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Localization and Quantitation of EBV-Associated Nuclear Antigen (EBNA) in Raji Cells
Short Communications Armed Forces Institute of Pathology, Washington, and Department of Tumor Biology, Karolinska Institutet, Stockholm
Localization and Quantitation of EBV-Associated Nuclear Antigen (EBNA) in Raji Cells 1) Lokalisation und quantitative Messung eines Epstein-Barr-Virus-assoziierten nuklearen Antigens (EBNA) in Raji-Zellen G. F. BAHR 2), U. MIKEL, and G. KLEIN With 5 Figures· Received October 9, 1974· Accepted November 15, 1974
Key words: Chromatin - Electron microscopy- EBV-associated nuclear antigen - Quantitation
Summary Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was localized to chromatin fibers of Raji cells. When EBNA was labeled with the horseradish-peroxidase technique of Sternberger a strong reaction covered most fiber surfaces as observed by electron microscopy. The dry mass of Raji chromatin increased after exposure to both EBV-positive and -negative serum, but the increase was significantly larger in the material treated with the positive serum.
Reedman and Klein (1973) have recently localized an EBV associated antigen in the nuclei of producer and non producer lymphoblastoid cell lines. In over 900/0 of the cells a strong anticomplement immunofluorescence (ACIF) reaction could be elicited, and the specificity of the antigen for 1) The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 2) This paper was given at the Postcongress Symposium on DNA and Pathology in Freiburg, Germany.
Quantitation of Antigen. 73
EBV was shown. Appley et al. (1973) extended this study to the ultrastructural level. Raji cells were swollen in a hypotonic medium, and a pellet was released from a spatula onto the surface of distilled water. The cells ruptured, but chromatin and chromosomes remained attached to the airwater interphase (Bahr, 1970), from which they were picked up on Formvar-coated grids for electron microscopy. The grids were exposed to serum of high titer against EBNA in the presence of complement, carefully washed, and the chromatin was stained with FITC-conjugated anti-human BIc! BIA globulin (Reedman and Klein, 1973). Grids so prepared were mounted in glycerol and the fluorescence photographed in appropriately filtered UV light. Glycerol was then removed by increasing concentrations of ethanol, and the grids were finally dried by the critical-point-drying method of Anderson (1951) without ever being subjected to the strong forces of surface tensoin in air-drying. Since the surface-spread preparations had been collected on locator-grids for electron microscopy, it was possible to find the nuclei previously photographed under the fluorescence microscope, in the electron microscope as well. Electron micrographs of unstained chromatin reflect the concentration, distribution, and extent of chromatin as image contrast (Zeitler and Bahr, 1962; Bahr, 1965, 1973), which correlated in each instance perfectly with the intensity, distribution, and extension of EBNA fluorescence from the same nucleus (Figs. I, 2). Moreover, since chromatin
Fig.
r
Fig. z
Fig. I. Immunofluorescence of EBNA in surface-spread Raji nuclear chromatin. X 1,600, AFIP Neg. 74-1781. Fig. 2. The same nucleus as in Fig. I seen at low magnification by transmission electron microscopy. The fibrous nature of chromatin is evident. X 1,600, AFIP Neg. 74-1782.
74 . G. F. Bahr, U. Mikel, and G. Klein
Fig. 3. Higher power view of the rim of a surface-spread Raji nucleus after critical-point drying. The average fiber has a diameter of 200 A. X 35,000, AFIP Neg. 74-1783.
