Quantitative AMA1 Protein ELISA Protocol

Three Nunc-immuno maxisorp plates were coated with 2 μg/mL Oxford, NIH, or WRAIR AMA1 protein in DPBS. The same independent standard curve (10 points) was run on each plate in duplicate, using a previously described high-titer anti-AMA1 human serum reference sample [14 (link)]. Otherwise, the same ELISA method for this experiment was performed on all three plates in parallel: plates were left at RT overnight, then washed 6 times with PBS containing 0.05% Tween 20 (PBS/T) and blocked for 1 h with Casein block solution (Pierce). After another wash step, samples were added to each plate for 2 h. Plates were washed again and alkaline phosphatase-conjugated goat anti-human IgG (γ-chain) (Sigma) diluted 1:1000 in Casein block solution was added for 1 h, before development with p-nitrophenylphosphate substrate (Sigma) diluted in diethanolamine buffer (Thermo Fisher Scientific). Optical density at 405 nm (OD₄₀₅) was read using a microplate reader (Biotek) and Gen5 v1 software.