High-throughput screening requires assays that have flexibility to test large numbers of specimens while being accurate to ensure reproducibility across all specimens and variables tested. Previously, we used a low-throughput, cell-based assay to identify compounds with antiviral activity against polioviruses. In this report, we report the development and implementation of a high-throughput automation platform for the identification of compounds with antiviral activity against polioviruses. The platform uses off-the-shelf automated equipment combined with a modified assay, with minimal changes to existing laboratory space. We evaluated automation systems from Hudson Robotics Inc., Agilent Technologies, and a microplate reader from PerkinElmer during the platform design. Optimization for high throughput was focused on bulk reagent additions, serial dilutions, microplate washing and measuring results from the tens-to-hundreds of microplates. We evaluated the automated cell-based assay for selectivity, sensitivity, accuracy, precision, and reproducibility. This platform can be applied to screen novel antivirals against polioviruses and non-polio enteroviruses.

Virus neutralization assays, widely used to detect and quantify antibodies induced by virus infection, are considered the gold standard for enterovirus serology testing. Conventional microneutralization assays have been used to assess enterovirus D68 (EV-D68) seroprevalence. While manual or automated 96-well assays are valuable, higher-density assays that increase throughput provide the opportunity to more efficiently screen large, population-based serology collections, as well as to test sample sets against multiple virus strains on the same plate or within the same run. Here, automation was implemented for bulk reagent dispensing, serial dilutions, and luminescence measurement to develop a 384-well enterovirus microneutralization assay that increases overall testing throughput, maintains the reproducibility of the standard 96-well assay, and reduces sample volume usage. EV-D68 strains Fermon, 14-18953, and 18-23087 were used to evaluate the automated 384-well microneutralization assay and compare to the conventional 96-well assay. Sensitivity and specificity were evaluated using pooled human sera and positive and negative control antisera. The Lower Limit of quantitation (LLOQ) was the same as for the 96-well assay and coefficients of variations (CV) of 7.35%, 5.97%, and 2.85% for the three EV-D68 strains respectively, were well below the typical goal of 20% CV for accuracy. Z-factor analysis yielded results of 0.694, 0.638, and 0.852, for the three EV-D68 strains respectively, indicating a high level of precision, reliability, and robustness. Intra-assay (7.25%) and inter-assay (7.12%) variability were well below 20% CV. Moreover, the 96-well and 384-well versions of the assay were highly concordant, with a 0.955 correlation coefficient in titers obtained for 50 sera tested. Validation of this automated 384-well microneutralization will support its use in large serology screens assessing the presence of EV-D68 neutralizing antibodies in human populations.