Time course for the interaction of an IGFR therapeutic antibody with its target. The cells are treated with either a vehicle control (far left picture) or the therapeutic IGFR antibody for 20 minutes (middle left picture), 30 minutes (middle right picture) or 2 hours (far right picture). All the treatments were then fixed, permeabilised and stained with Hoechst 33342 (blue nuclei) and another IGFR antibody (red staining). The therapeutic antibody was visualised with an anti-human alexa 488 secondary antibody (green staining).

High Content Analysis offers a very powerful tool for the development of therapeutic antibodies. The real advantage of therapeutic antibodies from a screening perspective is that they can easily be visualised in cell assays by simply using an appropriately fluorescently-labelled secondary antibody.

In the above example, we performed a dual labelled experiment where we detected both the therapeutic antibody and its target. While the dual labelling of the IGFR receptor might seem at first superflous, the critical difference between the therapeutic and the other IGFR antibody is that the latter is added after the cells are fixed and not, unlike the therapeutic, during the treatment phase of the experiment. The images above show complete co-localisation (yellow staining profile) of the therapeutic antibody with its target receptor at all time points post treatment. This result is important because it shows that the antibody causes the IGFR receptor to be internalised (note the change to punctate staining at 30 minutes) and then degraded without recycling at 2 hours. The complete absence of either green or red staining (with the exception of the control) during the time course of the experiment indicates that new receptor is not created within 2 hours nor is there any therapeutic antibody dissociated from its target.