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Isolation of Wilm's tumor antigens by chelation
411
Clinica Chimica Acta, 61 (1975) 411-414 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CCA 7137
ISOLATION
OF WILM’S TUMOR ANTIGENS
JOHN W. BEIERLE, Department of Southern (Received
BY CHELATION
KIM S. WISE, GARY N. TRUMP and SAMUEL
E. ALLERTON
of Microbiology and Immunology and Department of Biochemistry, California School of Dentistry, Los Angeles, Calif. 90007 (U.S.A.)
January
University
6, 1975)
Summary A procedure for ethylenediaminetetraacetate extraction of minced tumor was assessed as a method for isolating Wilm’s tumor antigens. An was detected by immunodiffusion using an adsorbed antiserum to this This antigen was also found in ethylenediaminetetraacetate extracts of cultures of nephroblastoma cells.
Wilm’s antigen extract. in vitro
Many tumor antigens appear to be located at the cell periphery [ 11. Since chelating agents have been used for cell dispersion [2] and removal of cell surface materials [ 31, ethylenediaminetetraacetate (EDTA) might therefore disengage surface antigens from tumors such as nephroblastomas. Phytic acid extraction has been reported effective in isolating Wilm’s tumor-specific antigens [ 41, although use of aqueous homogenates has proven unsuccessful [5]. We have previously isolated mucins from tumor tissues with EDTA [ 3,6,7] and have also observed similar substances in the sera of tumor-bearing patients [8] . These serum components are assumed to be secretion products, which may also include tumor-associated antigens. Thus, it was of interest to develop a method for isolating Wilm’s tumor antigens in yields stifficient for immunological and physical-chemical identification. Portions of Wilm’s tumors were obtained after surgery and kept on ice or frozen. The tissues were minced, washed in saline, and placed in 0.02% disodium-EDTA in 0.1 M calcium-and-magnesium-free phosphate buffered saline (pH 6.8). One ml of this EDTA solution was used per gram of tumor tissue. Extraction was performed at room temperature for two hours with continuous stirring. The mixture was then centrifuged for 15 minutes at 20 000 X g and the turbid supernate, the “EDTA extract”, was saved.
312
A serial monolayer culture of nephroblastoma cells (Ccl-31, TuWi) was obtained from the American Type Culture Collection, Rockville, Md. Cells were grown to confluency in Blake flasks using Eagle’s Minimum Essential Medium supplemented with 10% newborn calf serum. Con~uent cells were washed four times in situ with Hank’s Basal Salt Solution and incubated for 10 minutes at 37°C in the EDTA extractant. The detached cells were then triturated into a single cell suspension and their viability determined by trypan blue dye exclusion. The cells were immediately cent~fuged at 400 X g for 15 minutes, and the supemate was dialyzed versus water and lyophilized. Young male New Zealand white rabbits were injected in each footpad with 1 ml of EDTA tumor extract (20 mg total protein) emulsified with 1 ml of Freund’s complete adjuvant. Intraderm~ injections of the same composition and volume, were repeated at 3-week intervals, in multiple sites along the back. Four to five months later, potent antisera were obtained by injecting the rabbits with 0.5 ml (8-10 mg protein) of tumor extract in the ear vein. Two such injections were made followed by a week’s interval before final bleedings. Blood was obtained by venipuncture and serum was collected after clot retraction. Immunoglobulins were isolated from other serum proteins by precipitation with 50% saturated ammonium sulfate at pH 7.5 (purified antiserum). Immunoglobulins were diluted to 10 mg protein/ml in phosphate buffered saline. An equal volume of adsorbing solution or suspension (20-100 mg protein/ml of serum) was added to the diluted antiserum and incubated at 37°C for 2 hours, followed by 18 hours at 4°C. The resulting supemate was then concentrated $-fold. Adsorbing materials were normal