Cyanogen bromide activated

AgB purification was carried out following the protocol described by Oriol et al. [25 (link)], with some modifications. Briefly, parasite proteins from individual E. granulosus HF were precipitated by sodium acetate (5 mM, pH 5.0) and the resultant material was resuspended in phosphate-buffered saline (PBS) containing 20 μM 3,5-di-tert-butyl–4-hydroxytoluene (BHT). The HF parasite enriched fraction, obtained from individual cysts, was subjected to immunoaffinity chromatography using primary IgG antibodies against the recombinant forms of AgB8/1, AgB8/2 and AgB8/4 (see “Immunoblot” section). Antibodies were separately coupled to cyanogen bromide-activated Sepharose 4B resin (GE Healthcare) and the previously prepared HF material was passed through the columns. Bound AgB from each column was eluted with 100 mM tris-glycine pH 2.5, pooled together to reconstitute the original HF sample, then dialyzed against PBS/BHT and concentrated on Amicon Ultra-15 centrifugal filter device, MWCO 3 kDa (Millipore). Resultant purified AgB from individual cysts was kept separated and analyzed on SDS-PAGE 12% (S1B Fig). AgB concentration was determined using a Qubit quantitation fluorometer and Quant-iT reagents (Life Technologies).