Article Figures & Data
Figures
- FIG. 1.
Expression and purification of recombinant PvRII protein. (A) Recombinant PvRII was expressed as an insoluble protein in E. coli BL21(DE3) and electrophoresed using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Lanes: 1, BL21(DE3) with pet28a; 2, Bio-Rad low-range protein marker; 3, whole lysates; 4, soluble fraction; 5, insoluble fraction of pet28a-PvRII. (B) The expression of PvRII was confirmed by Western blotting (15% SDS-PAGE) with anti-His monoclonal antibody and horseradish peroxidase-conjugated anti-mouse immunoglobulin G. Lanes 1, Bio-Rad low-range protein marker; 2, BL21(DE3); 3, BL21(DE3) with pet28a; 4, pet28a-PvRII; 5, the soluble fraction of pet28a-PvRII; 6, the insoluble fraction of pet28a-PvRII. (C) Expressed PvRII was purified from the insoluble fraction with an Ni-NTA column under 8 M urea denaturing conditions. Lanes: 1, Bio-Rad low-range protein marker; 2, purified PvRII; 3, insoluble fraction of PvRII; 4, flowthrough. Arrows in panels B and C indicate the 20.4-kDa PvRII.
- FIG. 2.
Selection of binders to PvRII. Each well of the ELISA plate was precoated with recombinant PvRII (1 μg/well), and then antibodies from each phage (1010 CFU/well) were added to each well. Bound phages were detected with horseradish peroxidase-conjugated anti-M13 mouse antibody. Anti-HBV pre-S1 scFv (22) was used as an irrelevant antibody, and pooled sera of the six patients with P. vivax malaria were used for the reactivity of the purified recombinant PvRII. The results are the average of triplicate assays.
- FIG. 3.
Purification and analysis of the binding kinetics of soluble scFvs. (A) ScFvs were expressed in the form of soluble proteins without a p3 phage protein fused to the C-terminal portion of scFv. Monomeric scFvs were purified with an Ni-NTA affinity column using fast-protein liquid chromatography and subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. (B) The binding kinetics of the scFvs were measured using surface plasmon resonance on a BIAcore biosensor instrument. The graph shows an overlay plot of the sensorgrams obtained for SFDBII92 at six different concentrations (0.5 to 1 μM) against immobilized PvRII.
- FIG. 4.
Erythrocyte-binding assay. PvDBPII was expressed on the surface of COS-7 cells using the pDE-GFP surface display system. Transfected COS-7 cells were observed under confocal microscopy and reacted with a 10% erythrocyte suspension for rosette formation (A and C, pDE; B and D, pDE-PvDBPII). The microscope magnification is ×200. Interactions with the receptors of erythrocytes and PvDBPII were observed as rosettes (arrows).
- FIG. 5.
EBIA. Transfected COS-7 cells with pDE-PvDBPII were incubated with various concentrations of soluble scFvs (2 to 100 μg/ml) for 2 h at 37°C. After washing, a 10% erythrocyte suspension was added for rosette formation. The binding inhibition activity was scored after counting the rosettes in 20 fields at a magnification of ×100. An irrelevant scFv was used as a negative control. The error bars correspond to standard errors.
Tables
- TABLE 1.
Analysis of the CDR and human subgroup assignments for the three anti-PvRII scFvs in this studya
Clone and region Sequence for: Family CDR1 CDR2 CDR3 VL SFDBII12 GFTFGDYAMETH GISWNSGSIG GMETAANYYYYYGMETDV VκIII SFDBII58 GFTFGDYGMETH GISWNSGSIG DIYSSSWYAAFDI VκI SFDBII92 GFTFSSYWMETS NIKQDGSEKY DCSSTSCYGYYYGMETDV VλI VH SFDBII12 RASQSVSSSYLA GASSRAT QQYGSSRLT VHIII SFDBII58 RASQSISYHLN AASTLQS QKYNSAPLT VHIII SFDBII92 SGSSSNIGSNTVN SNNQRPS AAWDDSLNGWV VHIII ↵a VH and VL were searched against the Kabat database using the BLAST algorithm, and the scFv groups were analyzed via the site at http://acrmwww.biochem.ucl.ac.uk/abs/chothia.html.