consists almost entirely of 200-.1\ chromatin fibrils (Fig. 3), it was concluded that the EBV-associated antigen is located in chromatin in a rather widely distributed manner. The present report concerns itself with the direct localization of EBNA by methods for electron microscopy. In a first series of experiments the ACIF reaction was replaced by ferritin-labeled goat antihuman antibody ~lC (from Cappel Laboratories, Inc., Downingtown, Pa.). No meaningful results could be obtained with this reaction, however, since both positive and negative serum, as well as unreacted chromatin, were found to bind anti-~lc both diffusely and in patches. Some nonspecificity associated with ferritin labeling can often be suppressed by dipping the object into 5% albumin between each procedural step. In our case this measure abolished all affinity for anti-~H; from the chromatin, but binding on both sides of the support membrane continued, as could be clearly seen in electron-microscopic stereopairs. Sternberger has developed an immunocytochemical method for the identification of cellular antigens that is reputed to be about 1,000 times more sensitive than the fluorescent-antibody method (Sternberger, 1972). For our purposes, this method involves the following steps: a) Human anti-
Quantitation of Antigen. 75
serum to EBNA is exposed to surface-spread chromatin from nonproducin Raji cells, fixing added complement in the process; b) rabbit-antihuman globulin is exposed to the complex; and the following are added: sheep antirabbit IgG, purified soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP, made in the rabbit), 3,3' diamino-benzidine (DAB), H 2 0 2 , and osmium tetroxide. In analogy to the previous observation that antibody-fluorescence labeling is coincident with chromatin structure, a strong reaction to the Sternberger labeling method was observed on and along every chromatin fibril, resulting in encrustation and caking of chromatin strands when positive anti-EBNA serum was used (Fig. 4). EBNA-negative serum, as defined by the anticomplement fluorescence test, also reacted with most chromatin fibrils, but this reaction was less strong on visual evaluation (Fig. 5). In order to decide to what extent the sensitivity of the Sternberger procedure had led to reactions not specifically related to EBNA and therefore had invalidated this approach, a quantitative analysis of the electron-microscopic material was carried out. The basis of quantitative measurements with the electron microscope is the fact that contrast and object dry mass
Fig. 4. Chromatin fibers after reacting with complement-fixing, EBNA-positive antiserum, Sternberger's peroxidase marker (Sternberger, 1972), and osmium tetroxide. The fibers are heavily encrusted with reaction product. X 35,000, AFIP Neg. 74-1784.
7 6 . G. F. Bahr, U. Mikel, and G. Klein
Fig. 5. Chromatin fibers treated as in Figure 4 but with a negative antiserum rather than positive. There is positive reaction, but much lighter, as confirmed by quantitative evaluation (Tab. I). X 35,000, AFIP Neg. 74-r785.
are related in ways that can be used to determine the realtive and absolute mass of an object (Bahr, I965, I973). The application of this principle to the above experiments rendered the following results. Table I shows in its last column that positive serum produces in two instances a marked increase in the binding of the specific marker to the EBNA-positive cells. Serum from which nonspecific general antinuclear antibodies have been adsorbed with aid of EBV-negative cells (HBT-3) shows a weaker reaction than untreated positive serum. There is some reservation about this observation, however, because two different positive sera have been used for experiments 5 and 8. Osmium tetroxide alone or after treatment of nuclei with positve serum produces a I5% decrease in mass. In agreement with earlier observations on the reaction of OS04 with organic substances (Bahr, I 9 55); the metal oxid contributes significantly, however, to the mass of reacting materials when it is reduced by DAB. The quantity of reacting anti-EBNA-antibody bound to chromatin is not measurable, nor does its presence measurably influence the reaction of OS04 with chromatin.
Quantitation of Antigen . 77 Table 1 Experiment
BSSx
- Serum JH -~
+ Serum NM
- Serum + JH
+ Serum+ AG
..- . - - . - . - - - - - - -..- - . - .
-- -
Com- PAP':· plement -_
.... _ .
Relative Weight ++
OS04
-----------
+
167
+
2 3
+
+ +
4
+ 6
+ +
7
+
8 - - - - - - - - - _..
_ _ .. _ -
-
16 7
+
+
21 3
+
+
+
33 1
+
+
+
472
+
+
279
+
+
288
+
+ -
-_
34 1 ....
