A Pseudovirus‐Based Method for the Simultaneous Quantitation of Neutralization Antibodies Against All Three Poliovirus Serotypes

Poliovirus is characterized by three antigenically distinct serotypes that do not elicit cross-neutralizing antibodies. In the final stages of poliovirus eradication, the gold-standard conventional neutralization test (cNT) for detecting serum neutralizing antibodies (NAbs) is highly restricted due to biosafety concerns. To address this, we developed a high-throughput, tri-color pseudovirus-based neutralization assay (PBNA) for the simultaneous quantification of NAbs against all three poliovirus serotypes. We generated pseudoviruses by co-transfecting cells with P1 plasmids, a replication plasmid, and a T7 RNA polymerase plasmid. By optimizing P1 expression, sensitive cell selection (HEK 293T), and plasmid transfection ratios (3:3:1 for P1, replicon, and T7 plasmids), we produced high-titer pseudoviruses (>29-fold increase in titers). Based on high-titer pseudovirus encoding distinct fluorophores (E2, eGFP, and RFP), the PBNA was established, which was optimized for a 12 h incubation period, 4 × 10⁴ cells per well, and 1500 TCID₅₀/mL of pseudovirus. It demonstrated high sensitivity, strong serotype specificity, and excellent reproducibility. Furthermore, the PBNA and cNT exhibited excellent congruency (<i>r</i> > 0.88, all serotypes). The tri-color PBNA provides a safe, rapid, and alternative to the cNT, making it an invaluable tool for large-scale serosurveillance, novel vaccine evaluation, and fundamental virological investigations in the post-eradication era.