PvRII erythrocyte binding assay
COS-7 cells were transfected by Lipofectamine with 1-2 μg of pHVDR22 DNA, a plasmid kindly provided by L. Miller which expresses region II of the DBP of P. vivax on the cell surface as a chimera with the HSV gD protein. Duffy Fy (a-b+) erythrocytes were washed three times in RPMI 1640, resuspended to a hematocrit of 1% in 1 ml of complete DMEM with polyanions at concentrations of 0, 1, 10, 100, and 1000 μg/ml. This suspension was swirled over aspirated COS-7 cells 40-60 h after transfection and allowed to settle over 2 h at 37°C. The COS-7 cells were then washed three times with 2 ml of PBS to remove nonadherent erythrocytes. The number of adherent erythrocyte rosettes was scored in 20 randomly chosen fields at a magnification of 40 using an inverted microscope. Percentage inhibition of binding was determined by dividing the number of rosettes at each polyanion concentration by the percentage of rosettes at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.
PvDBP peptide BSA-heparin ELISA
Polyvinyl chloride 96-well microtiter plates were coated with 5 μg/ml heparin-albumin (Sigma) in a volume of 100 μl per well in 50 mM Tris-HCl pH 7.5 wash buffer overnight at room temperature. Plates were washed three times with wash buffer and blocked for 2 h at room temperature with 1% BSA in wash buffer at 400 ul per well. For polyanion blocking experiments, WT or mutant DBP polyanion binding site peptides were diluted to 10 μg/ml in a final concentration of polyanions at 0, 0.1, 1, 10, 100, or 1,000 μg/ml in wash buffer and added at 100 μl per well for 2 h. For controls, the DBP peptides were added at 10 μg/ml in 0.01, 0.15, 0.5, 1.0, and 2.0 M NaCl 50 mM Tris HCl pH 7.5 (data not shown). Plates were washed three times with wash buffer. Chicken anti-DYKDDDDK epitope antibody (Aves Labs, OR) at 1:5000 in 1% BSA wash buffer was added at 100 μl per well for 1 h at room temperature. Rabbit anti-chicken horseradish peroxidase antibody (Jackson ImmunoResearch) was added at 1:5000 in 1% BSA wash buffer at 100 μl per well for 1 h at room temperature. Plates were washed three times with wash buffer. The reaction was developed with 100 μl per well of 2% 3,3′,5,5′-tetramethylbenzidine 0.1 M NaOAc containing 0.001% hydrogen peroxide for about 5 min. The reaction was stopped with 100 μl per well of 1 M phosphoric acid. Absorbance measurements were made at 450 nm on a Biotek 133 microtiter plate reader. Percentage inhibition of binding was determined by dividing the absorbance at each polyanion concentration by the absorbance at 0 μg/ml of the polyanion, multiplying by 100 and subtracting this value from 100.
RANTES BSA-heparin ELISA
The same ELISA format used for the PvDBP Peptide-BSA-heparin ELISA described above was used for a competitive ELISA to detect RANTES binding to heparin, and competitors to this binding. Wells were coated with BSA-heparin, blocked with BSA and washed. A volume of 100 μl of 5 nM RANTES in wash buffer supplemented with 0, 0.5, 5, 50, 500 or 5000 nM peptide was added to triplicate wells. After incubation for 1.5 h at room temperature, the plates were washed in wash buffer and 100 μl of biotinylated anti-RANTES monoclonal antibody (R&D Systems) was added at 1:500 in 0.1% BSA wash buffer. After 1 h at room temperature, the plates were washed and 100 μl of streptavidin-horseradish peroxidase (Jackson ImmunoResearch) was added at 1:2000 in 0.1% BSA wash buffer. After 1 h at room temperature, the plates were washed. The reaction was developed as above. Percentage inhibition of binding was determined by dividing the absorbance at each peptide concentration by the absorbance at 0 nM of the peptide, multiplying by 100 and subtracting this value from 100.