Int. Archs Allergy appl. Immun. 54: 255-261 (1977)
Preparation and Properties of a Specific Antiserum Against Human Liver1 Anastasios A. Mihas and Jerry G. Spenney Division of Gastroenterology, Department of Medicine, University of Alabama in Birmingham and Birmingham V.A. Hospital, Birmingham, Ala.
Introduction Various autoantibodies have been de scribed in the sera of patients with primary biliary cirrhosis and chronic active hepatitis [4, 14], These associations raise the ques tion of autoimmune etiological factors in these liver diseases. These antibodies, how ever, are neither organ, nor species specific, and are generally regarded as diagnostically useful, but probably epiphenomena [6], A search for organ-specific antibodies in liver disease has continued but with limited suc cess [15]. 1 This work was supported by NIH grants CA19196 and AM17315 and V.A. projects 805901 ,02 .
Previous work from this laboratory [12] has reported the purification and characteri zation of a liver-specific antigen. This anti gen was demonstrated in the sera of several patients with liver disease and its diagnostic significance was speculated [1, 16, 17]. The specificity of the results from antisera raised in rabbits injected with mouse liver extracts are uncertain when applied to human mate rial. We have also isolated and purified an antigen from human liver. This material would then be the most appropriate antigen for producing antihuman liver sera (AHLS). In this article, we report the development and properties of antisera obtained by im munization of rabbits with a purified soluble fraction obtained from human liver.
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Abstract. Immunization of rabbits with a soluble human liver fraction incorporated into Freund’s adjuvants produced antibodies that reacted with human liver extracts as well as extracts of other human organs. Absorption of the antihuman liver sera (AHLS) with lyophilized human serum, heart, and kidney abolished the reactions with other organs but did not remove the liver antibodies. The existence of at least two different liver antigens could be shown by gel diffusion precipitation experiments. One of these antigens could be removed by absorption of the antisera with chicken liver extracts. The AHLS did not crossreact with liver extracts of other species. Comparison of the AHLS with a mouse liver anti serum described previously indicated that these antisera detected closely related but non identical liver antigens.
Mihas/Spenney
256
Preparation of Organ Extracts (Liver, Kidney, Spleen, Heart, Pancreas Adrenals, Lung, Stomach, Colon, Thyroid) Human organs appearing histologically normal were obtained at autopsy within 8 h after death. The organs were washed and the connective tissue and blood vessels were removed as thoroughly as possible. To obtain saline extracts (50% w/v), tis sue suspensions were made by homogenizing minced tissue in 0.9% NaCl in a VirTis homogenizer. The pancreatic extracts were prepared with the addition of i-aminocaproic acid (EACA) at 0.6% w/v, and aprotinin (20 Kallikrcin inactivator units/ml) to prevent autolysis. The homogenates were centrifuged at 12,000 g for 30 min and the supernatant was collected and stored at -20 °C until used. Liver Fractionation The 12,000 g liver extract was centrifuged at 100,000 g for 60 min in a Spinco L2-65 ultracen trifuge. The supernatant was mixed with equal volumes of saturated (NH4)2S 04 and after incuba tion for 2 h at 4 °C, the mixture was centrifuged at 12,000 g for 30 min at 4 °C. The sediment was dissolved in distilled water and freed of (NH4)2S04 by dialysis against 0.01 M phosphate buffer, pH 7.2. 40 ml of the redisolved (NH4),S 0 4 precipitate (‘soluble liver fraction’) containing 50 mg of protein/ml, were applied to a DEAE-cellulose (What man) column (2.6 X 60 cm) and fractions were eluted by a linear gradient prepared from 0-1 M NaCl in 0.01 M phosphate buffer, pH 7.2. Protein concentration of the eluates was determined by UV absorption at 280 nm. Fractions of the second peak were pooled, dialyzed against H20 and lyophilized. Polyacrylamides Gel Electrophoresis Electrophoresis was performed on 10% poly acrylamide gels containing 0.1% sodium dodccyl sulfate (SDS) according to the method of Laemmli [8], except that the samples were treated with 1% SDS at room temperature approximately 10 min before application to the gels. Coomassie brilliant blue R was used for visualization of proteins while the periodic acid Schiff (PAS) stain [7] was used for sugar residues.
