Enzyme-linked immunosorbent assays (ELISAs) are used to evaluate binding of broadly neutralizing antibodies (bNAbs) and polyclonal sera to native-like HIV-1 Env SOSIPs. Methods for immobilizing SOSIPs on plates differ, which can lead to variable or, in some cases, misleading results. Three methods used to immobilize SOSIPs were compared to determine how antigen immobilization methods affect Env conformation and ELISA results. HIV-1 SOSIPs were directly coated on polystyrene plates, captured by a monoclonal antibody against a C-terminal affinity tag, or randomly biotinylated and coated on a streptavidin plate. Binding of bNAbs with known epitopes were compared for each immobilization method. Binding of bNAbs tar... More
Enzyme-linked immunosorbent assays (ELISAs) are used to evaluate binding of broadly neutralizing antibodies (bNAbs) and polyclonal sera to native-like HIV-1 Env SOSIPs. Methods for immobilizing SOSIPs on plates differ, which can lead to variable or, in some cases, misleading results. Three methods used to immobilize SOSIPs were compared to determine how antigen immobilization methods affect Env conformation and ELISA results. HIV-1 SOSIPs were directly coated on polystyrene plates, captured by a monoclonal antibody against a C-terminal affinity tag, or randomly biotinylated and coated on a streptavidin plate. Binding of bNAbs with known epitopes were compared for each immobilization method. Binding of bNAbs targeting the V1V2, V3, CD4 binding site, and gp120/gp41 interface was comparable for all antigen immobilization methods. However, directly coated HIV-1 SOSIP ELISAs showed detectable binding of 17b, a CD4-induced antibody that binds a V3 epitope that is concealed on closed prefusion Env trimers in the absence of added CD4, whereas antibody-immobilized and randomly biotinylated Env-coated ELISAs did not show detectable binding of 17b in the absence of CD4. We conclude direct coating of HIV-1 SOSIPs on ELISA plates can result in exposure of CD4-induced antibody epitopes, suggesting disruption of Env structure and exposure of epitopes that are hidden in the closed, prefusion trimer.
