Polyclonal antibodies targeting defined epitopes on cancer biomarker MUC16

Measurements of the peptide epitope CA125 are crucial in ovarian cancer care. Despite its value as a tool in disease management, CA125 is an imperfect biomarker, with rates of false positive and false negative response that preclude its use as a screening tool in the general population. The monoclonal antibodies that perform capture and recognition in the CA125 test-OC125 and M11-were developed using complex targets as immunogens and recognize epitopes that have been located on mucin16 (MUC16) but otherwise remain undefined at the molecular level. We hypothesized that new antibodies recognizing MUC16 peptides of known sequence could enable the development of assay platforms that overcome the limitations of the current CA125 test. Here, we report the development and characterization of three sets of polyclonal antibodies that recognize known MUC16 peptides. Peptides that appear several times within MUC16's highly conserved tandem repeat region were used to immunize two sets of rabbits. Affinity-isolated antibodies were characterized by enzyme-linked immunosorbent assay (ELISA) and Western blot. All three peptides were successful antigens, as indicated by the ability of the resulting polyclonal antibodies to bind individually expressed proteins from the MUC16 tandem repeat region. In particular, the polyclonal antibodies raised against peptide 2 (ELGPYTLDRNSLYV) bound to all tandem repeats tested. Peptide 2 antibodies were able to detect intact MUC16 from ovarian cancer cells in a surface plasmon resonance (SPR) assay. In flow cytometry experiments, peptide 2 antibodies bind to MUC16-positive cells (OVCAR3) and do not bind to MUC16-negative cells (OVCAR8). The binding pattern of these polyclonal antibodies opens the possibility of developing new monoclonal antibodies recognizing known epitopes within the tandem repeat region. These reagents may eventually complement or replace the monoclonal antibodies used in the current clinical CA125 test.