Determination of Radiolabeled Antibody Binding Affinity

The binding of chDAB4 to the previously published La/SSB epitope (Tran et al. 2002 (link)) was performed as described (Staudacher et al. 2018 ). The Lindmo binding assay (Lindmo et al. 1984 (link)) was used to determine the immunoreactive fraction (IRF) of radiolabeled preparations of chDAB4. EL4 lymphoma cells were fixed and permeabilized using 10% neutral buffered formalin and methanol as previously described (Al-Ejeh et al. 2009a ; Al-Ejeh et al. 2009b (link)), resuspended in PBS with 1% FCS and serially diluted in duplicate 0.5 mL aliquots at cell numbers ranging from 5.5 × 10⁷ to 3.4 × 10⁶. Then 0.5 mL of 50 ng/mL radiolabeled antibody in 1% FCS was added to the cell suspensions. After incubation at 4 °C overnight, cells were centrifuged at 300 g, and 0.5 mL of the supernatant from each sample was placed in a separate tube and the radioactivity in the cell pellets and the supernatant was measured using the Hidex Automatic Gamma Counter.