Competitive Radiolignd Cell Binding Assay

A competitive radioligand cell binding assay was performed in order to determine the equilibrium binding dissociation constant of 10H6 at pH 7.4. Briefly, ~10⁶ LS174T cells were suspended into 1 mL of PBS/1% BSA at pH 7.4 containing various concentrations ranging from 0.01–1000 nM of unlabeled 10H6 + 1 nM ¹²⁵I-10H6. Cells were incubated for 90 min at 4°C to reach equilibrium. Cells were pelleted by centrifugation at 1500 x g for 5 min and washed three times with PBS/1% BSA, pH 7.4. Bound antibody was measured by counting gamma radiation of cell pellets.