Oncology 31: 17-21 (1975)
Tumor-Specific Antigen Binding Activity of Human Serum S. De Carvalho Belmont Medical Clinic, Bellflower, Calif.
Key Words. Tumor-specific antigen • TSA serum-binding factor Abstract. A factor is revealed in the serum of both normal and cancer-bearing individ uals which binds circulating tumor-specific antigens. This bond does not appear to be immunologic.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
In the course of extensive studies on the immunodiagnostic detection of tumor specific antigens (TSA) in the serum of patients with active neoplastic diseases (1) by heterologous (equine) specific antibodies (2), it has become apparent that the serum of several hundred individuals tested, both from normal and cancer patients, was able to produce lines of precipitation in agar with TSA but never with similar preparations from normal organs and tissues (2) (fig. 1). Quantitatively, the difference between cancer and normal individuals was not very marked. When serial log2 dilutions of cancer serum and noncancer serum were run in parallel in a linear-pattern agar diffusion plate against a TSA preparation containing 1 mg% of protein (3), it was found that cancer sera reached a maximum dilution of 2~s of TSA and noncancer sera a dilution of 2“6. Since many noncancer sera reacted below 2“5, it was felt that this variation is probably more individual than disease-related. The lines of precipitation in agar are unusually wide, ill-demarcated and fuzzy, though strong (fig. 2). Several batches of commercial preparations of 7-globulin from pools of human serum failed constantly to show any reaction with TSA. The TSAbinding serum factor is thermostable (56 °C for 1 h) and resists prolonged peri ods of eutectic freezing (up to 3 months at - 2 0 °C). The exact nature of this factor was not yet established. Studies are under way to attempt to identify this factor and to disclose the dynamics of the serum TSA-binding capacity. The possible interference of the serum TSA-binding ability with detection of TSA in the serum by specific heterologous precipitating antibody was studied in the following experiments.
De Carvalho
18
An original preparation of TSA containing 1 mg% of protein was concen trated isosmolarly with polyacrylamide (4) to 4 mg% and two types of serial dilutions from 4 to 0.2 mg% protein TSA obtained, one in normal human serum (NHS) and another in phosphate-buffered saline (PBS), pH 7.2 (fig. 3). In circu lar patterns, horse antitumor multivalent hyperimmune serum (batch HTU9C" s N '2) was placed in the central well and allowed to react simultaneous ly with NHS, with TSA (1 mg%) and with each one of the dilutions of NHS + TSA and PBS + TSA. The serum TSA-binding capacity is revealed and measured in this experi ment in which the horse antiserum (HTU9) reacted with 0.2 mg% TSA in PBS but only with 2 mg% of TSA in human serum. Relatively to the reactant horse serum, human serum masks TSA by a factor of 10 over the buffer. The requirement of at least ten times as much TSA in serum as in buffer for the detection by the horse antiserum seems to indicate that level as the saturated serum TSA-binding capacity beyond which TSA is left free for reaction with horse antiserum. However, the fact that most cancer sera still naturally bind additional TSA would indicate not only that there is an unsaturated TSA-binding capacity but
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Fig. 1. The center well contains highly concentrated TSA (4 mg%). Peripheral wells contain alternatively a human serum from a normal source, 2, 4 and 6, and from a cancer patient, 1, 3 and 5. Besides sharp lines of ‘reaction’ there are strong diffuse bands. The alternate arrangement of the peripheral wells gives rise to reactions of identity and non identity.