Immunization of Rabbits The Iyophilized material obtained from peak No. 2 of the DEAE-cellulose column was dis solved in distilled water in a concentration of 5 mg/ml and emulsified with an equal volume of Freund’s complete adjuvant. 2 ml of the emulsion were injected subcutaneously on the backs of two male albino rabbits so that there were two rows of 10 injections. 3, 6 and 8 weeks after the initial im munization the rabbits were injected with the lyophilized material emulsified in incomplete Freund’s adjuvant. Blood samples were obtained from the marginal ear vein and the sera were sep arated and stored to -70 °C until used. Double Diffusion Gel Precipitation Immunodiffusion was carried out on glass slides 2X3 in, covered with 0.9% agarose in barbi tal buffer, pH 7.2. Circular wells 5 mm in diame ter were cut at 5 mm distance and in some cases at 2 mm distance. All antisera were used undilut ed. Protein content of the various antigens was routinely determined by the method of Lowry et at. [9], using bovine serum albumin as a standard. Diffusion was allowed to proceed at room tempera ture for 24-48 h or at 4 °C for 48-72 h. Precipitin lines were stained with amido black. A bsorption Procedures The antisera were made liver-specific by ab sorbing 1-ml aliquots of antiserum by 20-30 mg protein of the following Iyophilized preparations: pooled human serum, human kidney extract, hu man heart extract and in some experiments chick en liver extract. In each case the mixture was in cubated overnight at 4 °C and the supernatant was separated at 12,000 g for 30 min.
Results
Ion Exchange Chromatography Figure 1 illustrates the results of DEAEcellulose chromatographic fractionation of the ‘soluble liver fraction’ (see ‘Materials and Methods'). Liver-specific antigen could only be detected in the second peak by double-diffusion against a liver-specific an-
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Materials and Methods
Liver-Specific Antibody
257
Fraction
Fig. 1. Diethylaminoethyl cellulose chroma tography of ‘soluble liver fraction’. Starting buff er was 0.01 M phosphate, pH 7.2. A linear gra dient of 0-1 M NaCl was added to the starting buffer at fraction No. 80.
tiserum produced by injecting allogenic liver extracts in CBA mice [12]. There was a significant degree of purifi cation as judged by polyacrylamide gel elec trophoresis in the SDS system. The gel electrophoretograms showed that the proteins of the peak No. 2 from DEAE were resolved in nine discrete bands while the gels ob tained from the crude liver extract exhibited over 30 bands (fig. 2).
Fig. 2. Acrylamide gel electrophoretogram of ‘soluble liver fraction’ (A) and DEAE peak No. 2 (B). 100 ng of protein was applied on each gel.
Extracts of other organs (heart, kidney, ad renal, lung and pancreas) produced only one faint precipitin line which appeared to be identical for all organs including liver. The remaining two precipitin lines obtained with liver extracts were not found in other organs indicating unique hepatic antigens
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Immunodiffusion Studies The antisera were tested against normal human serum in varying concentrations and gave one or two faint lines of precipitation. These precipitin lines with human serum constituents could be abolished by absorp tion of the antisera with 20 mg of lyophilized pooled human serum/ml. Therefore, in all subsequent tests antisera were routinely absorbed in this manner prior to use. Nor mal (preimmune) rabbit serum failed to re act with either normal human serum or ex tracts of different human organs. In tests with saline extracts of liver at a protein concentration of 25 mg/ml, these AHLS yielded three lines of precipitation.
Fig. 3a, b. Reactions of the AHLS (central well) with human organ extracts by double diffu sion in 0.9°/o agarose. Peripheral wells contain ex tract (20-25 mg/ml) of (1) liver, (2) heart, (3) kid ney, (4) pancreas, (5) lung, and (6) colon.
