Detecting T. cruzi Proteins in Trypomastigote EVs

To confirm the presence of T. cruzi-specific proteins in EV secreted by trypomastigotes, Western blots using anti-trans-sialidase (mAb39) antibody were performed, following the methodology described by Retana Moreira et al. in 2021^{13 (link)}. For the analysis, 30 µg of EV of trypomastigotes were resolved by SDS-PAGE, transferred to PVDF membranes (Trans-Blot Turbo Midi PVDF Transfer Packs, Bio-Rad Laboratories, USA) for 40 min at 40 V in a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories, USA), and blocked overnight with 5% non-fat milk in PBS-0.1% Tween 20. The membranes were then washed in PBS-0.1% Tween 20 and incubated overnight at 4 °C with the primary antibody anti-TS mAb 39 (1:1000) (produced in mice). After the incubation, the membranes were washed in PBS-0.1% Tween 20 and incubated for 1 h with secondary antibody goat anti-mouse IgGs conjugated with peroxidase (1:1000) (Agilent Technologies, Santa Clara, CA, USA). The reaction was visualized using Clarity ECL Western substrate (BioRad, Hercules, CA, USA) in a ChemiDoc Imaging System (BioRad, Hercules, CA, USA).