Development of novel blocking ELISA based on a monoclonal antibody targeting conserved epitopes on VP6 for evaluating...

Rotavirus (RV) is a major pathogen causing viral diarrhea in infants and various young animals, with Group A rotavirus (RVA) being the most prevalent. Porcine rotavirus A (PoRVA) causes severe gastroenteritis in piglets. VP6, a conserved immunogenic protein of PoRVA, serves as an ideal target for developing diagnostic assays and vaccines. Current indirect ELISAs for PoRVA antibody detection are prone to false positives, however, blocking ELISA (bELISA) offers superior specificity, hence the importance of developing VP6-targeting blocking mAbs. In this study, a highly expressing CHO-K1 cell line for VP6 was constructed, yielding VP6 with good immunogenicity. Subsequently, one mAb-3B3 with blocking activity was prepared using hybridoma technology. Molecular docking and molecular dynamics simulations determined that the binding between mAb-3B3 and VP6 is reliable and stable, and identified the conserved key interacting amino acids Tyr159, Thr367, and Val236, which provides a structural basis for the design of novel vaccines. Based on mAb-3B3, a bELISA method was first developed. ROC curve analysis (50 negative and 50 positive sera) determined a cutoff value of 23.23%, yielding an AUC of 0.9973 and 98.04% sensitivity and specificity. This bELISA can clearly distinguish between positive and negative serum. Moreover, the assay displayed no cross-reactivity with antisera against six common pig pathogens. This assay demonstrated 95.65% concordance with the indirect fluorescent antibody test among 186 clinical samples, validating its practical application. With high specificity, sensitivity, and reproducibility, this bELISA applies to clinical diagnosis of PoRVA and assessment of vaccine-induced immunity, aiding effective control of RVA.