Neutralizing Antibody Assay for CHIKV and SFV

Assessment of neutralizing antibodies in mice was performed using a modified luciferase based assay (described in [31 (link)]). CHIKV and SFV clones expressing NanoLuc luciferase (nLuc) (Promega, Madison, WI) in-frame between the capsid and E3 proteins were engineered as previously described [32 (link)]. Neutralization was performed by incubating heat-inactivated serum diluted 1:20 with 5x10³ PFU of either CHIKV or SFV expressing nLuc overnight at 4°C. Confluent BHK-21 in 96 well plates were then infected in triplicate with serum: virus mixture for one hour, followed by washing and addition of fresh media. Five hours post-infection, media was discarded and cells were lysed and analyzed for luciferase expression using the Nano-Glo luciferase system (Promega). Data are expressed as fold-neutralization using normal mouse serum for normalization. Plaque reduction neutralization 50% (PRNT₅₀) assay in BHK-21 cells was used for determination of neutralizing titers in human samples (described in [33 (link)]).