Antibodies against rRBP2-P1 were produced by Thermo Fisher Scientific (USA). Briefly, two rabbits were immunized intramuscularly with 0.50 mg of rRBP2-P1 emulsified with Freund’s complete adjuvant on day 0, followed by three boosts of the antigen emulsified with incomplete Freund’s adjuvant on days 14, 42, and 56. The serum was collected on day 70. Serum was also used for rabbit IgG purification by protein G Sepharose (GE Healthcare), according to the standard protocol. The specificity of serum for rRBP2-P1 was confirmed by Western blotting against recombinant P. vivax RBPs (rRBPs) (13 (link)). For this, 1 ng of each rRBP was separated on SDS-PAGE gels and transferred to the membrane. The membrane containing proteins was probed with rabbit anti-rRBP2-P1 IgG (1 μg/ml), followed by goat anti-rabbit HRP (0.2 μg/ml; Millipore). Reactions were detected with chemiluminescence. Coomassie-stained SDS-PAGE of the same rRBP samples (1 μg/lane) was used to demonstrate that each sample contained a protein (see Fig. S1 in the supplemental material).
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