In some experiments, protease inhibitor cocktails (Cat P2714, Sigma-Aldrich, St Louis, MO; Cat 1836170, Roche Diagnostics), or addition of non-ionic detergents (Triton X-110 or Triton X-114) were added according to manufacturer's recommended protocol: 4 hr at 25uC. The recombinant antigens were stored at 220uC and used as immunogens for in vitro and in vivo up to one week after production. Protein expression of the full-length product (as evidenced by immunoblot using the anti-HA Cterminal tag mAb) was quantitated against an IVTT-produced recombinant P. falciparum (PF14_0051) protein of known concentration expressing the same N-terminal 6xHis and C-terminal HA tags. Immunoblot images were acquired in TIF format and analysed using the AnalySIS LS software (version 5.0; Soft Imaging Systems GmbH, Germany).