-
-
x BSS = basic salt solution in which the EBNA-positive and -negative sera are diluted (Reedman and Klein, 1973). + Nonspecific affinity to nuclear proteins eliminated by adsorption with chromatin from human breast carcinoma (HBT-3) cells (Bassin et aI., 1972). >IPAP includes the steps of rabbit antihuman IgG, sheeep anti rabbit IgG, and DAB with H202. ++ Relative mass (in arbitrary units) of Raji nuclei by electron microscopy is 167. This mass did not change when it was treated with positive serum and complement. Unspecific mass increase from PAP reagents and OS04 was 28% (213-167 = 46; line 3 minus I). Unspecific mass increase from negative serum, complement, PAP, and OS04 is 71% (331-46-167 = 1I8; line 4 minus 3). Specific binding from positive serum amounted to 84% mass increase (472-118-46-167 = 141; lines 5 minus 4) over the original nuclear mass after correction for unspecific binding. Unspecific mass increase from complement and PAP was 67% (279-167 = II2; line 6 minus I). In order to eliminate unspecific affinities of antiserum to chromatin, positive and negative sera were adsorbed with nuclei from HBT-3 cells. The unspecific uptake remained 72%, nevertheless (288-167 = I21; line 7 minus I). Specific uptake from positive serum treated with HBT-3 cells was 32% (341-167-121 = 53; lines 8 minus 7) over the original mass after correction for unspecific binding.
We conclude that the complement-fixing EBV-associated antigen is widely distributed throughout the nucleus. It occurs on chromatin fibers at sites so close to each other that available immunocytochemical techniques cannot distinguish them. There is reactivity (antibodies) in negative serum to nuclear sites of Raji cells that could not be eliminated by adsorption with chromatin from EBV-negative HBT-3 cells. This adsorbed negative
78 . G. F. Bahr, U. Mikel, and G. Klein
serum gives a relatively strong reaction with the sensitive Sternberger procedure and may signal the presence of some nonadsorbed antinuclear antibodies. Dipping of the mounted nuclei in a normal sheep serum between each procedural step as recommended by Sternberger produces a slight mass increase with the full reaction but abolishes all differences of positive to negative antiserum. What has been said about interphase chromatin applies in equal measure to chromosomes. The condensation of chromatin into chromosomes makes each reaction appear subjectively stronger, however.
References Reedman, B. M., and Klein, G.: Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer II, 499-520 (1973) Appley, M., Klein, G., and Bahr, G. F.: Localizing the EBV-associated nuclear antigen on chromatin fibers of Burkitt's lymphoma (Raii) nuclei. J. BioI. 59, 10 a (1973) Bahr, G. F.: Human chromosome fibers. Exp. Cell Res. 62, 39-49 (1970) Anderson, T.: Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. II, 13, 130-134 (1951) Zeitler, E., and Bahr, G. F.: A photometric procedur.e for weight determination of submicroscopic particles. J. app\. Physics 33, 847-853 (1962) Bahr, G. F.: The determination of the dry mass in populations of isolated particles. Lab. Invest. 14,955-977 (19 6 5) Bahr, G. F.: Determining the dry mass of small biologic objects by quantitative electron microscopy. In: Micromethods in Molecular Biology, V. Neuhoff, Ed., p. 257. SpringerVerlag, Berlin (1973) Sternberger, L.: Personal communications. The authors are grateful to Dr. Sternberger for a discussion of his PAP technique Sternberger, L.: The unlabeled-antibody-Peroxidase and the quantitative-immunouranium methods in light and electron immunohistochemistry. In: Techniques of Biochemical and Biophysical Morphology, D. Glick and R. M. Rosenbaum, Eds., p. 67-88. John Wiley and Sons, New York (1972) Bahr, G. F.: Continued studies about fixation with osmium tetroxide. Exp. Cell Res. 9. 277- 28 5 (1955) Bassin, R. H., Plata, E. J., Gervin, B., Mattern, C. F., Haapala, D. K., and Chu, E. W.: Isolation of a continuous epithelioid cell line, HBT-3, from a human breast carcinoma. Proc. Soc. expo BioI. Med. 141, 673-680 (1972) G. Bahr, M. D., U. Mikel, Armed Forces Institute of Pathology, Washington, 20306, U.S.A. G. Klein, M. D., Department of Tumor Biology, Karolinska Institutet, S-r04 or Stockholm 60, Sweden
D.
