ELISA for Cytophilic IgG1 and IgG3

The standard operating procedures developed by the African Malaria Network Trust was used to assess cytophilic IgG1 and IgG3 concentrations by enzyme linked immunosorbent assay (ELISA) to a panel of recombinant proteins, as described previously [13 (link)]. Briefly, recombinant proteins (0.1 µg/well) diluted in phosphate buffered saline (PBS) were coated on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered-milk 0.1% PBS-Tween 20. Plasma samples were diluted 1:50 for all recombinant proteins. Peroxidase conjugated anti-human IgG1 (NL16 clone) diluted 1:2000 and anti-human IgG3 (ZG4 clone) diluted 1:5000 (Skybio, France) were used for revealing the reaction with 3,3′,5,5′-tetramethylbenzidine (TMB) as substrate. Standard curves were established using human IgG1 and IgG3 purified proteins (Binding Site, France) to determine the concentration of specific antibodies. Each point was tested in duplicate.