Tumor-Specific Antigen Binding Activity of Human Serum
19
Fig. 2. The center well contains horse antitumor hyperimmune 7 -globulin. Wells 1 and 4 contain unabsorbed tumor antigens with multiple line reactivity. Well 2 contains extract of normal organs and tissues showing identity of reaction with one line of the unabsorbed tumor antigens. Wells 3 and 6 contain buffered solutions; well 5 contains normal human serum. The ‘reaction’ between TSA (4) and serum (5) is not as clear-cut; it is noncontiguous with the residual antinormal activity of the center well, but it is partly contiguous with its antitumor activity.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
that the immunologic reaction with the heterologous antibody is able to dis sociate the serum-binding factor from TSA and reveal the tumor antigen. To what quantitative extent this dissociation occurs is not known. The reaction of human serum with TSA is instantaneous, requires no in cubation and produces a turbidity which cannot be centrifuged at or below 14,000 £ even though it shows a cloudiness in agar. This fact plus the non-7-globulin nature of the TSA-binding factor seem to indicate that we are not in the presence of an antigen-antibody reaction. It is known that positively charged biologic and synthetic polymers can produce ‘precipitation’ lines in agar with basic proteins (5). Similar binding of foreign proteins exists as a form of archyphylectic immunity before the emergence of the 7-globulin-secreting immuno cyte (6). Purified hepatitis B surface antigen is known to ‘cross-react’ with serum proteins such as apolipoprotein, lactoferrin, albumin and oq-T-glycoprotein by nonspecific absorption (7). Further examples of nonspecific precipitation in immunodiffusion tests and its mechanisms are discussed extensively by Crowle (8). In the the system proposed for the detection of circulating tumor antigens
De Carvalho
20
(1), the technique calls for quantitative absorption in the agar plate of whatever reactivity the horse antitumor serum may depict against antigens from normal tissues. This is accomplished by loading the agar through the center well with an isosmolarly concentrated pool of NHS. The possibility that this residual NHS could react with TSA in the cancer serum from the opposing well and thus produce an artifact does not exist. The previous experiments show that TSA is already bound in its own cancer serum and only heterologous immune serum, but not the isologous nonimmune serum will react with it. The characteristics of the specific lines of reaction lend further support to this possibility. A third element of interference, easy to rule out, is the presence of allotypic serum reactions. Characterization of this binding factor may be very important for the inter actions between the dual immune system. Cooperband's (9) results point to an a2 -globulin as a natural immunosuppressant of T cells. The biologic significance of the serum TSA-binding factor, its nature, non specificity, and its antigen-masking effect (7), hence its role in the host-tumorimmunologic conflict present yet another challenge to the cancer immunologist.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Fig. 3. Polyacrylamide rod imbibition stages following immersion in TSA solution. The rods increase 70-fold their volume by absorbing water and solute isosmolarly from the protein solution.
Tumor-Specific Antigen Binding Activity of Human Serum
21
References 1
2
3 4 5
6 7
8 9
De Carvalho, S.: Detection of neoantigens in the serum of patients with active neo plastic diseases by the absorption-immunodiffusion method. Oncology 27: 193-234 (1973). De Carvalho, S.: In vitro properties of a specific heterologous anti-leukemic and anti tumor cytotoxic antibody and their bearing on its application in vivo. Expl. molec. Path. 2: suppl., pp. 150-178 (1963). De Carvalho, S.: Preparation of antigens specific of human breast carcinoma by an immunochromatographic method. Nature, Lond. 203: 1186-1188 (1964). Curtain, C.: Concentrating protein solution. Nature, Lond. 203: 1380 (1964). Mora, P.T. and Young, B.C.: Reversible blocking of T2 bacteria bacteriophage anti serum with polyelectrolytes. J. biol. Chem. 237: 1870-1875 (1962). Papermaster, B.W.; Condie, R.M.; Finstad, T.K.; Good, R.A., and Gabrielsen, A.E.: Phylogenetic development of adaptive immunity. Fed. Proc. 22: 1152-1155 (1963). Goudeau, A.; Houwen, B., and Daukert, T.: Cross-reactions of human serum-proteins with HBsAg. Lancetii: 1325 (1974). Crowle, A.T.: Immunodiffusion, pp. 174-179 (Academic Press, New York 1973). Cooperband, S.R.: Science 159: 1243 (1968).
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Dr. S. De Carvalho, Belmont Medical Clinic, Bellflower, C4 90706 (USA)
Tumor-Specific Antigen Binding Activity of Human Serum S. De Carvalho Belmont Medical Clinic, Bellflower, Calif.
