Antibody binding assay to screen mouse and human antibodies:
As shown in Figure 2, the antibody screening procedure was successfully applied to 12 different RTK, using either red-labeled antispecies conjugates or red-labeled antitag antibodies. The assay window ranges from 3 to 34 depending on the concentration and antibody affinity.
The same assay format was used to determine the affinity constant (KD) of the previously screened antibodies by increasing the concentration of primary antibody in the presence of a saturating concentration of Red-labeled secondary antibody. The KD values obtained were in the nanomolar range and in good agreement with the antibody specifications.
Moreover, taking advantage of the nondestructive HTRF measurement, we were able to record the fluorescence signal evolution at different time points during one hour and therefore calculate the Kon constant. In the same way, we also measured the Koff rate by adding a large excess of unlabeled antibody at the equilibrium.