antibody titers (Pearson r = 0.846). This correlation was further confirmed across multiple PoRV genotypes-including G4P[23], G5P[23], and G9P[7]-in a controlled vaccine trial. The assay also showed high analytical sensitivity, no cross-reactivity with other common porcine pathogens, and excellent intra- and inter-assay reproducibility. Epitope mapping localized the mAb16 binding site to the VP5 subunit of VP4, providing a structural basis for its broad genotype across-reactivity. Collectively, this cELISA provides a sensitive, specific, and high-throughput alternative to conventional NT for PoRV serosurveillance and vaccine immunogenicity assessment.