Econo pac

The inclusion bodies were solubilized (6 m GuHCl, 0.2 m NaCl, 11.5 mm NaH₂PO₄, 10 mm Tris, 10 mm BetOH, pH 7.8) and harvested by centrifugation at 10 900 g for 30 min at 4 °C. The pellet was resuspended in buffer solubilization and incubated with Ni Sepharose high-performance resin (GE Healthcare Life Sciences, Uppsala, Sweden) for 1 h at 4 °C. The resin was collected by centrifugation at 1360 g for 5 min and the recombinant protein was purified in Econo-Pac^® chromatography columns (Bio-Rad Laboratories), according to the manufacturer's instructions, using elution buffer (10 mm phosphate buffer, 8 m urea, 10 mm imidazole, 10 mm BetOH, pH 4.3). Purified P1–P5 antigen were subjected to successive dialysis against elution buffer, containing 6 m, 4 m and 2 m urea for 3 h in each buffer at 4 °C. Then, the antigen was dialysed in a dialysis sack (molecular weight cut-off 12 000 Da, Sigma-Aldrich) against phosphate-buffered saline (PBS, 2.1 mm NaH₂PO₄, 8.3 mm Na₂HPO₄, 0.15 m NaCl, pH 7.3), also for 3 h, at 4 °C. Lastly, P1–P5 antigen were recovered, and the protein concentration was determined by the colorimetric bicinchoninic acid method (Thermo Fisher Scientific). The multiepitope antigen (P1–P5) was used to standardize an enzyme-linked immunoassay, named ELISA IgG anti-SmME (ELISA-based immunoenzymatic test, using a Schistosoma mansoni multiepitope antigen).

Liposomes stability on storage was evaluated in freshly prepared liposomal suspensions under different conditions: at 4 °C for 90 days and at 37 °C for 48 h, in citrate buffer (10 mM citrate buffer, 145 mM NaCl, pH 6.0) with or without 1% (w/v) bovine serum albumin (BSA—Merck, Darmstadt, Germany). Aliquots were collected, non-encapsulated drug was removed by size exclusion chromatography using a desalting column of 1000 Dalton cutoff (Econo-Pac^®, BIO-RAD, Hercules, CA, USA), and a complete characterization of stable liposomes was repeated.