(fig. 3a, b). Absorption tests confirmed the above findings. Following absorption of the AHLS with kidney (30 mg/ml) and heart (25 mg/ml) extracts, the two lines related to the liver antigen persisted, while the lines common to all other organs were removed. Conversely, when the antisera were ab sorbed with human liver extracts (40 mg/ ml), both lines related to liver antigens were abolished also. These results could be ob
Mihas/Spenney
tained only when human liver extracts or crude liver homogenates were used as ab sorbents. Particulate fractions of human liv er (12,000 g pellet) failed to remove the precipitin lines related to liver antigens. This indicated that the liver antigens detect ed by the AHLS were present only in the soluble liver protein fraction. The antisera tested against the original antigenic material (peak No. 2 from the DEAE-cellulose chromatography) gave only one precipitin line which fused with one of the two lines of human liver extract in a complete identity pattern. Previous studies with a liver-specific antigen in the BALB/C mouse had shown that this antigen was pre sent in all mammalian species tested, but was absent from frog and chicken liver ex tracts. The AHLS was further absorbed with chicken liver extracts (20 mg/ml) and this resulted in complete removal of one of the two precipitin lines obtained with hu man liver extracts. The second line still cross-reacted with the precipitin line ob tained from the DEAE-fraction (peak No. 2) in a complete identity fashion (fig- 4). These experiments indicated that the AHLS was specific for liver tissue and most likely for a single antigen - or a group of antigens with very similar immunochemical properties - of the soluble liver protein fraction. The species specificity of the reaction of the antisera to human liver extract was then examined. Saline liver extracts from dog, rabbit, rat, guinea pig and mouse were test ed with the AHLS. Antigenic activity could be detected in no mammalian species tested other than in man. Figure 5 shows the reac tions of human liver extract with AHLS and monospecific antimouse liver serum
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
258
Liver-Specific Antibody
259
the antisera react with different antigenic molecules or alternatively with different ‘re gions’ (peptides) of the same molecule.
Fig. 4. Center well contains AHLS absorbed with kidney, heart, and chicken liver extract. Peri pheral wells: (1) human liver extract; (2) DEAE peak No. 2.
■
1 o (p1 *rr) o
Fig. 5. Center well: human liver extract (30 mg/ml). Peripheral wells: 1, 3 and 5 contain AHLS, 2, 4 and 6 contain AMLS.
(AMLS) produced by immunization of CBA mouse with liver extracts from BALB/C mouse. It is obvious that although there is cross-reactivity between the two antisera, the lines of precipitation show an incom plete identity pattern, indicating that each of
The results presented in this paper indi cate the existence of a liver-specific antigen in the soluble protein fraction of the liver. The reactions thus far observed were limit ed to human liver and negative results were obtained with liver extracts from several mammalian species other than man. In ad dition to the organ-specific antigen(s), the DEAE human liver fraction contained anti gens that were nonorgan specific. Antigens of the latter type could be demonstrated in several human organs and in the blood. Strictly liver-specific antibodies could be obtained by removing the nonorgan-specific antibodies by absorptions. Following ab sorption these antibodies were detecting only a liver-specific antigen similar to that reported previously in the BALB/C mouse. The incomplete identity reaction, as indicat ed by spur formation, when AHLS and AMLS sera reacted with human liver ex tracts leads us to postulate that both anti bodies are reacting with very similar or the same antigenic material but the reaction in volves different ‘regions’ (polypeptides) of the same molecule. This view, i.e., that the antigen reported in this paper is the same or very closely related to the previously de scribed antigen in the mouse (12] is further supported by the results obtained after ab sorption of the AHLS with chicken liver ex tracts. In these experiments one of the pre cipitin lines obtained with human liver ex tract was completely removed, but not the second line which fused in a complete ident
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
oc
Discussion
260
ity reaction with the DEAE fraction. Since chicken liver extracts were found previously to lack the mouse antigen it may be as sumed that the precipitin line observed with human liver extract following this absorp tion was due to this antigen. Organ specificity of liver antigens have been the subject of several reports in the lit erature. Most investigators, however, gave much attention to particulate liver fractions and to a family of remarkably thermostable antigens referred to as BE antigens for the resistance to boiling and ethanol precipita tion [2, 13]. Relatively little work has been done with antigens prepared from the solu ble liver protein fraction [3, 10]. Very re cently the isolation and characterization of a liver-specific macrolipoprotein was reported [11]. This protein showed a complete or gan-specificity, but incomplete species speci ficity as indicated by antibody assays and im munofluorescence studies [5], The physico chemical and immunological properties of this protein make it very unlikely that it is similar or identical to the antigen reported in this paper. They may belong, however, to a family of organ-specific antigens of the soluble liver protein fraction with great di agnostic and prognostic significance. Their occurrence in the sera of several patients with both acute and chronic liver disease lends credence to this view.
