Purification of antigens from urine of a sarcoma patient by affinity chromatography

J. F. Huth, R. K. Gupta, D. L. Morton

Research output: Contribution to journalArticle

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 injecion 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 antigen(s) was labeled with 125I 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 500,000 dalton range.

Original languageEnglish (US)
Pages (from-to)237-247
Number of pages11
JournalJournal of Surgical Oncology
Volume18
Issue number3
StatePublished - 1981

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Affinity Chromatography
Sarcoma
Urine
Antigens
Radioimmunoassay
Liver
Serum
Freund's Adjuvant
Ammonium Sulfate
Neoplasm Antigens
Sepharose
Immunoglobulins
Immune Sera
Polyacrylamide Gel Electrophoresis
Cultured Cells
Neoplasms
Rabbits
Injections

ASJC Scopus subject areas

  • Oncology
  • Surgery

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Purification of antigens from urine of a sarcoma patient by affinity chromatography. / Huth, J. F.; Gupta, R. K.; Morton, D. L.

In: Journal of Surgical Oncology, Vol. 18, No. 3, 1981, p. 237-247.

Research output: Contribution to journalArticle

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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 injecion 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 antigen(s) was labeled with 125I 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 500,000 dalton range.",
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