Horseradish peroxidase conjugated goat anti human igg γ chain specific

ELISA was performed as described previously [11 (link)]. Briefly, 96-well Nunc-Immuno Maxisorp plates (Thermo Scientific) were coated with 0.25 μg recombinant M. ulcerans 18 kDa shsp per well, washed with washing buffer (dH₂O, 0.01% Tween 20) and incubated with blocking buffer (5% non-fat dry milk in PBS). Subsequently, plates were incubated with human blood serum samples (1:100 diluted). After washing, horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, SouthernBiotech) was added. Plates were washed and developed with TMB Microwell Peroxidase Substrate (KPL). The reaction was stopped using 0.16 M sulfuric acid. The absorbance was measured at 450 nm in a Tecan Sunrise microplate reader. All samples were tested in duplicates and mean values were calculated.
The cut-off value for positivity (OD₄₅₀ = 0.25) was determined by testing serum samples with a range of ODs in ELISA by Western Blot analysis (S1 Fig). Sero-conversion/reversion was defined as a change in OD (ΔOD) between baseline and follow up samples of at least ±0.3.

Western Blot analysis was performed as described [11 (link)]. Shortly, 15 μg of recombinant M. ulcerans 18 kDa shsp or P. falciparum AMA-1 were separated on NuPAGE Novex 4–12% Bis-Tris ZOOM Gels with 1.0 mm IPG well (Invitrogen) under reducing conditions. After electrophoresis, proteins were transferred onto nitrocellulose membranes using an iBlot Gel Transfer Device (Invitrogen). Membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween 20 and cut into strips. Strips were then incubated with human blood sera (1:1000 dilution), washed with 0.3 M PBS containing 1% Tween 20 and thereafter incubated with horseradish peroxidase conjugated goat anti-human IgG (γ-chain specific, Southern Biotech). After a second washing step, bands were visualized by chemiluminescence using ECL Western Blotting substrate (Pierce).