human plasma, whole blood, and EDTA extracts and homogenates of normal human kidney. The reaction of unadsorbed purified antiserum with the tumor EDTA extract or normal human serum resulted in many precipitin bands after Ouchterlony double diffusion. Preimmunization rabbit serum did not precipitate with either the extract or normal human serum by this procedure. In contrast, a single precipitin line formed when antiserum, adsorbed with pooled human plasma and normal adult kidney EDTA homogenates, was allowed to react with the EDTA extract (Fig. 1A). This reaction could not be eliminated by further exposure of the antiserum to these adsorbants or to phosphate buffered saline homogenates of normal human kidney or whole human blood types. Antibody to Forssman antigen and to A and B blood groups, Rh+, failed to react in double diffusion tests with the tumor extract, over a wide series of dilutions. The anti-EDTA tumor extract did not react in radial diffusion tests with Abelev’s ~pha-fetoprotein 191. Further, neither human e~thropoietin nor hyaluronic acid, which are found in high concentrations in the sera of Wilm’s patients [ 10,111 reacted. EDTA extraction of the Wilm’s cell cultures was found to leave over 95% of the cells viable. This extract was concentrated to 109 cell equiv~en~/ml and reacted with the adsorbed antiserum. A line of complete identity was observed between this cultured cell extract and the tumor extract (Fig. 1B). Thus, the use of EDTA resulted in release of immunologically identical antigens from Wilm’s tumors and a cultured Wilm’s cell line. It appears, therefore, that chelation may prove useful in isolating such membrane-bound antigens.
413
Fig. 1. Immunodlffusion reactions of adsorbed purified rabbit antiserum to Wllm’s tumor ED TA exl xacts. A. Adsorbed antiserum reactions with the immunogen and normal plasma and kidney exti :acts. C:enter wall: antiserum adsorbed with pooled human plasma and normal adult human kidney EDTA sz&act (R6). Peripheral wells (clockwise from top): Wilm’s tumor EDTA extract (WTE). normal human ki dney e:xtract (NHKE), WTE repeat, pooled normal human plasma (C), WTE. The Wllm’s tumor extract wasd lluted 1 : 5 with phosphate buffered saline. B. Adsorbed antiserum reactions with the tumor an.d TuH Ii cell culture EDTA extracts. Center well: adsorbed antiserum (R5). Peripheral wells (clockwise from top): WTE. EDTA extract of cultured Wllm’s tumor cells (WT CELL EXT.), WTE. normal human Lplasm ia (Cl. WTE.
414
Acknowledgements The technical expertise of Mrs Veronica Routt is gratefully acknowledged. This work was supported by: a grant from the New York Cancer Research Institute, Inc. (J.W.B.), NIH Training Grant DE 0094-07 (K.S.W.), NIH Program Grant DE 02847 (S.E.A. and J.W.B.), NIH Cancer Center Grant 1 PO1 CA14089 (S.E.A.), and an NIH Career Development Award 1 K04 CA 70675 (J.W.B.). References 1 2 3 4 5 6 7 8 9 10 11
P. Gold. M. Gold and S.O. Freedman, Cancer Res., 28 (1971) 1279 L. Weiss, Int. Rev. Cytol.. 9 (1960) 187 J.W. Beierle, Science, 161 (1968) 798 P. Burtin and M.C. Gendron, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 2051 E. Linder, Int. J. Cancer, 4 (1969) 232 S.E. Allerton, J.W. Beierle. D.R. Powars and L.A. Bavetta, Cancer Res., 30 (1970) K. Wise, J. Beierle, S. Allerton and D. Powars. Fed. Proc.. 30 (1971) 1279 D.R. Powars. S.E. Allerton. J.W. Beierle and B.B. Butler, Cancer, 29 (1972) 1597 G.I. Abelev. Prog. Exp. Tumor Res., 7 (1965) 104 W.G. Thurman. H. Grabstald and P.H. Lieberman, Arch. Intern. Med.. 117 (1966) B.S. Morse and M. Nussbaum, Am. J. Med., 42 (1967) 996
679
280
Clinica Chimica Acta, 61 (1975) 411-414 @ Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
SHORT COMMUNICATION CCA 7137
ISOLATION
OF WILM’S TUMOR ANTIGENS
JOHN W. BEIERLE, Department of Southern (Received
BY CHELATION
KIM S. WISE, GARY N. TRUMP and SAMUEL
E. ALLERTON
of Microbiology and Immunology and Department of Biochemistry, California School of Dentistry, Los Angeles, Calif. 90007 (U.S.A.)
