Figure 4. Characterization of pH sensitivity for Sso7d-ev-hFc.

(A) End-point assay to determine pH sensitivity for Sso7d-ev-hFc. Yeast cells displaying Sso7d-his-hFc were incubated with 2 µM hFc-biotin and the yeast-hFc complexes were dissociated in buffers at pH 7.4 and pH 4.5. Undissociated hFc remaining on yeast surface was detected using strep-PE. A cell sample where no dissociation step was carried out after hFc labeling at pH 7.4, and unstained cells were used as controls. (C) ELISA for determination of the apparent K_D of binding between hFc and Sso7d-ev-hFc, at pH 7.4 and pH 4.5. hIgG (2 µg/ml) was immobilized on a microtiter plate and incubated with twelve different concentrations of soluble Sso7d-ev-hFc. hIgG-bound Sso7d-ev-hFc was detected using an anti-his-alkaline phosphatase conjugated antibody, and p-nitrophenyl phosphate as the substrate. Error bars indicate the standard deviation of absorbance measurements at 405 nm.