Antibody Response to Leishmania Antigens

Antibody production was investigated before and after infection, during the same period of the splenocyte cultures. For this, rSMP-3 and L. infantum SLA (0.5 and 1.0 µg per well, respectively) were added to the plates for 16 h at 4 °C. After blocking with phosphate buffer saline (PBS 1×) plus 0.05% Tween 20 (PBS-T), individual serum samples were diluted 1:100 in PBS-T, and incubation was processed for 1 h at 37 °C. After washing, the anti-mouse IgG1 and IgG2a horseradish-peroxidase conjugated antibodies (Sigma-Aldrich) were diluted 1:5000 and 1:10,000, respectively, in PBS-T and added to the wells. A new incubation was processed for 1 h at 37 °C, at which time the plates were washed and reactions were developed using H₂O₂, ortho-phenylenediamine, and citrate-phosphate buffer pH 5.0, for 30 min, in the dark. Reaction interruption was performed using 2 N H₂SO₄, and optical density (O.D.) values were read in an ELISA microplate spectrophotometer (Molecular Devices, Spectra Max Plus, Sunnyvale, CA, USA), at 492 nanometers. The ratios between IgG2a and IgG1 levels were calculated as described elsewhere [87 (link)].