References 1 Arakawa, Y.; Bull, D.; Schott, C., and David son, C.: Experimental and clinical study of a liver specific antigen. (Abstract.) Clin. Res. 18: 53 (1974). 2 Auer, I. O. and Milgrom, F.: Studies on ther mostable liver-specific antigens. Int. Archs Al lergy appl. Immun. 42: 871-882 (1972).
3 Dorncr, M.; Simon, E. J., and Miescher, P. A.: Studies on a liver specific antigen. Fed. Proc. Fed. Am. Socs. exp. Biol. 21: 43 (1962). 4 Dumonde, D. C.: Tissue-specific antigens. Adv. Immunol. 5: 245-412 (1966). 5 Hopf, U.; Meyer zum Buschenfelde, K. H.. and Freundenberg, J.: Liver-specific antigens of different species. II. Localization of a mem brane antigen at cell surface of isolated hepatocytes. Clin. exp. Immunol. 16: 117-124 (1974). 6 Hrobo, T.: Mechanism and role of immuno logical tolerance. Monogr. Allergy, vol. 3 (Kar ger, Basel 1968). 7 Korn, E. D. and Wright, P. L.: Macromolecular composition of amoeba plasma membrane. J. biol. Chem. 248: 439-447 (1973). 8 L a e m m l i , U. K.: Cleavage of structural prote ins during the assembly of the head of bacter iophage T4. Nature, Lond. 227: 680-685 (1970). 9 Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenolreagent. J. biol. Chem. 193: 965 (1951). 10 Meyer zum Buschenfelde, K. H.: Untersuchun gen über die immunbiologische Bedeutung löslicher Leberproteine. Z. ges. exp. Med. 148: 131-163 (1968). 11 Meyer zum Buschenfelde, K. H. and Miescher, P. A.: Liver specific antigens. Purification and characterization. Clin. exp. Immunol. 10: 89-102 (1972). 12 Mihas, A.; Hirschowitz, B., and Saccomani, G.: Purification and characterization of a liver specific antigen. J. Immun. 116: 1228-1236 (1976). 13 Milgrom, F.; Maide Tuggac, Z., and Witebsky, E.: Organ-specific antigens of liver, testicle and pituitary. J. Immun. 94: 157-163 (1965). 14 Paronetto, F.; Schaffner, F.; Mutter, R., el al.: Circulating antibodies to bile ductular cells in various liver diseases. J. Am. med. Ass. 187: 503-506 (1964). 15 Schumacher, K. und Kock, W.: Untersuchun gen zur Spezifität antizytoplasmatischer Anti körper bei chronisch progressiver Hepatitis. Klin. Wschr. 46: 925 (1968). 16 Smith, J. B. and Iverson, G. M.: Occurrence of
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Mihas/Spenney
261
Liver-Specific Antibody
Received: September 23, 1976 Correspondence to: Dr. Anastasios A. Mihas, Division of Gastroenterology, Department of Medicine, University of Alabama, Birmingham, AL 35294 (USA)
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
liver-specific antigen in adult human serum. Clin. exp. Immunol. 12: 1-4 (1972). 17 Smith, J. 1L and Lewellyn, F. B.: a,Fetoprotein and liver-specific antigen in viral hepatitis type B. Archs intern. Med. 133: 437-439 (1974).
