High-affinity binding of TTCF by antibodies generated from plasmablasts and memory B cells. Saturation binding experiments were performed to determine the affinities of recombinant antibodies. TTCF antigen was labeled with europium, which emits a strong fluorescent signal at 615 nm on incubation with a chelating reagent. Antibodies were immobilized in a 96-well plate and incubated with TTCF-europium (100nM to 4pM) for 2 hours at 37°C. Fluorescence counts at 615 nm were recorded and KD calculated by the use of nonlinear regression analysis. A control antibody (clone 8.18.C5) that was also produced in CHO-S cells was included in all experiments. (A) Recombinant TTCF Abs 1 and 2 were generated from TTCF tetramer-positive plasmablasts (donor 1). (B) TTCF Abs 3, 4, and 5 originated from TTCF tetramer-positive memory B cells of 3 different donors.
