Development of an in-cell ELISA for the titration of porcine reproductive and respiratory syndrome virus using monoclonal antibodies against its nucleocapsid protein.

In-cell ELISA provides objective, reproducible PRRSV titration superior to conventional CPE assays.

This study describes the development of an in-cell ELISA method for quantifying porcine reproductive and respiratory syndrome virus (PRRSV) using HRP-conjugated monoclonal antibodies targeting the nucleocapsid protein. PRRSV remains a significant economic threat to global swine production, and conventional TCID assays relying on cytopathic effect observation suffer from subjectivity and poor reproducibility. The novel in-cell ELISA is performed directly in 96-well culture plates without cell harvesting, using MARC-145 cells. The optimized assay achieved 98.39% specificity and 100% sensitivity with an S/N cut-off of 1.50, demonstrated excellent diagnostic accuracy (ROC AUC of 0.9985), and showed no cross-reactivity with porcine epidemic diarrhea virus. The method was validated for antiviral drug screening applications using 25(OH)D as a test compound. This objective and reproducible alternative to conventional CPE-based titration methods offers significant advantages for PRRSV quantification in research and diagnostic settings.

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