Preparation and Properties of a Specific Antiserum Against Human Liver1 Anastasios A. Mihas and Jerry G. Spenney Division of Gastroenterology, Department of Medicine, University of Alabama in Birmingham and Birmingham V.A. Hospital, Birmingham, Ala.
Introduction Various autoantibodies have been de scribed in the sera of patients with primary biliary cirrhosis and chronic active hepatitis [4, 14], These associations raise the ques tion of autoimmune etiological factors in these liver diseases. These antibodies, how ever, are neither organ, nor species specific, and are generally regarded as diagnostically useful, but probably epiphenomena [6], A search for organ-specific antibodies in liver disease has continued but with limited suc cess [15]. 1 This work was supported by NIH grants CA19196 and AM17315 and V.A. projects 805901 ,02 .
Previous work from this laboratory [12] has reported the purification and characteri zation of a liver-specific antigen. This anti gen was demonstrated in the sera of several patients with liver disease and its diagnostic significance was speculated [1, 16, 17]. The specificity of the results from antisera raised in rabbits injected with mouse liver extracts are uncertain when applied to human mate rial. We have also isolated and purified an antigen from human liver. This material would then be the most appropriate antigen for producing antihuman liver sera (AHLS). In this article, we report the development and properties of antisera obtained by im munization of rabbits with a purified soluble fraction obtained from human liver.
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Abstract. Immunization of rabbits with a soluble human liver fraction incorporated into Freund’s adjuvants produced antibodies that reacted with human liver extracts as well as extracts of other human organs. Absorption of the antihuman liver sera (AHLS) with lyophilized human serum, heart, and kidney abolished the reactions with other organs but did not remove the liver antibodies. The existence of at least two different liver antigens could be shown by gel diffusion precipitation experiments. One of these antigens could be removed by absorption of the antisera with chicken liver extracts. The AHLS did not crossreact with liver extracts of other species. Comparison of the AHLS with a mouse liver anti serum described previously indicated that these antisera detected closely related but non identical liver antigens.
Mihas/Spenney
256
Preparation of Organ Extracts (Liver, Kidney, Spleen, Heart, Pancreas Adrenals, Lung, Stomach, Colon, Thyroid) Human organs appearing histologically normal were obtained at autopsy within 8 h after death. The organs were washed and the connective tissue and blood vessels were removed as thoroughly as possible. To obtain saline extracts (50% w/v), tis sue suspensions were made by homogenizing minced tissue in 0.9% NaCl in a VirTis homogenizer. The pancreatic extracts were prepared with the addition of i-aminocaproic acid (EACA) at 0.6% w/v, and aprotinin (20 Kallikrcin inactivator units/ml) to prevent autolysis. The homogenates were centrifuged at 12,000 g for 30 min and the supernatant was collected and stored at -20 °C until used. Liver Fractionation The 12,000 g liver extract was centrifuged at 100,000 g for 60 min in a Spinco L2-65 ultracen trifuge. The supernatant was mixed with equal volumes of saturated (NH4)2S 04 and after incuba tion for 2 h at 4 °C, the mixture was centrifuged at 12,000 g for 30 min at 4 °C. The sediment was dissolved in distilled water and freed of (NH4)2S04 by dialysis against 0.01 M phosphate buffer, pH 7.2. 40 ml of the redisolved (NH4),S 0 4 precipitate (‘soluble liver fraction’) containing 50 mg of protein/ml, were applied to a DEAE-cellulose (What man) column (2.6 X 60 cm) and fractions were eluted by a linear gradient prepared from 0-1 M NaCl in 0.01 M phosphate buffer, pH 7.2. Protein concentration of the eluates was determined by UV absorption at 280 nm. Fractions of the second peak were pooled, dialyzed against H20 and lyophilized. Polyacrylamides Gel Electrophoresis Electrophoresis was performed on 10% poly acrylamide gels containing 0.1% sodium dodccyl sulfate (SDS) according to the method of Laemmli [8], except that the samples were treated with 1% SDS at room temperature approximately 10 min before application to the gels. Coomassie brilliant blue R was used for visualization of proteins while the periodic acid Schiff (PAS) stain [7] was used for sugar residues.
