Nunc immuno microwell 96

Wells, (Nunc-Immuno™ MicroWell™ 96, Thermo Scientific, Denmark) were coated with L. donovani promastigote lysate (10 μg protein per well) in 20 mM carbonate-bicarbonate buffer (pH 9.25) overnight at 4 °C, washed in PBS and incubated with 200 μl/well blocking solution (5% fetal calf serum, 0.05% Tween-20 and PBS pH7.2) for 30 min at 37 °C. Volunteer sera samples (diluted 1:200 and 1:400 in the same blocking buffer − 200 μl/well) were incubated in duplicate for two hours at 37 °C. Thereafter, Protein-A conjugated Horseradish Peroxidase (HRP, Jackson ImmunoResearch Laboratories, INC. PA, USA) conjugated secondary antibodies were added and plates were incubated for 1 h at 37 °C. The plates were rinsed with PBS-T (155 mM NaCl, 160 mM Na₂HPO₄, 4 mM KH₂PO₄; pH 7.4, and 0.05% Tween) followed by addition of ABTS substrate (Sigma-Aldrich, St. Louis, USA). The absorbance at 405–190 nm was measured in a SPECTROstar^® Nano microplate reader (BMG LABTECH, Germany).

To estimate the exposure of volunteers to Ph. orientalis bites, anti-saliva IgG antibodies in volunteers' sera were measured via ELISA. Microtiter plate wells (Nunc-Immuno™ MicroWell™ 96, Thermo Scientific, Denmark) were coated with Ph. orientalis salivary gland homogenate (SGH, corresponding to 0.2 gland/well, prepared as previously described (Vlkova et al., 2011 (link)) and diluted in coating buffer (Carbonate-Bicarbonate, pH 9) and incubated overnight at 4 °C. The wells were then washed with 100 μl of PBS-T and incubated with 100 μl of blocking/diluting solution (2% [w/v] low fat dry milk in PBS-T) for 60 min at 37 °C to block free binding sites. Thereafter, sera were diluted 1:50 and 1:100 in diluting solution/coating buffer and incubated in duplicate for 90 min at 37 °C. Secondary antibody, α-human IgG HRP (Sigma) diluted 1:1000 was added in a total of 100 μl and incubated for 45 min at 37 °C. Lastly the unbound antibodies were washed six times using PBS-T three minutes per wash. 100 μl of orthophenylenediamine and H₂O₂ in McIlwain phosphate-citrate buffer (pH 5.5) were used as a substrate solution. After 5 min incubation in the dark the reaction was stopped by adding of 100 μl of 10% H₂SO₄. Absorbance was measured with a Multiskan RC ELISA reader (Labsystems) at 492 nm wavelength.