January
University
6, 1975)
Summary A procedure for ethylenediaminetetraacetate extraction of minced tumor was assessed as a method for isolating Wilm’s tumor antigens. An was detected by immunodiffusion using an adsorbed antiserum to this This antigen was also found in ethylenediaminetetraacetate extracts of cultures of nephroblastoma cells.
Wilm’s antigen extract. in vitro
Many tumor antigens appear to be located at the cell periphery [ 11. Since chelating agents have been used for cell dispersion [2] and removal of cell surface materials [ 31, ethylenediaminetetraacetate (EDTA) might therefore disengage surface antigens from tumors such as nephroblastomas. Phytic acid extraction has been reported effective in isolating Wilm’s tumor-specific antigens [ 41, although use of aqueous homogenates has proven unsuccessful [5]. We have previously isolated mucins from tumor tissues with EDTA [ 3,6,7] and have also observed similar substances in the sera of tumor-bearing patients [8] . These serum components are assumed to be secretion products, which may also include tumor-associated antigens. Thus, it was of interest to develop a method for isolating Wilm’s tumor antigens in yields stifficient for immunological and physical-chemical identification. Portions of Wilm’s tumors were obtained after surgery and kept on ice or frozen. The tissues were minced, washed in saline, and placed in 0.02% disodium-EDTA in 0.1 M calcium-and-magnesium-free phosphate buffered saline (pH 6.8). One ml of this EDTA solution was used per gram of tumor tissue. Extraction was performed at room temperature for two hours with continuous stirring. The mixture was then centrifuged for 15 minutes at 20 000 X g and the turbid supernate, the “EDTA extract”, was saved.
312
A serial monolayer culture of nephroblastoma cells (Ccl-31, TuWi) was obtained from the American Type Culture Collection, Rockville, Md. Cells were grown to confluency in Blake flasks using Eagle’s Minimum Essential Medium supplemented with 10% newborn calf serum. Con~uent cells were washed four times in situ with Hank’s Basal Salt Solution and incubated for 10 minutes at 37°C in the EDTA extractant. The detached cells were then triturated into a single cell suspension and their viability determined by trypan blue dye exclusion. The cells were immediately cent~fuged at 400 X g for 15 minutes, and the supemate was dialyzed versus water and lyophilized. Young male New Zealand white rabbits were injected in each footpad with 1 ml of EDTA tumor extract (20 mg total protein) emulsified with 1 ml of Freund’s complete adjuvant. Intraderm~ injections of the same composition and volume, were repeated at 3-week intervals, in multiple sites along the back. Four to five months later, potent antisera were obtained by injecting the rabbits with 0.5 ml (8-10 mg protein) of tumor extract in the ear vein. Two such injections were made followed by a week’s interval before final bleedings. Blood was obtained by venipuncture and serum was collected after clot retraction. Immunoglobulins were isolated from other serum proteins by precipitation with 50% saturated ammonium sulfate at pH 7.5 (purified antiserum). Immunoglobulins were diluted to 10 mg protein/ml in phosphate buffered saline. An equal volume of adsorbing solution or suspension (20-100 mg protein/ml of serum) was added to the diluted antiserum and incubated at 37°C for 2 hours, followed by 18 hours at 4°C. The resulting supemate was then concentrated $-fold. Adsorbing materials were normal human plasma, whole blood, and EDTA extracts and homogenates of normal