Determination of Immunoreactivity of 64Cu-Immunoconjugates

The immunoreactivity of each ⁶⁴Cu-labelled immunoconjugate was determined by a conventional saturation assay according to the method recommended by Denoël et al. [28 (link)]. Briefly, increasing concentrations of BT474 cells (1.10⁶, 2.10⁶, 5.10⁶ and 10.10⁶ cells per tube) were incubated in 0.5 mL of binding media (25 mM HEPES pH 7, containing 0.2% of BSA, completed with Dulbecco’s Modified Eagle’s Medium (DMEM) F12 (1/1, v/v) glutamax) with 0.60 pmol of the appropriate immunoconjugate (6.8–27.4 GBq/µmol) in a final volume of 1 mL. After 30 min of gentle shaking, the samples were centrifuged at 460× g for 8 min at 4 °C. The supernatants were removed, the cell pellets were washed with PBS containing 0.2% BSA and centrifuged again at 460× g for 8 min. The supernatants and pellets collected were recovered separately for radioactivity counting using a γ-counter (Wallac 1480 Wizard^® 3‘‘, Perkin Elmer, Villebon sur Yvette, Every, France). The IRF was determined by performing a rectangular hyperbolic fit (one site-specific binding, GraphPad Prism 9.4.1) of the binding curve obtained by plotting B/(B+S) as a function of cell concentration, where B and S are the pellet and supernatant activities counted, respectively. IRF was obtained by extrapolating the quadratic hyperbola value at infinite antigen concentration. All experiments were performed in triplicate.