The total shed material was 2-fold diluted with 200 mM ammonium acetate (pH 6.5) and loaded onto a Sepharose CL-4B column (1 × 40 cm, GE Healthcare, Piscataway, NJ) preequilibrated with 100 mM ammonium acetate (pH 6.5). The column was eluted with the equilibration buffer, in a flow rate of 0.2 mL/min using a peristaltic pump (GE Healthcare). Fractions (N = 80) of 1 mL were collected and then screened by chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) as described elsewhere [20 (link)], using anti-T. cruzi membrane polyclonal antibody (mouse) or anti-Alpha Gal purified from sera of chronic Chagasic patients (human Ch anti-αGal), as described [21 (link)]. The most reactive fractions being pooled and concentrated in a vacuum centrifuge and then resuspended in filtered PBS for further analysis by nanoparticle tracking analysis (NTA) as previously described [20 (link), 29 , 30 (link)].
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