Key Words. Tumor-specific antigen • TSA serum-binding factor Abstract. A factor is revealed in the serum of both normal and cancer-bearing individ uals which binds circulating tumor-specific antigens. This bond does not appear to be immunologic.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
In the course of extensive studies on the immunodiagnostic detection of tumor specific antigens (TSA) in the serum of patients with active neoplastic diseases (1) by heterologous (equine) specific antibodies (2), it has become apparent that the serum of several hundred individuals tested, both from normal and cancer patients, was able to produce lines of precipitation in agar with TSA but never with similar preparations from normal organs and tissues (2) (fig. 1). Quantitatively, the difference between cancer and normal individuals was not very marked. When serial log2 dilutions of cancer serum and noncancer serum were run in parallel in a linear-pattern agar diffusion plate against a TSA preparation containing 1 mg% of protein (3), it was found that cancer sera reached a maximum dilution of 2~s of TSA and noncancer sera a dilution of 2“6. Since many noncancer sera reacted below 2“5, it was felt that this variation is probably more individual than disease-related. The lines of precipitation in agar are unusually wide, ill-demarcated and fuzzy, though strong (fig. 2). Several batches of commercial preparations of 7-globulin from pools of human serum failed constantly to show any reaction with TSA. The TSAbinding serum factor is thermostable (56 °C for 1 h) and resists prolonged peri ods of eutectic freezing (up to 3 months at - 2 0 °C). The exact nature of this factor was not yet established. Studies are under way to attempt to identify this factor and to disclose the dynamics of the serum TSA-binding capacity. The possible interference of the serum TSA-binding ability with detection of TSA in the serum by specific heterologous precipitating antibody was studied in the following experiments.
De Carvalho
18
An original preparation of TSA containing 1 mg% of protein was concen trated isosmolarly with polyacrylamide (4) to 4 mg% and two types of serial dilutions from 4 to 0.2 mg% protein TSA obtained, one in normal human serum (NHS) and another in phosphate-buffered saline (PBS), pH 7.2 (fig. 3). In circu lar patterns, horse antitumor multivalent hyperimmune serum (batch HTU9C" s N '2) was placed in the central well and allowed to react simultaneous ly with NHS, with TSA (1 mg%) and with each one of the dilutions of NHS + TSA and PBS + TSA. The serum TSA-binding capacity is revealed and measured in this experi ment in which the horse antiserum (HTU9) reacted with 0.2 mg% TSA in PBS but only with 2 mg% of TSA in human serum. Relatively to the reactant horse serum, human serum masks TSA by a factor of 10 over the buffer. The requirement of at least ten times as much TSA in serum as in buffer for the detection by the horse antiserum seems to indicate that level as the saturated serum TSA-binding capacity beyond which TSA is left free for reaction with horse antiserum. However, the fact that most cancer sera still naturally bind additional TSA would indicate not only that there is an unsaturated TSA-binding capacity but
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Fig. 1. The center well contains highly concentrated TSA (4 mg%). Peripheral wells contain alternatively a human serum from a normal source, 2, 4 and 6, and from a cancer patient, 1, 3 and 5. Besides sharp lines of ‘reaction’ there are strong diffuse bands. The alternate arrangement of the peripheral wells gives rise to reactions of identity and non identity.
