Methyl crystal violet solution

Heat-inactivated (56 °C for 30 min) serum samples were serially diluted and incubated with approximately 60 PFU of wild-type SARS-CoV-2 (2019-nCoV/Victoria/1/2020), for 1 h at 37 °C in 5% CO₂. Samples were then incubated with Vero E6 [Vero 76, clone E6 (ECACC 85020206), European Collection of Authenticated Cell Cultures, UK] monolayers in 24-well plates (Nunc, ThermoFisher Scientific, Loughborough, UK) under MEM (Life Technologies, California, USA) containing 1.5% carboxymethylcellulose (Sigma), 5% (v/v) foetal calf serum (Life Technologies) and 25 mM HEPES buffer (Sigma). After incubation, at 37 °C for 96 h, plates were fixed overnight with 20% (w/v) formalin/PBS, washed with tap water, and stained with methyl crystal violet solution (0.2% v/v) (Sigma). The neutralising antibody titres were defined as the serum dilutions resulting in a 50% reduction relative to the total number of plaques counted without antibody by using Probit analysis written in R programming language for statistical computing and graphics. An internal positive control for the PRNT assay was run using a sample of human MERS convalescent serum known to neutralise SARS-CoV-2 (National Institute for Biological Standards and Control, UK).

Samples were diluted in serum-free MEM containing antibiotic/antimycotic (Life Technologies) and incubated in 24-well plates (Nunc, ThermoFisher Scientific, Loughborough, UK) with Vero E6 cell monolayers. Virus was allowed to adsorb at 37 ^oC for 1 h, then overlaid with MEM containing 1.5% carboxymethylcellulose (Sigma), 4% (v/v) foetal bovine serum (Sigma) and 25 mM HEPES buffer (Life Technologies). After incubation at 37 °C for 5 days, they were fixed overnight with 20% (w/v) formalin/PBS, washed with tap water and stained with methyl crystal violet solution (0.2% v/v) (Sigma).

Monoclonal antibody was serially diluted and incubated with ~70 plaque forming units of wild-type SARS-CoV-2 (2019-nCoV/Victoria/1/2020), for 1 h at 37°C in a humidified box. The virus/antibody mixture was then allowed to absorb onto monolayers of Vero E6 [(ECACC 85020206, European Collection of Authenticated Cell Cultures, UK] for 1 h at 37 °C in a humidified box. Overlay media [MEM (Life Technologies, California, USA) containing 1.5% carboxymethylcellulose (Sigma), 5% (v/v) fetal calf serum (Life Technologies) and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer (Sigma)] was added and the 24-well plates were incubated in a humidified box at 37 °C for 5 days. Plates were fixed overnight with 20% (w/v) formalin/PBS, washed with tap water and stained with methyl crystal violet solution (0.2% v/v) (Sigma). The neutralizing antibody titers were defined as the amount of antibody (µg mL⁻¹) resulting in a 50% reduction relative to the total number of plaques counted without antibody, by performing a Spearman–Kärber analysis⁴⁰ using Microsoft Excel v2016. An internal positive control for the PRNT assay was run using a sample of human MERS convalescent serum known to neutralize SARS-CoV-2 (National Institute for Biological Standards and Control, United Kingdom).