Immunization and Antibody Purification Protocol for Plasmodium falciparum HRP2

DHP, potassium hexacyanoferrate(iii), and potassium chloride were purchased from Sigma-Aldrich (St. Louis, USA). potassium hexacyanoferrate(ii) trihydrate, sulfuric acid (98%), and buffer saline solution (PBS) of pH 7.00 were from Merck (Darmstadt, Germany). Purified polyclonal Ab-PfHRP2, Ag-PfHRP2, and human serum samples were obtained from Leônidas and Maria Deane Institute (Oswaldo Cruz Foundation, Manaus, Brazil). The biomolecules preparation process consisted of the recombinant expression of Plasmodium falciparum HRP2 in Escherichia coli BL21 pLysS (Invitrogen), and the purification was performed using affinity chromatography nickel columns (QIAGEN), following the manufacturer instructions.^{28 (link)} Later, mice were immunized with the purified recombinant protein (4 bi-weekly 10 μg doses). Serum samples were collected, and reactivity was analyzed by indirect enzyme-linked immunosorbent assay (ELISA). Antibodies were purified using the protein G sepharose (Sigma) resin column following the manufacturer's recommendations.
All the other reagents employed were of analytical grade and were used without further purification. Aqueous solutions were prepared by using purified water from a Milli-Q system.