Tumor-Specific Antigen Binding Activity of Human Serum
19
Fig. 2. The center well contains horse antitumor hyperimmune 7 -globulin. Wells 1 and 4 contain unabsorbed tumor antigens with multiple line reactivity. Well 2 contains extract of normal organs and tissues showing identity of reaction with one line of the unabsorbed tumor antigens. Wells 3 and 6 contain buffered solutions; well 5 contains normal human serum. The ‘reaction’ between TSA (4) and serum (5) is not as clear-cut; it is noncontiguous with the residual antinormal activity of the center well, but it is partly contiguous with its antitumor activity.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
that the immunologic reaction with the heterologous antibody is able to dis sociate the serum-binding factor from TSA and reveal the tumor antigen. To what quantitative extent this dissociation occurs is not known. The reaction of human serum with TSA is instantaneous, requires no in cubation and produces a turbidity which cannot be centrifuged at or below 14,000 £ even though it shows a cloudiness in agar. This fact plus the non-7-globulin nature of the TSA-binding factor seem to indicate that we are not in the presence of an antigen-antibody reaction. It is known that positively charged biologic and synthetic polymers can produce ‘precipitation’ lines in agar with basic proteins (5). Similar binding of foreign proteins exists as a form of archyphylectic immunity before the emergence of the 7-globulin-secreting immuno cyte (6). Purified hepatitis B surface antigen is known to ‘cross-react’ with serum proteins such as apolipoprotein, lactoferrin, albumin and oq-T-glycoprotein by nonspecific absorption (7). Further examples of nonspecific precipitation in immunodiffusion tests and its mechanisms are discussed extensively by Crowle (8). In the the system proposed for the detection of circulating tumor antigens
De Carvalho
20
(1), the technique calls for quantitative absorption in the agar plate of whatever reactivity the horse antitumor serum may depict against antigens from normal tissues. This is accomplished by loading the agar through the center well with an isosmolarly concentrated pool of NHS. The possibility that this residual NHS could react with TSA in the cancer serum from the opposing well and thus produce an artifact does not exist. The previous experiments show that TSA is already bound in its own cancer serum and only heterologous immune serum, but not the isologous nonimmune serum will react with it. The characteristics of the specific lines of reaction lend further support to this possibility. A third element of interference, easy to rule out, is the presence of allotypic serum reactions. Characterization of this binding factor may be very important for the inter actions between the dual immune system. Cooperband's (9) results point to an a2 -globulin as a natural immunosuppressant of T cells. The biologic significance of the serum TSA-binding factor, its nature, non specificity, and its antigen-masking effect (7), hence its role in the host-tumorimmunologic conflict present yet another challenge to the cancer immunologist.
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Fig. 3. Polyacrylamide rod imbibition stages following immersion in TSA solution. The rods increase 70-fold their volume by absorbing water and solute isosmolarly from the protein solution.
Tumor-Specific Antigen Binding Activity of Human Serum
21
References 1
2
3 4 5
6 7
8 9
De Carvalho, S.: Detection of neoantigens in the serum of patients with active neo plastic diseases by the absorption-immunodiffusion method. Oncology 27: 193-234 (1973). De Carvalho, S.: In vitro properties of a specific heterologous anti-leukemic and anti tumor cytotoxic antibody and their bearing on its application in vivo. Expl. molec. Path. 2: suppl., pp. 150-178 (1963). De Carvalho, S.: Preparation of antigens specific of human breast carcinoma by an immunochromatographic method. Nature, Lond. 203: 1186-1188 (1964). Curtain, C.: Concentrating protein solution. Nature, Lond. 203: 1380 (1964). Mora, P.T. and Young, B.C.: Reversible blocking of T2 bacteria bacteriophage anti serum with polyelectrolytes. J. biol. Chem. 237: 1870-1875 (1962). Papermaster, B.W.; Condie, R.M.; Finstad, T.K.; Good, R.A., and Gabrielsen, A.E.: Phylogenetic development of adaptive immunity. Fed. Proc. 22: 1152-1155 (1963). Goudeau, A.; Houwen, B., and Daukert, T.: Cross-reactions of human serum-proteins with HBsAg. Lancetii: 1325 (1974). Crowle, A.T.: Immunodiffusion, pp. 174-179 (Academic Press, New York 1973). Cooperband, S.R.: Science 159: 1243 (1968).
Downloaded by: Lund University Libraries 130.235.66.10 - 1/1/2019 8:43:34 PM
Dr. S. De Carvalho, Belmont Medical Clinic, Bellflower, C4 90706 (USA)
