Anti-PfRH5 monoclonal antibodies were described previously5 (link). Hybridomas expressing 9AD4, QA1 and QA5 were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma) supplemented with 4 mM L-glutamine (Sigma), 0.01 M HEPES (Life Technologies), 100 U penicillin and 0.1 mg/ml streptomycin (Sigma), and 20% fetal calf serum (Gibco). They were then transferred into CD Hybridoma medium (Life Technologies) with glutamine, penicillin, and streptomycin. The cells were harvested after 7-10 days. The cell culture supernatant was exchanged into 20 mM phosphate pH 7.0 with a tangential flow filtration device (Pall).
The sample was then loaded onto a HiTrap Protein G HP column (GE Healthcare), eluted in 0.1 M glycine-HCl (pH 3.0), and immediately neutralised with 0.1 M Tris (pH 8.0). The sample was exchanged into 100 mM phosphate (pH 6.4), 300 mM NaCl, 2 mM EDTA, 5 mM L-cysteine (pH 6.4), and 1.5 mM β-mercaptoethanol using PD-10 columns (GE Healthcare).
Antibody fragments were generated by addition of papain agarose (Sigma), and overnight incubation at 37°C. The papain agarose was removed by centrifugation, and the sample loaded onto a HiTrap Protein A HP column (GE Healthcare). The material that did not bind to the column was gel filtered on a Superdex 200 16/60 column (GE Healthcare) in 20 mM HEPES (pH 7.5) and 150 mM NaCl.