Polio virus neutralisation assay

To evaluate the immune response elicited by vaccination, poliovirus neutralisation assays were performed at the CDC as previously published by WHO^41,42,43. Briefly, all samples were diluted 1 in 4, mixed with 80–100 CCID₅₀ of each poliovirus (Sabin 1, 2 or 3) and incubated for 3 hours at 35 °C, then added to HEp-2c cells (7,500 cells per well) in 96-well plates. Plates were incubated at 35 °C, 5% CO₂ for 5 days at which point cells were stained with crystal violet and CPE measured by optical density at 570 nm. Neutralisation titres were reported as reciprocal 50% endpoint titres determined by the Spearman-Karber method⁴⁴. Positive samples are defined as having ≥ 3.0 log₂ titre with an assay background of 2.5 log₂ titre.