c.,
Localization and Quantitation of EBV-Associated Nuclear Antigen (EBNA) in Raji Cells 1) Lokalisation und quantitative Messung eines Epstein-Barr-Virus-assoziierten nuklearen Antigens (EBNA) in Raji-Zellen G. F. BAHR 2), U. MIKEL, and G. KLEIN With 5 Figures· Received October 9, 1974· Accepted November 15, 1974
Key words: Chromatin - Electron microscopy- EBV-associated nuclear antigen - Quantitation
Summary Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was localized to chromatin fibers of Raji cells. When EBNA was labeled with the horseradish-peroxidase technique of Sternberger a strong reaction covered most fiber surfaces as observed by electron microscopy. The dry mass of Raji chromatin increased after exposure to both EBV-positive and -negative serum, but the increase was significantly larger in the material treated with the positive serum.
Reedman and Klein (1973) have recently localized an EBV associated antigen in the nuclei of producer and non producer lymphoblastoid cell lines. In over 900/0 of the cells a strong anticomplement immunofluorescence (ACIF) reaction could be elicited, and the specificity of the antigen for 1) The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense. 2) This paper was given at the Postcongress Symposium on DNA and Pathology in Freiburg, Germany.
Quantitation of Antigen. 73
EBV was shown. Appley et al. (1973) extended this study to the ultrastructural level. Raji cells were swollen in a hypotonic medium, and a pellet was released from a spatula onto the surface of distilled water. The cells ruptured, but chromatin and chromosomes remained attached to the airwater interphase (Bahr, 1970), from which they were picked up on Formvar-coated grids for electron microscopy. The grids were exposed to serum of high titer against EBNA in the presence of complement, carefully washed, and the chromatin was stained with FITC-conjugated anti-human BIc! BIA globulin (Reedman and Klein, 1973). Grids so prepared were mounted in glycerol and the fluorescence photographed in appropriately filtered UV light. Glycerol was then removed by increasing concentrations of ethanol, and the grids were finally dried by the critical-point-drying method of Anderson (1951) without ever being subjected to the strong forces of surface tensoin in air-drying. Since the surface-spread preparations had been collected on locator-grids for electron microscopy, it was possible to find the nuclei previously photographed under the fluorescence microscope, in the electron microscope as well. Electron micrographs of unstained chromatin reflect the concentration, distribution, and extent of chromatin as image contrast (Zeitler and Bahr, 1962; Bahr, 1965, 1973), which correlated in each instance perfectly with the intensity, distribution, and extension of EBNA fluorescence from the same nucleus (Figs. I, 2). Moreover, since chromatin
Fig.
r
Fig. z
Fig. I. Immunofluorescence of EBNA in surface-spread Raji nuclear chromatin. X 1,600, AFIP Neg. 74-1781. Fig. 2. The same nucleus as in Fig. I seen at low magnification by transmission electron microscopy. The fibrous nature of chromatin is evident. X 1,600, AFIP Neg. 74-1782.
74 . G. F. Bahr, U. Mikel, and G. Klein
Fig. 3. Higher power view of the rim of a surface-spread Raji nucleus after critical-point drying. The average fiber has a diameter of 200 A. X 35,000, AFIP Neg. 74-1783.
