ELISA for Recombinant NTS-DBL1α Antibodies

For the immune sera set, antibodies against the recombinant NTS-DBL1α were measured by ELISA as previously described [26 (link)]. Maxisorp plates (Nunc, Rosklid) were coated overnight at 4 °C with 1 μg of the recombinant antigen dissolved in 15 mM Na₂CO₃ and 35 mM NaHCO₃ (pH 9.6). Plates were washed three times with PBS containing 0.1 % Tween 20 (PBST) and then blocked with 1 % bovine serum albumin (BSA) in PBST for 1 h at room temperature and subsequently washed three times with PBST. All samples were titrated by six serial dilutions in PBS to determine concentration dependency, and they were incubated in triplicate for 1 h at room temperature. Plates were washed three times with PBST, then bound IgG was measured by incubation for 1 h at room temperature with alkaline phosphatase-conjugated goat anti-human IgG (Sigma) diluted 1:1000 in PBS. Plates were washed three times with PBST and developed with SigmaFast p-nitrophenyl phosphate tablets (Sigma) for 15 min. The optical density (OD) was measured at 405 nm in an ELISA reader (Multiskan™ GO Microplate Spetrophotometer, Thermo Scientific). A control pool of six non-malaria exposed Swedish donors was included, as well as control wells without serum (background). Seropositivity was determined based on the 1:500 dilutions as the mean OD₄₀₅ plus two standard deviations of the Swedish control pool.