Identification of an immunoreactive glycoprotein in the urine of sarcoma patients

Tumor-associated antigens (TAA) can be detected in the urine of sarcoma patients by a variety of assays. This study was designed to correlate antigen activity in three assays: complement fixation assay (CFA), enzyme-linked immunosorbent assay (ELISA), and Western blot (WB). This study identifies the antibody class responsible for TAA identification in these assays and characterizes the nature of the antigen. One hundred eighty-nine urine samples from eight sarcoma patients with known levels of TAA in CFA were tested in ELISA and WB. Allogeneic anti-TAA containing sera (1 degree antibody) from a sarcoma patient was reacted with urine samples followed by detection with alkaline phosphatase-linked goat anti-human IgG and IgM (2 degree antibody in both assays). Reactivity in CFA correlated to IgM reactivity in ELISA and WB (chi 2 test, P less than 0.001). No correlation was found to IgG reactivity in either assay. Reactivity in WB vs ELISA was also highly correlated for IgM (P less than 0.001). TAA was visualized in WB as a distinct pattern of repeating bands, with most bands being detected in the range 30,000-60,000 Da. The separation between bands approximated 2500-3000 Da, suggesting a molecule composed of repeating subunits. This study suggests that the antigen is glycoprotein in nature, and that the detecting antibody is of the IgM class.