96-well half-area plates were coated with 100 μg/mL heparin or MAD2-6 IgA, incubated at 37°C for 1 hour, and blocked with 1% BSA in PBS. Serially diluted P14-P19 PfCSP peptides and recombinant PfCSP (see table S2 for sequence information) were added to the plate and incubated for 1 hour at room temperature. Next, 1:500 goat anti-human IgG Fc-alkaline phosphatase (Southern Biotech 2048-04) was added to the plate for a 1 hour incubation, followed by p-nitrophenyl phosphate (p-NPP) substrate (Sigma). The plates were read with the EnSpire plate reader (Perkin Elmer) at 405 nm after 1 hour.
In competition experiments, plates were coated with 100 μg/mL MAD2-6 IgA or IgG overnight at 4°C and blocked with 1% BSA in PBS. A mixture of heparin (Fresenius Kabi 401586J) at a range of concentrations and 0.5 μg/mL of biotinylated P17 was added to the plates. Next, streptavidin-alkaline phosphatase (Southern Biotech 7100-04) was added to the plate, and the plates were developed and read after 30 minutes as described above.
In competition experiments, plates were coated with 100 μg/mL MAD2-6 IgA or IgG overnight at 4°C and blocked with 1% BSA in PBS. A mixture of heparin (Fresenius Kabi 401586J) at a range of concentrations and 0.5 μg/mL of biotinylated P17 was added to the plates. Next, streptavidin-alkaline phosphatase (Southern Biotech 7100-04) was added to the plate, and the plates were developed and read after 30 minutes as described above.