Ligandtracer instrument

Real-time in vitro binding and retention measurements of the radiolabeled conjugates were performed at room temperature using LigandTracer instruments (Ridgeview Instruments AB, Uppsala, Sweden) on CD44v6-positive A431, H314 and UM-SCC74B cells. In vitro binding specificity measurements were performed on LigandTracer instruments using MDA-MB-231 cells as negative controls. LigandTracer Grey was used for ¹²⁵I-AbD19384 and LigandTracer White was used for ¹²⁴I-AbD19384. Binding traces using several subsequent concentrations (0–90 nM) were obtained for at least 1 h per concentration, followed by a dissociation measurement for at least 15 h. The shapes of the real-time binding curves produced in LigandTracer were compared in the evaluation software TraceDrawer 1.6.1 (Ridgeview Instruments AB, Vänge, Sweden). Through kinetic fitting using a 1:1 and 1:2 models, the dissociation equilibrium constant K_D (corresponding to the apparent affinity), the association rate constant k_a and the dissociation rate k_d were obtained.

Binding affinities of the radiolabeled EVD to living SK-RC-52 renal carcinoma cells were measured using the LigandTracer instrument (Ridgeview Instruments). The TraceDrawer Software (Ridgeview Instruments AB) was used for evaluating the kinetics (31 (link)). The binding and dissociation kinetics were measured at room temperature. After a background measurement, increasing concentrations of radiolabeled EVD (1.8 and 5.4 nM) were added to the cells to determine the binding kinetics. After the association phase, the cell media was replaced and the retention in the dissociation phase was measured. The real time association and dissociation data were fitted into a one-to-one Langmuir binding model using the TraceDrawer Software. Both association and dissociation rates were determined, and the equilibrium dissociation constant was calculated.