ELISA for Human IgG Subclasses

The ELISA to detect the IgG subclasses was performed as previously described [10 (link)]. The sera were diluted 1:100 and evaluated for each IgG subclass using mouse monoclonal antibodies to human IgG subclasses (IgG1 clone 8c/6-39; IgG2 clone HP-6014; IgG3 clone HP-6050; IgG4 clone HP-6025) (Sigma, St. Louis, MO) according to the manufacturer instructions. Specifically, the secondary antibody dilutions were 1:1.000, 1:15.000, 1:40.000 and 1:60.000 for IgG 1, IgG 2, IgG 3 and IgG 4 respectively. The cut-off value was obtained by testing 6 different negative control sera from individuals never exposed to malaria from Belo Horizonte, Brazil. Monoclonal antibody binding was detected with OPD substrate tablets (Thermo Fisher Scientific, USA). The mean optical density value at 492 nm±3 SD (VersaMax^TM Microplate Reader) for duplicate determinations in negative sera was used as the cut-off value for different subclasses and peptides.