Goat anti mouse total igg conjugated to alkaline phosphatase

MaxiSorp plates (Thermo Fisher Scientific) were coated overnight at 4 °C with CIDR (IT4var07), CyRPA, IMX-DogTag, or SnoopLigaseΔC at 1 μg/mL in coating buffer (15 mM sodium carbonate with 35 mM sodium bicarbonate, pH 9.6). The His₆-tag of CIDR (IT4var07) and SnoopLigaseΔC had been removed using MBP-TEV protease. IMX-DogTag had no purification tag, while CyRPA has a C-tag. Plates were washed six times with PBS/T (PBS with 0.5% Tween 20) and blocked with PBS/T with 10% skim milk for 1 h at 25 °C. Plates were washed as previously with PBS/T and incubated with duplicates of 3-fold serially diluted serum samples for 2 h at 25 °C. Following a wash step as before with PBS/T, goat anti-mouse total IgG conjugated to alkaline phosphatase (Sigma-Aldrich) (1:3,000 dilution in PBS/T) was added to the plate and incubated for 1 h at 25 °C. After a final wash step with PBS/T, p-nitrophenylphosphate (Sigma-Aldrich) at 1 mg/mL diluted in diethanolamine buffer (1 M diethanolamine, pH 9.8, Thermo Scientific) was used as a developing substrate. A₄₀₅ was obtained using a SpectraMAX M3 plate-reader (Molecular Devices). The endpoint titer is defined as the x-axis intercept of the dilution curve at an absorbance value greater than the mean A₄₀₅ plus five standard deviations for a serum sample from a naïve mouse at a serum dilution of 1:100.

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.