Immunization of Rabbits The Iyophilized material obtained from peak No. 2 of the DEAE-cellulose column was dis solved in distilled water in a concentration of 5 mg/ml and emulsified with an equal volume of Freund’s complete adjuvant. 2 ml of the emulsion were injected subcutaneously on the backs of two male albino rabbits so that there were two rows of 10 injections. 3, 6 and 8 weeks after the initial im munization the rabbits were injected with the lyophilized material emulsified in incomplete Freund’s adjuvant. Blood samples were obtained from the marginal ear vein and the sera were sep arated and stored to -70 °C until used. Double Diffusion Gel Precipitation Immunodiffusion was carried out on glass slides 2X3 in, covered with 0.9% agarose in barbi tal buffer, pH 7.2. Circular wells 5 mm in diame ter were cut at 5 mm distance and in some cases at 2 mm distance. All antisera were used undilut ed. Protein content of the various antigens was routinely determined by the method of Lowry et at. [9], using bovine serum albumin as a standard. Diffusion was allowed to proceed at room tempera ture for 24-48 h or at 4 °C for 48-72 h. Precipitin lines were stained with amido black. A bsorption Procedures The antisera were made liver-specific by ab sorbing 1-ml aliquots of antiserum by 20-30 mg protein of the following Iyophilized preparations: pooled human serum, human kidney extract, hu man heart extract and in some experiments chick en liver extract. In each case the mixture was in cubated overnight at 4 °C and the supernatant was separated at 12,000 g for 30 min.
Results
Ion Exchange Chromatography Figure 1 illustrates the results of DEAEcellulose chromatographic fractionation of the ‘soluble liver fraction’ (see ‘Materials and Methods'). Liver-specific antigen could only be detected in the second peak by double-diffusion against a liver-specific an-
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Materials and Methods
Liver-Specific Antibody
257
Fraction
Fig. 1. Diethylaminoethyl cellulose chroma tography of ‘soluble liver fraction’. Starting buff er was 0.01 M phosphate, pH 7.2. A linear gra dient of 0-1 M NaCl was added to the starting buffer at fraction No. 80.
tiserum produced by injecting allogenic liver extracts in CBA mice [12]. There was a significant degree of purifi cation as judged by polyacrylamide gel elec trophoresis in the SDS system. The gel electrophoretograms showed that the proteins of the peak No. 2 from DEAE were resolved in nine discrete bands while the gels ob tained from the crude liver extract exhibited over 30 bands (fig. 2).
Fig. 2. Acrylamide gel electrophoretogram of ‘soluble liver fraction’ (A) and DEAE peak No. 2 (B). 100 ng of protein was applied on each gel.
Extracts of other organs (heart, kidney, ad renal, lung and pancreas) produced only one faint precipitin line which appeared to be identical for all organs including liver. The remaining two precipitin lines obtained with liver extracts were not found in other organs indicating unique hepatic antigens
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Immunodiffusion Studies The antisera were tested against normal human serum in varying concentrations and gave one or two faint lines of precipitation. These precipitin lines with human serum constituents could be abolished by absorp tion of the antisera with 20 mg of lyophilized pooled human serum/ml. Therefore, in all subsequent tests antisera were routinely absorbed in this manner prior to use. Nor mal (preimmune) rabbit serum failed to re act with either normal human serum or ex tracts of different human organs. In tests with saline extracts of liver at a protein concentration of 25 mg/ml, these AHLS yielded three lines of precipitation.
Fig. 3a, b. Reactions of the AHLS (central well) with human organ extracts by double diffu sion in 0.9°/o agarose. Peripheral wells contain ex tract (20-25 mg/ml) of (1) liver, (2) heart, (3) kid ney, (4) pancreas, (5) lung, and (6) colon.
