ELISA for Anti-Leishmania Antibody Detection

ELISA was performed to detect anti-Leishmania antibodies in sera samples [19 (link)]. The flat-bottom 96-well microplates (Corning, USA) were coated with 100 μL/well of purified antigen at a concentration of 5 μg/mL in 0.1 M carbonate/bicarbonate (pH 9.6) buffer by overnight incubation at 4°C. Unbound antigens were removed by washing the plate five times in phosphate buffered saline-Tween 20 (PBST, pH 7.4 containing 0.05% Tween 20). Blocking was performed with 200 μL of 3% nonfat skimmed milk in PBST for 2 hours at room temperature. Then, the wells were washed, 5 times with washing buffer. Diluted serum samples (1 : 100 in PBST) were applied to the plates and incubated for 1 hour at room temperature. The plates were washed as before and 100 μL of 1 : 4000 dilution of horseradish peroxidase-conjugated goat anti-human IgG (Sigma, USA) was added to the plates and incubated for 1.5 hours at room temperature. The plates were then washed as before and incubated with substrate (100 μL/well of 0.4 mg/mL OPD, 0.3% H₂O₂ in 0.1 M citrate buffer, pH 5) for 20 minutes. The reaction was stopped by using 1 N H₂SO₄. The absorbance at 490 nm was checked with a microplate reader (Bio-Tek, ELx800). Positive sera from VL-confirmed cases were applied in each plate. The cutoff point was fixed at 2SD above the mean of control samples.