human kidney. The reaction of unadsorbed purified antiserum with the tumor EDTA extract or normal human serum resulted in many precipitin bands after Ouchterlony double diffusion. Preimmunization rabbit serum did not precipitate with either the extract or normal human serum by this procedure. In contrast, a single precipitin line formed when antiserum, adsorbed with pooled human plasma and normal adult kidney EDTA homogenates, was allowed to react with the EDTA extract (Fig. 1A). This reaction could not be eliminated by further exposure of the antiserum to these adsorbants or to phosphate buffered saline homogenates of normal human kidney or whole human blood types. Antibody to Forssman antigen and to A and B blood groups, Rh+, failed to react in double diffusion tests with the tumor extract, over a wide series of dilutions. The anti-EDTA tumor extract did not react in radial diffusion tests with Abelev’s ~pha-fetoprotein 191. Further, neither human e~thropoietin nor hyaluronic acid, which are found in high concentrations in the sera of Wilm’s patients [ 10,111 reacted. EDTA extraction of the Wilm’s cell cultures was found to leave over 95% of the cells viable. This extract was concentrated to 109 cell equiv~en~/ml and reacted with the adsorbed antiserum. A line of complete identity was observed between this cultured cell extract and the tumor extract (Fig. 1B). Thus, the use of EDTA resulted in release of immunologically identical antigens from Wilm’s tumors and a cultured Wilm’s cell line. It appears, therefore, that chelation may prove useful in isolating such membrane-bound antigens.
413
Fig. 1. Immunodlffusion reactions of adsorbed purified rabbit antiserum to Wllm’s tumor ED TA exl xacts. A. Adsorbed antiserum reactions with the immunogen and normal plasma and kidney exti :acts. C:enter wall: antiserum adsorbed with pooled human plasma and normal adult human kidney EDTA sz&act (R6). Peripheral wells (clockwise from top): Wilm’s tumor EDTA extract (WTE). normal human ki dney e:xtract (NHKE), WTE repeat, pooled normal human plasma (C), WTE. The Wllm’s tumor extract wasd lluted 1 : 5 with phosphate buffered saline. B. Adsorbed antiserum reactions with the tumor an.d TuH Ii cell culture EDTA extracts. Center well: adsorbed antiserum (R5). Peripheral wells (clockwise from top): WTE. EDTA extract of cultured Wllm’s tumor cells (WT CELL EXT.), WTE. normal human Lplasm ia (Cl. WTE.
414
Acknowledgements The technical expertise of Mrs Veronica Routt is gratefully acknowledged. This work was supported by: a grant from the New York Cancer Research Institute, Inc. (J.W.B.), NIH Training Grant DE 0094-07 (K.S.W.), NIH Program Grant DE 02847 (S.E.A. and J.W.B.), NIH Cancer Center Grant 1 PO1 CA14089 (S.E.A.), and an NIH Career Development Award 1 K04 CA 70675 (J.W.B.). References 1 2 3 4 5 6 7 8 9 10 11
P. Gold. M. Gold and S.O. Freedman, Cancer Res., 28 (1971) 1279 L. Weiss, Int. Rev. Cytol.. 9 (1960) 187 J.W. Beierle, Science, 161 (1968) 798 P. Burtin and M.C. Gendron, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 2051 E. Linder, Int. J. Cancer, 4 (1969) 232 S.E. Allerton, J.W. Beierle. D.R. Powars and L.A. Bavetta, Cancer Res., 30 (1970) K. Wise, J. Beierle, S. Allerton and D. Powars. Fed. Proc.. 30 (1971) 1279 D.R. Powars. S.E. Allerton. J.W. Beierle and B.B. Butler, Cancer, 29 (1972) 1597 G.I. Abelev. Prog. Exp. Tumor Res., 7 (1965) 104 W.G. Thurman. H. Grabstald and P.H. Lieberman, Arch. Intern. Med.. 117 (1966) B.S. Morse and M. Nussbaum, Am. J. Med., 42 (1967) 996
679
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