4.3. Carbonyl Index Quantitation of Parasite Protein Fractions

Spectrophotometry and immune dot-blot methods were applied to measure the carbonyl index. For this purpose, an aliquot of the BSA fatty acid-free solution (5 μg/μL, Sigma) was derivatized with a DNPH probe, and their carbonyl index was measured in alkaline medium at 450 nm [41]. Its value corresponds to the basal carbonyl index in our conditions (non-oxidized BSA). In parallel, an aliquot of the BSA solution was oxidized with 10 mM FeSO₄ by incubation at 37 °C for 2 h. In both cases, basal and oxidized derived BSA was precipitated with 10% TCA for 15 min and the pellets obtained were washed with 85% acetone for 30 min and centrifuged at 4 °C, 7000 rpm for 5 min. Supernatants were discarded, and the pellets were dissolved in PBS. Subsequently, oxidized and non-oxidized (basal) BSA solutions were quantified with the Bradford assay for normalization at 1 µg/µL [40], and quantification of the carbonyl index values by the alkaline medium at 450 nm.

Next, stoichiometric mixtures of the standard proteins (oxidized and basal) were completed to obtain standards solutions with intermediates carbonyl index values. These standard solutions were used to build the protein carbonylated calibration curve of the dot-blot assay. For its purpose, all standards solutions were derivatized using 6% SDS for 2 min and 10 mM DNPH in 2 N HCl for 10 min, and the reaction was stopped by employing Tris/Glycerol supplemented with β-mercaptoethanol 85:15 [22,42] ( all reagents from Sigma).

Afterward, 200 ng of each standard were spotted by triplicate on the PVDF membrane (GE Healthcare, Pittsburgh, PA, USA), blocked with PBS-5% milk for 2 h, incubated with Anti-DNP primary antibody (Merck) (1:5000) for 2 h, followed by incubations with Anti-Rabbit secondary antibody (Thermo Fischer Scientific, Burlington, ONT, Canada) (1:5000) for 1 h.

Membranes were washed with PBS-Milk-Tween 20, PBS-milk and PBS for 5 min and revealed by chemiluminescence using Kit-Nova for 3 min [11]. The images were captured by a ChemiDoc System (Biorad, Hercules, CA, USA) and the intensity of the spots was analyzed using the Image Lab 6.0 software (Biorad). Replicates were built at different days to evaluate repeatability, reproducibility and linearity of carbonylated protein calibration curves.

To quantify carbonyl index values in samples from parasite proteins at different stages (rings, trophozoites and schizonts), they were DNPH-derivatized, diluted ten folds in PBS, spotted on PVDF membranes, and incubated with the antibodies replicating the methodology described above.

Data were analyzed with GraphPad Prism 6.0 (GraphPad, San Diego, CA, USA) and presented as the mean ± standard error. C.I.s data, repeatability and reproducibility were analyzed by Student’s t-test using the parametric test in one-tailed. Statistical significance between groups was established at p < 0.05.