Premium
Purification of antigens from urine of a sarcoma patient by affinity chromatography
Author(s) -
Huth James F.,
Gupta Rishab K.,
Morton Donald L.
Publication year - 1981
Publication title -
journal of surgical oncology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.201
H-Index - 111
eISSN - 1096-9098
pISSN - 0022-4790
DOI - 10.1002/jso.2930180304
Subject(s) - antigen , urine , affinity chromatography , antibody , titer , antiserum , microbiology and biotechnology , chemistry , medicine , immunology , biology , biochemistry , enzyme
Abstract Previous publications from our laboratory have demonstrated the presence of antigens in the urine of cancer patients detectable by the complement fixation (CF) assay. The purpose of this study was to develop a methodology for purification of these tumor‐associated antigen(s) using xenogeneic antisera as a specific immunoabsorbent. Urine possessing high antigen titer by CF was collected from sarcoma patients. Xenoantiserum to the urinary antigens was prepared by injection of concentrated, dialyzed urine mixed with complete Freund's adjuvant into rabbits. Control xenoantisera were obtained by injection of pooled human normal urine in an identical manner. The antisarcoma urine xenoantisera were absorbed with human normal liver and insolubilized human normal serum to remove cross‐reacting antibodies to normal human components. The immunoglobulin fraction of the xenoantisera was isolated by ammonium sulfate precipitation and covalently linked to Sepharose 4B beads. Urine from a sarcoma patient was reacted with the immunobeads, and the affinity absorbed antigen(s) were eluted with 3.5 M potassium thiocyanate. The affinity purified urinary antigen had a CF titer of 1:128 against xenoantisera that had been quantitatively absorbed with human normal liver cells and the insolubilized normal human serum. Absorption of the xenoantiserum with cultured allogeneic sarcoma cells removed all activity against the affinity purified antigen(s). The results suggest that the cultured sarcoma cells expressed antigens immunologically similar to those present in the urine of sarcoma patients, and that these antigens can be isolated from urine of cancer patients and purified by affinity chromatography. The affinity purified antigens(s) was labeled with 125 I by the vapor phase chloramine‐T method, and a radioimmunoassay was developed. By radioimmunoassay, antisarcoma urine xenoantiserum absorbed with human normal liver and insolubilized serum had a titer of 1:4000 against the affinity purified antigen. Xenoantisera to pooled normal human urine treated similarly showed no reactivity to the affinity purified antigen. Polyacrylamide gel electrophoresis of the radiolabeled antigen(s) showed a single peak in the 40,000‐ to 50,000‐ dalton range.