(fig. 3a, b). Absorption tests confirmed the above findings. Following absorption of the AHLS with kidney (30 mg/ml) and heart (25 mg/ml) extracts, the two lines related to the liver antigen persisted, while the lines common to all other organs were removed. Conversely, when the antisera were ab sorbed with human liver extracts (40 mg/ ml), both lines related to liver antigens were abolished also. These results could be ob
Mihas/Spenney
tained only when human liver extracts or crude liver homogenates were used as ab sorbents. Particulate fractions of human liv er (12,000 g pellet) failed to remove the precipitin lines related to liver antigens. This indicated that the liver antigens detect ed by the AHLS were present only in the soluble liver protein fraction. The antisera tested against the original antigenic material (peak No. 2 from the DEAE-cellulose chromatography) gave only one precipitin line which fused with one of the two lines of human liver extract in a complete identity pattern. Previous studies with a liver-specific antigen in the BALB/C mouse had shown that this antigen was pre sent in all mammalian species tested, but was absent from frog and chicken liver ex tracts. The AHLS was further absorbed with chicken liver extracts (20 mg/ml) and this resulted in complete removal of one of the two precipitin lines obtained with hu man liver extracts. The second line still cross-reacted with the precipitin line ob tained from the DEAE-fraction (peak No. 2) in a complete identity fashion (fig- 4). These experiments indicated that the AHLS was specific for liver tissue and most likely for a single antigen - or a group of antigens with very similar immunochemical properties - of the soluble liver protein fraction. The species specificity of the reaction of the antisera to human liver extract was then examined. Saline liver extracts from dog, rabbit, rat, guinea pig and mouse were test ed with the AHLS. Antigenic activity could be detected in no mammalian species tested other than in man. Figure 5 shows the reac tions of human liver extract with AHLS and monospecific antimouse liver serum
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
258
Liver-Specific Antibody
259
the antisera react with different antigenic molecules or alternatively with different ‘re gions’ (peptides) of the same molecule.
Fig. 4. Center well contains AHLS absorbed with kidney, heart, and chicken liver extract. Peri pheral wells: (1) human liver extract; (2) DEAE peak No. 2.
■
1 o (p1 *rr) o
Fig. 5. Center well: human liver extract (30 mg/ml). Peripheral wells: 1, 3 and 5 contain AHLS, 2, 4 and 6 contain AMLS.
(AMLS) produced by immunization of CBA mouse with liver extracts from BALB/C mouse. It is obvious that although there is cross-reactivity between the two antisera, the lines of precipitation show an incom plete identity pattern, indicating that each of
The results presented in this paper indi cate the existence of a liver-specific antigen in the soluble protein fraction of the liver. The reactions thus far observed were limit ed to human liver and negative results were obtained with liver extracts from several mammalian species other than man. In ad dition to the organ-specific antigen(s), the DEAE human liver fraction contained anti gens that were nonorgan specific. Antigens of the latter type could be demonstrated in several human organs and in the blood. Strictly liver-specific antibodies could be obtained by removing the nonorgan-specific antibodies by absorptions. Following ab sorption these antibodies were detecting only a liver-specific antigen similar to that reported previously in the BALB/C mouse. The incomplete identity reaction, as indicat ed by spur formation, when AHLS and AMLS sera reacted with human liver ex tracts leads us to postulate that both anti bodies are reacting with very similar or the same antigenic material but the reaction in volves different ‘regions’ (polypeptides) of the same molecule. This view, i.e., that the antigen reported in this paper is the same or very closely related to the previously de scribed antigen in the mouse (12] is further supported by the results obtained after ab sorption of the AHLS with chicken liver ex tracts. In these experiments one of the pre cipitin lines obtained with human liver ex tract was completely removed, but not the second line which fused in a complete ident
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
oc
Discussion
260
ity reaction with the DEAE fraction. Since chicken liver extracts were found previously to lack the mouse antigen it may be as sumed that the precipitin line observed with human liver extract following this absorp tion was due to this antigen. Organ specificity of liver antigens have been the subject of several reports in the lit erature. Most investigators, however, gave much attention to particulate liver fractions and to a family of remarkably thermostable antigens referred to as BE antigens for the resistance to boiling and ethanol precipita tion [2, 13]. Relatively little work has been done with antigens prepared from the solu ble liver protein fraction [3, 10]. Very re cently the isolation and characterization of a liver-specific macrolipoprotein was reported [11]. This protein showed a complete or gan-specificity, but incomplete species speci ficity as indicated by antibody assays and im munofluorescence studies [5], The physico chemical and immunological properties of this protein make it very unlikely that it is similar or identical to the antigen reported in this paper. They may belong, however, to a family of organ-specific antigens of the soluble liver protein fraction with great di agnostic and prognostic significance. Their occurrence in the sera of several patients with both acute and chronic liver disease lends credence to this view.
