FIGURE 8.

Binding of Band 3 fragments with PvTRAg38. A, solid phase ELISA. The plate was coated with fixed concentration (0.5 μm) of untagged PvTRAg38 and reacted with increasing concentrations (0–2 μm) of histidine-tagged recombinant Band 3 fragments. The plate was developed with mouse anti-His₆ monoclonal antibody as described in the text. Histidine-tagged bacterial thioredoxin from D. desulfuricans was used as negative control. B, specificity of binding of Band 3 fragments to PvTRAg38 by competition in solid phase ELISA. Increasing concentrations of untagged PvTRAg38 were mixed with a fixed concentration (0.5 μm) of histidine-tagged Band 3 fragments and allowed to react with immobilized GST-PvTRAg38 (50 nm) for 4 h at 37 °C. Bound recombinant histidine-tagged Band 3 fragments were detected with mouse anti-His₆ monoclonal antibody as described in the text. Binding in the absence of untagged PvTRAg38 was taken as percentage control for rest of the concentrations. The mean ± S.D. value of absorbance from three experiments is plotted. C, specificity of binding of Band 3 fragments to PvTRAg38 by competition in rosetting assay. The RE4-PvTRAg38 transfected CHO-K1 cells were preincubated with different concentrations (0–10 μm) of Band 3 fragments and then allowed to interact with human erythrocytes. The results are expressed as relative binding to positive control (binding of untreated PvTRAg38 transfected cells with RBCs). The mean and ± standard deviation of three experiments are reported. D–F, SPR analysis of PvTRAg38 interaction with Band 3 fragments B3F1 (D), B3F3 (E), and B3F6 (F). These Band 3 fragments (B3F1, B3F3, and B3F6) were immobilized on the different cells of CM5 chip surface. Three different concentrations of PvTRAg38 (0.4, 0.8, and 1.3 μm) were injected at flow rate of 30 μl/min over the surface of immobilized B3F1, B3F3, and B3F6 in independent experiments. Sensogram curves show dose-dependent responses of PvTRAg38 binding with these Band 3 fragments.