Immunospot elispot analyzer

Samples were tested using a standard microneutralization assay for antibodies to oral poliovirus types 1, 2, and 3 Sabin strains (kindly provided from Dr. Steven Oberste, Polio and Picornavirus Laboratory Branch Chief, CDC). Protocols were adopted from established protocols at the Global Polio Specialized Laboratory, Centers for Disease Control and Prevention [44 ]. Briefly, 80–100 CCID50 of each poliovirus serotype and 2-fold serial dilutions of serum were combined and pre-incubated at 35°C, 5% CO₂ for 3 hours before addition of HEp-2(C) cells (50,000 cells per well) in 96-well tissue culture treated flat bottom plates (CytoOne). After incubation for 5 days at 35°C and 5% CO₂, plates were stained with Neutral Red (Sigma) and imaged by Immunospot ELISPOT Analyzer. Each specimen was run in duplicates, with parallel specimens from one study subject tested in the same assay run, and the neutralization titers estimated by the Spearman-Kärber method and reported as the reciprocal of the calculated 50% end point titer (e.g. serum dilution factor) and log2 transformed. Reference antiserum pool was used to monitor assay performance and variation. A serum sample was considered positive if antibodies were present at ≥1:8 dilution. Specimens with antibody titers <1:8 were considered seronegative and given a log2 value of 2.