Measuring Affibody-mcDM1 Conjugate Binding Affinity

To measure the binding affinity of ^99mTc-labeled affibody-mcDM1 conjugates to SKOV3 cells with high HER2 expression, a Ligandtracer Yellow instrument (Ridgeview Instruments, Vänge, Sweden) was used as described earlier [17 (link)]. In brief, one day before the experiment, 2 × 10⁶ cells were seeded to one side of an 89-mm petri dish and were incubated at 37 °C overnight. The measurements were performed at room temperature. Several concentrations (1, 2 and 5 nM) of the ^99mTc-labeled affibody-mcDM1 conjugates were added stepwise to measure the association phase. To measure the dissociation phase, the medium containing ^99mTc-labeled affibody-mcDM1 conjugates was exchanged to complete medium lacking conjugate. The data were collected, corrected for nuclide decay, and analyzed using Tracedrawer software (Ridgeview Instruments) for the equilibrium dissociation constants (K_D). Interaction map analysis (Ridgeview Diagnostics, Uppsala, Sweden) was performed to estimate the interaction heterogeneity.