Development and Validation of an Antigen-Capture ELISA for Quantitative Detection of Donkey-Origin Rotavirus:...

Rotavirus A (RVA) causes severe diarrhea in donkey foals, but rapid quantitative antigen assays for vaccine development and manufacturing control are lacking. We developed a VP6-based antigen-capture ELISA (acELISA) for donkey-origin RVA using rabbit anti-RVA polyclonal IgG as the capture antibody and horseradish peroxidase-conjugated mouse anti-VP6 monoclonal antibody 3F6 as the detector antibody. Assay conditions were optimized, and performance was evaluated through standard-curve analysis, specificity and repeatability testing, Western blot (WB) comparison, median tissue culture infectious dose (TCID₅₀), reverse transcription quantitative polymerase chain reaction (RT-qPCR), ultrafiltration membrane screening, and pilot-scale tangential flow filtration (TFF) monitoring. The optimized acELISA detected VP6 at 1 ng/mL and showed a linear range of 1-25 ng/mL, with no cross-reactivity with eight common equine pathogens, and intra- and inter-assay coefficients of variation below 10%. The acELISA estimates were consistent with Western blot densitometry. In process applications, the assay supported the selection of a 100 kDa membrane and monitored pilot-scale TFF, yielding 75.69% VP6 recovery and an 18-fold increase in antigen purity. This acELISA provides a specific and practical in-process tool for quantitative antigen monitoring in donkey rotavirus vaccine production and is intended to complement, rather than replace, infectivity- and genome-based assays.