Validation of Humanized Mouse Antibodies

Antibody therapy is being developed and tested as one of the most promising agents for treatment of various human diseases. As of March 2016, 350 antibody candidates are in clinical trials. Many of these antibodies have been taken from animals and “humanized” by genetic modification. Our experiment tests monoclonal antibodies that have been harvested from mouse hybridoma (spleen-derived) cells and cloned until the heavy and light chains of the antibody can be recognized by human cells. Because of this “humanization” procedure, basic antibody assays are needed to demonstrate that the binding, specificity and functional parameters of the antibodies are not lost during cloning. The purpose of this research is to perform this validation through assays. The antibodies are harvested from cell supernatants and purified using affinity chromatography. Then, the antibody fractions are tested for reactivity with human target protein PTP-Beta, via western blot and ELISA procedures. Cross-reactivity of the antibody is tested against human eta and cynomolgus beta proteins. The work presented in this poster describes results from one particular mouse antibody, R15, which has been humanized to functionally enhance endothelial survival. The goal is to generate a therapeutic antibody candidate that improves endothelium survival and stability