Figure 1. Insect cell-expressed FREP1 protein and its interaction with iRBC lysate detected by ELISA.

(a) The FREP1 was expressed and secreted from High Five cells, determined by 12% SDS-PAGE (left) and western blot assay (right). This result also demonstrated that anti-FREP1 antibody could specifically recognize FREP1. Lanes: 1,3: cell lysate; 2,4: culture medium. (b) ELISA signals were significantly different between Plasmodium falciparum-infected red blood cell (iRBC) lysate and uninfected RBC lysate. The lysate of iRBC and uninfected human RBC were used to coat the ELISA plate, followed by sequential incubation with recombinant insect cell expressed FREP1 protein, 1^st antibody, alkaline phosphatase-conjugated 2^nd antibody. The samples were developed by the addition of 100 μL of pNPP and OD₄₀₅ reading. The retained FREP1 in iRBC lysate-treated wells was significantly higher than in uninfected RBC lysate (p < 0.0002). (c) When the heat inactivated FREP1 protein (InactFREP1) replaced the functional FREP1, the binding between FREP1 and iRBC lysate disappeared (p < 0.001).