Anti-Pfs25 IgG antibodies in serum were measured by a standardized ELISA protocol using a reference serum, as previously described (52 (link)). Briefly, Nunc-Immuno maxisorp plates (Thermo Scientific, UK) were coated with Pfs25-Ctag O/N at 4°C. Plates were washed with PBS + 0.1% Tween-20 (PBS/T) six times and blocked for 1 h with 5% skimmed milk in PBS/T at room temperature (RT). Test serum samples were diluted as required in PBS/T, before 100 μL of sample were added to triplicate wells and incubated for 2 h at RT. Plates were washed as before and 100 μL goat anti-mouse total IgG conjugated to alkaline phosphatase (Sigma-Aldrich) diluted 1:3,000 in PBS/T was added to each well and incubated for 1 h at RT. Following a final wash in PBS/T, one p-nitrophenylphosphate (Sigma-Aldrich, UK) tablet was dissolved in 1× diethanolamine buffer (Thermo Scientific, UK) and 100 μL was added to each well as a developing substrate. The optical density (OD) of each well was read at 405 nm using an ELx800 absorbance microplate reader (Biotek, UK). A serially diluted standard reference serum with a known antibody titer was used to determine the antibody titer of individual samples [as previously reported (52 (link))]. The minimal detection limit (MDL) of the ELISA was 10 Antibody Unit. Samples which fell below this value were defined as having 1 AU in order to display them on a log scale graph.
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