Delineation of PfRh4 binding sites on CR1 and KD measurements by SPR. (A) Recombinant PfRh4 binds to sCR1 and CCP1-3 as shown by SPR. Duplicate injections of sCR1 and smaller CR1 fragments, all at a concentration of 5μM, were performed over a CM5 chip that was coupled with rPfRh4. A small fraction of inactive rPfRh4 present on the chip surface triggers nonspecific background binding for the first analyte samples to be assayed (CCP1-3 and sCR1), as manifested in imperfect reproducibility of the sensorgrams. After saturation of this nonspecific binding capacity, duplicate injections are of acceptable reproducibility (all other constructs). (B) Use of SPR to measure the dissociation constant (KD) of CCP1-3 for PfRh4. The left-hand panels show duplicate sensorgrams for a range of increasing CCP1-3 concentrations (1.0μM, 2.5μM, 5.0μM, 10.0μM, and 25.0μM, bottom to top) flowing over a CM5-chip surface with a loading of 410 RUs (i) and 1480 RUs (iii) of rPfRh4. The right-hand panels show plots of the RUs versus CCP1-3 on 2 different flow cells, coupled with 410 RUs (ii) and 1480 RUs (iv) of rPfRh4. The dashed vertical line indicates the KD fitted to both plots simultaneously. In all panels, blank-subtracted sensorgrams are shown.
