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Fig. 3 | Virology Journal

Fig. 3

From: A high-throughput screening assay to identify inhibitory antibodies targeting alphavirus release

Fig. 3

Optimizing parameters for CHIKV and MAYV anti-budding assays. Cells were bulk infected for 3.5 h with CHIKV or MAYV nLuc reporter virus, excess virus washed off, cells plated and incubated for 18 h with or without the addition of control inhibitor, and nLuc activity measured in 50 µL supernatant. A Different cell lines were tested for susceptibility to infection by CHIKV nLuc. Cells were infected in bulk and then removed from the plastic surface and counted. Thus, MOIs were calculated retrospectively and ranged from 0.2 (TZM) to 1.3 (U2OS). RD cells were used in subsequent assays to assess MOI ranges for CHIKV (B) and MAYV (C) reporter viruses using control inhibitors mAb C9 (0.5 µg/mL) or pooled sera (diluted 1:50) respectively. D RD cells were incubated with CHIKV nLuc virus for 2–3.5 h at 30 min increments, after which the assay was continued as described. E Post bulk infection with CHIKV nLuc, excess virus was washed off and 50 µL supernatant was removed from plated cells at 1 h intervals from 17 to 20 h to measure nLuc activity. F Pilot screen demonstrating suitability for HTS screening. Results are shown as percentage of nano-luciferase reading of wells containing no antibody. Infected cells with and without C9 on 10 test plates were subjected to Z′ analysis to determine variation and suitability for HTS systems. A Z′ factor of 0.5–1.0 is deemed sufficiently robust for HTS assays. All results shown carry a CV score of ≤ 15%

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