~ouRiv.4L OF SURGICAL RESEARCH 50,475-479 (1991) Identification of an lmmunoreactive Glycoprotein in the Urine of Sarcoma Patients JAMES F. HUTH, M.D., VANCE FOWLER, B.S., AND SUE TAYLOR, MS. Department of Surgery, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 Presented at the Annual Meeting of the Association for Academic Surgery, Houston, Texas, November 14-17,1990 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), en- zyme-linked immunosorbent assay (ELISA), and West- ern 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 (lo anti- body) from a sarcoma patient was reacted with urine samples followed by detection with alkaline phospha- tase-linked goat anti-human IgG and IgM (2’ antibody in both assays). Reactivity in CFA correlated to IgM reactivity in ELISA and WB (x2 test, P < 0.001). No correlation was found to IgG reactivity in either assay. Reactivity in WB vs ELISA was also highly correlated for IgM (P <c 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. Thesep- aration 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. C 1991 Academic Press. Inc. INTRODUCTION Immunoreactive substances found in the urine of sar- coma patients with measurable tumor burdens have previously been described [l]. It is inferred that these substances are tumor-associated antigens (TAA) since reactivity of urine from sarcoma patients has been dem- onstrated in both complement fixation assays (CFA) and enzyme-linked immunosorbent assays (ELISA) against sera from autologous and allogeneic sarcoma sources [2,3]. However, urine obtained from normal hu- man volunteers has not usually shown reactivity against these allogeneic serum sources, and autologous reactiv- ity is rarely seen in normal subjects. Further studies have shown that levels of antigen excretion vary consid- erably during the course of disease. Urinary antigen lev- els rise dramatically when patients harboring a known disease undergo cytotoxic chemotherapy or radiation therapy [4]. Following excision of primary tumors, anti- gen levels fall due to the removal of the antigen source. However, longitudinal studies of postoperative patients have shown that the return of detectable antigen in the urine is predictive of subsequent disease recurrence in the vast majority of patients [5]. Additional studies have demonstrated that a correla- tion exists between the presence of antigen in the urine and circulating immune complexes in the serum of these patients. Renal biopsies of melanoma and sarcoma pa- tients demonstrating the phenomenon of antigen excre- tion in the urine have shown immune complex deposi- tion in the glomerular basement membrane, suggesting that immune complex deposition may play a role in the transfer of TAA from the serum to the urine of these patients [6]. We have recently used an immunoblotting technique consisting of polyacrylamide gel electrophoresis (PAGE) and Western transfer followed by reaction with immunoprobes to immunologically identify the proteins responsible for the reactivity seen in the CFA and ELISA assays. This paper describes the techniques used for this isolation and compares reactivity to TAA by these various techniques, CFA, ELISA, and immu- noblot. MATERIALS AND METHODS One hundred eighty-nine urine samples obtained from eight sarcoma patients with known levels of TAA as de- termined by CFA were selected for further purification and analysis by ELISA, PAGE, and Western blot. These samples were centrifuged (17,OOOg, 10 min at 4OC), fil- tered, and concentrated loo-fold with a hollow fiber concentrator and a PM-10 membrane (10,000 MW cut- off) filtration cell (Amicon). The concentrate was dia- 475 0022-4804/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.