Standardized ELISA for Pfs25 Antibodies

ELISAs were performed against full-length Pfs25 protein using standardized methodology as previously described (29 (link), 30 (link)). Briefly ELISA plates were coated over-night with Pfs25 protein (2µg/mL, 50 µL per well). The plates were blocked with StartingBlock™ T20 (PBS) Blocking Buffer (ThermoFisher Scientific,UK) and the assay is performed by using a standard curve and internal controls from the reference serum. Unknown test serum samples from immunized volunteers are diluted and added in triplicate to the ELISA plate. After a two hour incubation period, the diluted sera were discarded, the plate was washed and a secondary polyclonal antibody against the γ–chain of human IgG conjugated to alkaline phosphatase (Sigma, UK) was added. After 1 hour incubation, followed by a wash step, the alkaline phosphatase substrate was added. The substrate is left to develop for 25 minutes and the absorbance at 405nm was read using a plate reader. A standard curve and Gen5 ELISA software v3.04 (BioTek, UK) was used to convert the OD405 of individual test samples into arbitrary units (AU). These responses in AU are reported in μg/mL following generation of a conversion factor by calibration-free concentration analysis (CFCA) as described in Supplementary Materials and Methods.