consists almost entirely of 200-.1\ chromatin fibrils (Fig. 3), it was concluded that the EBV-associated antigen is located in chromatin in a rather widely distributed manner. The present report concerns itself with the direct localization of EBNA by methods for electron microscopy. In a first series of experiments the ACIF reaction was replaced by ferritin-labeled goat antihuman antibody ~lC (from Cappel Laboratories, Inc., Downingtown, Pa.). No meaningful results could be obtained with this reaction, however, since both positive and negative serum, as well as unreacted chromatin, were found to bind anti-~lc both diffusely and in patches. Some nonspecificity associated with ferritin labeling can often be suppressed by dipping the object into 5% albumin between each procedural step. In our case this measure abolished all affinity for anti-~H; from the chromatin, but binding on both sides of the support membrane continued, as could be clearly seen in electron-microscopic stereopairs. Sternberger has developed an immunocytochemical method for the identification of cellular antigens that is reputed to be about 1,000 times more sensitive than the fluorescent-antibody method (Sternberger, 1972). For our purposes, this method involves the following steps: a) Human anti-
Quantitation of Antigen. 75
serum to EBNA is exposed to surface-spread chromatin from nonproducin Raji cells, fixing added complement in the process; b) rabbit-antihuman globulin is exposed to the complex; and the following are added: sheep antirabbit IgG, purified soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP, made in the rabbit), 3,3' diamino-benzidine (DAB), H 2 0 2 , and osmium tetroxide. In analogy to the previous observation that antibody-fluorescence labeling is coincident with chromatin structure, a strong reaction to the Sternberger labeling method was observed on and along every chromatin fibril, resulting in encrustation and caking of chromatin strands when positive anti-EBNA serum was used (Fig. 4). EBNA-negative serum, as defined by the anticomplement fluorescence test, also reacted with most chromatin fibrils, but this reaction was less strong on visual evaluation (Fig. 5). In order to decide to what extent the sensitivity of the Sternberger procedure had led to reactions not specifically related to EBNA and therefore had invalidated this approach, a quantitative analysis of the electron-microscopic material was carried out. The basis of quantitative measurements with the electron microscope is the fact that contrast and object dry mass
Fig. 4. Chromatin fibers after reacting with complement-fixing, EBNA-positive antiserum, Sternberger's peroxidase marker (Sternberger, 1972), and osmium tetroxide. The fibers are heavily encrusted with reaction product. X 35,000, AFIP Neg. 74-1784.
7 6 . G. F. Bahr, U. Mikel, and G. Klein
Fig. 5. Chromatin fibers treated as in Figure 4 but with a negative antiserum rather than positive. There is positive reaction, but much lighter, as confirmed by quantitative evaluation (Tab. I). X 35,000, AFIP Neg. 74-r785.
are related in ways that can be used to determine the realtive and absolute mass of an object (Bahr, I965, I973). The application of this principle to the above experiments rendered the following results. Table I shows in its last column that positive serum produces in two instances a marked increase in the binding of the specific marker to the EBNA-positive cells. Serum from which nonspecific general antinuclear antibodies have been adsorbed with aid of EBV-negative cells (HBT-3) shows a weaker reaction than untreated positive serum. There is some reservation about this observation, however, because two different positive sera have been used for experiments 5 and 8. Osmium tetroxide alone or after treatment of nuclei with positve serum produces a I5% decrease in mass. In agreement with earlier observations on the reaction of OS04 with organic substances (Bahr, I 9 55); the metal oxid contributes significantly, however, to the mass of reacting materials when it is reduced by DAB. The quantity of reacting anti-EBNA-antibody bound to chromatin is not measurable, nor does its presence measurably influence the reaction of OS04 with chromatin.
Quantitation of Antigen . 77 Table 1 Experiment
BSSx
- Serum JH -~
+ Serum NM
- Serum + JH
+ Serum+ AG
..- . - - . - . - - - - - - -..- - . - .
-- -
Com- PAP':· plement -_
.... _ .
Relative Weight ++
OS04
-----------
+
167
+
2 3
+
+ +
4
+ 6
+ +
7
+
8 - - - - - - - - - _..
_ _ .. _ -
-
16 7
+
+
21 3
+
+
+
33 1
+
+
+
472
+
+
279
+
+
288
+
+ -
-_
34 1 ....
