Figure 3
Production of reformatted nanobodies in transfected HEK-6E cells and comparative analyses of binding affinities.
HEK-6E cells were transfected with cDNA-expression constructs encoding nanobodies fused C-terminally to chimeric tags (c-myc-His6x or avi-His6x) or to the Fc domain of mouse IgG2c (mFc). Transfected cells were cultured in serum free medium for 6 d. Cell supernatants were clarified by centrifugation and analyzed by SDS-PAGE and Coomassie staining (top). Qualitative comparison of nanobody affinities was performed by ELISA using serial dilutions of monovalent nanobodies and wells coated with CDTa. After 20 minutes of incubation, wells were washed three times to remove unbound proteins and bound nanobodies were detected with a peroxidase-conjugated monoclonal antibody directed against the c-myc tag. A c-myc tagged nanobody directed against ARTC229 was used as negative control. Symbols used for the respective nanobodies and the calculated dissociation constants are indicated in the middle. Representative results are shown for five families of CDTa-specific nanobodies. Results for CDTb-specific families are shown in Supplementary Fig. S3.
