DOI: 10.5281/zenodo.17718642
title: Development of novel blocking ELISA based on a monoclonal antibody targeting conserved epitopes on VP6 for evaluating porcine rotavirus A

description: Explanation: This file contains all the raw data files for molecular dynamics simulations and binding free energy calculations of the 3B3 system.

Note: (1) Since this system is an antigen-antibody system, both of which belong to large protein systems, the trr and xtc files containing water generated during the molecular dynamics process have reached approximately 25GB. Therefore, the xtc trajectory files containing water are not provided here. Instead, the fit.xtc file (without water and ions, only containing the studied complex - antigen-antibody) that has undergone periodic correction and elimination of translation and rotation is provided. This file contains data of all frames during the dynamics process. All subsequent analyses are based on this fit.xtc file. (2) The binding free energy calculation is also performed based on this file. Since the intermediate files generated during the binding free energy calculation process have reached approximately 10GB, the intermediate files are also not provided. (3) If other researchers want to reproduce our work, for the binding free energy calculation part, they can refer to the post written by me to reproduce the binding free energy (http://bbs.keinsci.com/thread-53483-1-1.html). (4) As for the molecular dynamics simulation part, parameter files for all steps, mdp files, top files, itp files, and tpr files generated during the intermediate process have been provided. Researchers can directly run the md steps according to the provided tpr files to reproduce our work.
description: AbstractRotavirus (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 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. It achieved 95.65% concordance with indirect fluorescent antibody testing in 186 clinical samples, validating its practical application. With high specificity, sensitivity, and reproducibility, this bELISA is applicable for both clinical diagnosis of PoRVA and assessment of vaccine-induced immunity, aiding in the effective control of RVA.Key words: Porcine rotavirus-A, VP6, Blocking ELISA, Molecular docking, Molecular dynamics simulation