-
-
x BSS = basic salt solution in which the EBNA-positive and -negative sera are diluted (Reedman and Klein, 1973). + Nonspecific affinity to nuclear proteins eliminated by adsorption with chromatin from human breast carcinoma (HBT-3) cells (Bassin et aI., 1972). >IPAP includes the steps of rabbit antihuman IgG, sheeep anti rabbit IgG, and DAB with H202. ++ Relative mass (in arbitrary units) of Raji nuclei by electron microscopy is 167. This mass did not change when it was treated with positive serum and complement. Unspecific mass increase from PAP reagents and OS04 was 28% (213-167 = 46; line 3 minus I). Unspecific mass increase from negative serum, complement, PAP, and OS04 is 71% (331-46-167 = 1I8; line 4 minus 3). Specific binding from positive serum amounted to 84% mass increase (472-118-46-167 = 141; lines 5 minus 4) over the original nuclear mass after correction for unspecific binding. Unspecific mass increase from complement and PAP was 67% (279-167 = II2; line 6 minus I). In order to eliminate unspecific affinities of antiserum to chromatin, positive and negative sera were adsorbed with nuclei from HBT-3 cells. The unspecific uptake remained 72%, nevertheless (288-167 = I21; line 7 minus I). Specific uptake from positive serum treated with HBT-3 cells was 32% (341-167-121 = 53; lines 8 minus 7) over the original mass after correction for unspecific binding.
We conclude that the complement-fixing EBV-associated antigen is widely distributed throughout the nucleus. It occurs on chromatin fibers at sites so close to each other that available immunocytochemical techniques cannot distinguish them. There is reactivity (antibodies) in negative serum to nuclear sites of Raji cells that could not be eliminated by adsorption with chromatin from EBV-negative HBT-3 cells. This adsorbed negative
78 . G. F. Bahr, U. Mikel, and G. Klein
serum gives a relatively strong reaction with the sensitive Sternberger procedure and may signal the presence of some nonadsorbed antinuclear antibodies. Dipping of the mounted nuclei in a normal sheep serum between each procedural step as recommended by Sternberger produces a slight mass increase with the full reaction but abolishes all differences of positive to negative antiserum. What has been said about interphase chromatin applies in equal measure to chromosomes. The condensation of chromatin into chromosomes makes each reaction appear subjectively stronger, however.
References Reedman, B. M., and Klein, G.: Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer II, 499-520 (1973) Appley, M., Klein, G., and Bahr, G. F.: Localizing the EBV-associated nuclear antigen on chromatin fibers of Burkitt's lymphoma (Raii) nuclei. J. BioI. 59, 10 a (1973) Bahr, G. F.: Human chromosome fibers. Exp. Cell Res. 62, 39-49 (1970) Anderson, T.: Techniques for the preservation of three-dimensional structure in preparing specimens for the electron microscope. Trans. N. Y. Acad. Sci. Ser. II, 13, 130-134 (1951) Zeitler, E., and Bahr, G. F.: A photometric procedur.e for weight determination of submicroscopic particles. J. app\. Physics 33, 847-853 (1962) Bahr, G. F.: The determination of the dry mass in populations of isolated particles. Lab. Invest. 14,955-977 (19 6 5) Bahr, G. F.: Determining the dry mass of small biologic objects by quantitative electron microscopy. In: Micromethods in Molecular Biology, V. Neuhoff, Ed., p. 257. SpringerVerlag, Berlin (1973) Sternberger, L.: Personal communications. The authors are grateful to Dr. Sternberger for a discussion of his PAP technique Sternberger, L.: The unlabeled-antibody-Peroxidase and the quantitative-immunouranium methods in light and electron immunohistochemistry. In: Techniques of Biochemical and Biophysical Morphology, D. Glick and R. M. Rosenbaum, Eds., p. 67-88. John Wiley and Sons, New York (1972) Bahr, G. F.: Continued studies about fixation with osmium tetroxide. Exp. Cell Res. 9. 277- 28 5 (1955) Bassin, R. H., Plata, E. J., Gervin, B., Mattern, C. F., Haapala, D. K., and Chu, E. W.: Isolation of a continuous epithelioid cell line, HBT-3, from a human breast carcinoma. Proc. Soc. expo BioI. Med. 141, 673-680 (1972) G. Bahr, M. D., U. Mikel, Armed Forces Institute of Pathology, Washington, 20306, U.S.A. G. Klein, M. D., Department of Tumor Biology, Karolinska Institutet, S-r04 or Stockholm 60, Sweden
D.
c.,