Association between allo-immunoreactive and xeno-immunoreactive subunits of a glycoprotein tumor-associated antigen

Using allogeneic antibody, we previously described a tumor-associated antigen (TAA) in the urine of 68% of melanoma patients. The TAA was purified from urine of a melanoma patient and used as immunogen to develop a murine monoclonal antibody (AD1-40F4) and xenopolyclonal antibodies in a baboon. Sera from melanoma patients treated with whole melanoma cell vaccine were used as the source of human antibody to the glycoprotein antigen. Treatment with 2-mercaptoethanol and separation by sodium dodecyl sulfate/polyacrylamide gel electrophoresis resolved the high-molecular-mass glycoprotein TAA into smaller subunits. Immunoblot analysis indicates that the murine monoclonal antibody (AD1-40F4) recognized a 90-100-kDa subunit of the antigen while human anti-TAA antibodies primarily recognized a 65-kDa subunit in addition to the 90-100-kDa subunit. Baboon polyclonal antibodies recognized the same subunits plus a 120-kDa subunit. Blocking studies indicated that the murine monoclonal and baboon polyclonal antibodies recognized the closely related epitopes on the 90-100-kDa subunit, while human antibodies recognized an epitope entirely distinct from that recognized by the mouse antibody. These results demonstrate the epitope complexity associated with the high-molecular-mass glycoprotein TAA.