References 1 Arakawa, Y.; Bull, D.; Schott, C., and David son, C.: Experimental and clinical study of a liver specific antigen. (Abstract.) Clin. Res. 18: 53 (1974). 2 Auer, I. O. and Milgrom, F.: Studies on ther mostable liver-specific antigens. Int. Archs Al lergy appl. Immun. 42: 871-882 (1972).
3 Dorncr, M.; Simon, E. J., and Miescher, P. A.: Studies on a liver specific antigen. Fed. Proc. Fed. Am. Socs. exp. Biol. 21: 43 (1962). 4 Dumonde, D. C.: Tissue-specific antigens. Adv. Immunol. 5: 245-412 (1966). 5 Hopf, U.; Meyer zum Buschenfelde, K. H.. and Freundenberg, J.: Liver-specific antigens of different species. II. Localization of a mem brane antigen at cell surface of isolated hepatocytes. Clin. exp. Immunol. 16: 117-124 (1974). 6 Hrobo, T.: Mechanism and role of immuno logical tolerance. Monogr. Allergy, vol. 3 (Kar ger, Basel 1968). 7 Korn, E. D. and Wright, P. L.: Macromolecular composition of amoeba plasma membrane. J. biol. Chem. 248: 439-447 (1973). 8 L a e m m l i , U. K.: Cleavage of structural prote ins during the assembly of the head of bacter iophage T4. Nature, Lond. 227: 680-685 (1970). 9 Lowry, O. H.; Rosebrough, N. J.; Farr, A. L., and Randall, R. J.: Protein measurement with the Folin phenolreagent. J. biol. Chem. 193: 965 (1951). 10 Meyer zum Buschenfelde, K. H.: Untersuchun gen über die immunbiologische Bedeutung löslicher Leberproteine. Z. ges. exp. Med. 148: 131-163 (1968). 11 Meyer zum Buschenfelde, K. H. and Miescher, P. A.: Liver specific antigens. Purification and characterization. Clin. exp. Immunol. 10: 89-102 (1972). 12 Mihas, A.; Hirschowitz, B., and Saccomani, G.: Purification and characterization of a liver specific antigen. J. Immun. 116: 1228-1236 (1976). 13 Milgrom, F.; Maide Tuggac, Z., and Witebsky, E.: Organ-specific antigens of liver, testicle and pituitary. J. Immun. 94: 157-163 (1965). 14 Paronetto, F.; Schaffner, F.; Mutter, R., el al.: Circulating antibodies to bile ductular cells in various liver diseases. J. Am. med. Ass. 187: 503-506 (1964). 15 Schumacher, K. und Kock, W.: Untersuchun gen zur Spezifität antizytoplasmatischer Anti körper bei chronisch progressiver Hepatitis. Klin. Wschr. 46: 925 (1968). 16 Smith, J. B. and Iverson, G. M.: Occurrence of
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
Mihas/Spenney
261
Liver-Specific Antibody
Received: September 23, 1976 Correspondence to: Dr. Anastasios A. Mihas, Division of Gastroenterology, Department of Medicine, University of Alabama, Birmingham, AL 35294 (USA)
Downloaded by: King's College London 137.73.144.138 - 3/5/2018 7:33:27 PM
liver-specific antigen in adult human serum. Clin. exp. Immunol. 12: 1-4 (1972). 17 Smith, J. 1L and Lewellyn, F. B.: a,Fetoprotein and liver-specific antigen in viral hepatitis type B. Archs intern. Med. 133: 437-439 (1974).
