A serum pool collected from TZ adult donors with high anti- CHO-rPvs48/45 antibody titers was used for IgG purification, as described previously [72 (link), 73 (link)]. To prepare the antigen-Sepharose conjugate, CNBr-sepharose 4B (Amersham Bioscience AB, Uppsala, Sweden) was activated with 1 mM HCl. Then, 5 mg of the CHO-rPvs48/45 protein was dissolved in 1 mL of coupling buffer (0.1 M NaHCO3 containing 0.5 M NaCl, pH 8.0). The sera were diluted 5-fold with PBS (1x) containing 0.5 M sodium chloride and were mixed with the antigen-sepharose conjugate and stirred O/N gently at 4°C. The antigen-sepharose beads were then washed with 5 mL of TRIS (20 mM containing 0.5 M NaCl, pH 8.0) and then with 5 mL of TRIS (20 mM, pH 8.0). The bound antibody was eluted with a solution containing glycine (0.1 M, pH 2.5). The fractions (F1, F2, F3) were collected in TRIS solution (1 M, pH 8.0) to instantly neutralize the solutions before dialyzing them against phosphate buffer (0.1M, pH 7.0). The antibody (IgG) concentration of each fraction was determined by the absorbance of the solution at 280 nm [72 (link), 73 (link)]. In addition, ELISA was used to determine the recognition of rPvs48/45 by each of the purified antibody fractions.
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