Enzyme-Linked ImmunoSorbent Assay (ELISA) [15 (link),16 (link)] was performed to assess malaria antibody concentrations; Afro Immuno Assay (AIA) protocols were followed that are developed to standardize methods for evaluating malaria vaccines and sponsored by the African Malaria Network Trust (AMANET [www.amanet-trust.org ]). Briefly, standard curves were established using purified human IgG (Binding Site, France) to determine the concentration of specific antibodies. Each point was tested in duplicate.
Specific and total IgG were measured using recombinant proteins diluted in phosphate-buffered saline (PBS) for specific IgG or an anti-human IgG (Fab-specific, Sigma Aldrich, France) diluted in carbonate buffer for total IgG, both were coated at 0.1-μg/well on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered milk, 0.1% Tween, 20 PBS. Maternal and cord blood samples were diluted at 1:200 for all IgG-specific, recombinant proteins except for AMA1 (1:2,000) and total IgG (1:1,000,000). An anti-human IgG coupled with peroxidase (1:3,000, Caltag, UK) was used to reveal the reaction with TMB One (3, 3′, 5, 5′-tetramethylbenzidine, Mast Diagnostic, France). Plates were read at 450 nm.
Specific and total IgG were measured using recombinant proteins diluted in phosphate-buffered saline (PBS) for specific IgG or an anti-human IgG (Fab-specific, Sigma Aldrich, France) diluted in carbonate buffer for total IgG, both were coated at 0.1-μg/well on MaxiSorp Nunc plates (Thermo Fisher Scientific, Denmark) and blocked with 3% powdered milk, 0.1% Tween, 20 PBS. Maternal and cord blood samples were diluted at 1:200 for all IgG-specific, recombinant proteins except for AMA1 (1:2,000) and total IgG (1:1,000,000). An anti-human IgG coupled with peroxidase (1:3,000, Caltag, UK) was used to reveal the reaction with TMB One (3, 3′, 5, 5′-tetramethylbenzidine, Mast Diagnostic, France). Plates were read at 450 nm.