Znt. J . Cancer: 19, 305-316 (1977)
COMPARATIVE STUDIES ON EBV ANTIGENS BY IMMUNOFLUORESCENCE A N D IMMUNOPEROXIDASE TECHNIQUES R. STEPHENS, K. TRAUL,P. GAUDREAU, J. YEH, L. FISHER and S . A. MAYYASI The John L. Smith Memorial for Cancer Research, Pfizer Inc., Maywood, New Jersey 07607, USA
Three groups of EBV antigens, VCA, M A and EA, were compared by the techniques of electron microscopic immunoperoxidase (IP) and immunofluorescence (IF). P3HR-1 and EBV suparinfected Raji cells served as targets for labelled sera from patients with BL, N P C and IM or from healthy donors. 1251 peroxidase-labelled antibodies were also prepared to determine, autoradiographically, the penetration of the complex into the cell system, and to monitor the incubation and washing procedures. The development of a gentle sedimentation technique proved critical in handling the fragile target cells. VCA and M A were readily identified and localized by both procedures without significant modification of the basic techniques. lndentlfication of early antigens by IP required modification of the fixation method to include a brief treatment in acetone. The diffuse early antigen (EAD) was found to be associated with cellular ribosomes.
Molecular and serologic studies t o define the association of Epstein-Barr virus (EBV) (Epstein et al., 1964; Burkitt and Burchenal, 1967; Burkitt and Wright, 1970) with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and infectious mononucleosis (IM), have led to many interesting observations regarding the associated antigens (Henle and Henle, 1975; Klein, 1973; Hampar and Martos, 1973). While the role of EBV in all of these diseases is not absolutely defined, it continues to be the prime subject for etiological investigations. Immunofluorescence (IF) has proved t o be an invaluable tool in identifying and locating several of the groups of EBV-associated antigens (Fig. l), as well as in seroepidemiologic analyses. (Henle et al., 1968, 1971; Klein et al., 1966; Reedman and Klein, 1973; Ernberg et al., 1974). The more sensitive immunoelectron microscopic (IEM) techniques of immunoferritin (Hampar et al., 1970) and autoradiography (Inoue and Klein, 1970) have also been employed, but to a lesser extent. Generally the latter studies have been limited to antigens associated with larger structures such as intact virions or cell membranes. We initiated immunoperoxidase (IP) studies of several EBV antigens-virus capsid antigens (VCA), early antigens (EA) and membrane antigens (MA) -with the goal of refining knowledge about the localization of these antigens. Use of the IP techniques has been shown to have significant advan-
tages over IF methods in identifying the various antigenic sites because the IP procedure is more sensitive than IF and provides greater resolution. MATERIAL AND METHODS
Cell cultures
Burkitt-lymphoma-derived P3HR-1 and Raji cells were grown in RPMI 1640 medium supplemented with 8 % fetal calf serum. Changing of the cell culture medium took place every 5-7 days. Cell counts were made using the conventional trypan blue exclusion method and viability of the cells was usually maintained around 85-90 %. Preparation of EBV
EBV was prepared from tissue culture supernatant fluids of P3HR-1 cultures. Six days after the cultures had been set up, the fluids were pooled and the cells removed by centrifugation at 800 g for 15 rnin. The virus-containing supernatant was then centrifuged at 70,000 g for 90 min t o pellet the virus. The pellets were resuspended in a mixture of 50% fresh plus 50 % spent medium which had been previously filtered through a 0.22 pm pore size filter. All virus suspensions were stored at -95" C prior t o use. Induction of early antigen
Twenty-four-hour cultures of Raji cells were harvested, counted and inoculated into 2-oz plastic flasks. Each flask received 4 ml of medium (50% fresh, 50% spent as above) containing 6 x 1 0 5 (viable) cells per ml. One ml of appropriately diluted virus suspension was added t o each flask. The cultures were mixed by shaking and then incubated at 35" C for 42 h. The cells were harvested and washed gently with buffered saline. A small aliquot was used immediately to prepare acetone-fixed smears for immunofluorescence analysis while the remainder of the cell pellets were fixed as described below in the paragraphs dealing with immunoperoxidase procedures. Received: October 21, 1976 and in revised from December 9, 1976.
306
STEPHENS ET AL.
FIGURE 1 Observations of immunofluorescence reactions showing EB viral MA complete and segmented EAD ( c )and EAR (D) in P3HR-1 cells (A, B) and EBV superinfected Raji cells (c, D) x 320.
(A),
VCA
(B),
307
VISUALIZATION OF EBV ANTIGENS
Sera and serum conjugation
EBV-positive and -negative sera used for these studies were from healthy persons, or patients with nasopharyngeal carcinoma, Burkitt's lymphoma or infectious mononucleosis. Sera were stored undiluted in 1-ml aliquots at -95" C prior to use. Peroxidase conjugates were formed with 12 parts horse-radish peroxidase and 5 parts serum protein (Kurstak, 1971) after which the complex was purified by the Shabo (1972) sedimentation procedure. Iodination of peroxidase-conjugated globulins was accomplished by the chloramine T method (Hunter, 1967; Yeh et al., 1975). One hundred pl of peroxidase-conjugated immunoglobulins (75 pg/ml) were incubated for 1 min with 1 mCi of lZ5I2and 25 pl of chloramine T (4 mg/ml), after which 100 p1 of sodium metabisulfite (3.5 mglml) and 200 pl KI (2 mg/ml) were added. The mixture was eluted off a BioGel P-60 column (1 cm x10 cm, mesh 100-200) with 0.5 M phosphate buffer, PH 7.4, then 0.5-pI fractions were collected and each fraction was assayed for TCA precipitability. The specific activity of the peak fractions was found to contain 5 x lo4 cpm per ,ug conjugated protein. Fluorescence staining for VCA andlor EA
Following the indirect procedure of Henle (1966), acetone-fixed smears were allowed t o incubate for 30 min with appropriate dilutions (8-16 units of antibody) of either NPC, IM, or BL sera. EBVnegative serum was used at 1:lO dilution. After incubation, the smears were rinsed (three times) in PBS and stained with 16 units of fluorescein-labelled (goat) anti-human IgG serum. After the cell smears
had been rinsed again three times in PBS they were counterstained with Evans' blue dye and allowed to air dry. The smears were mounted in glycerol containing 10% PBS, and the nature and quality of fluorescence was based on the evaluation of 300-500cells using a Reichert Zetopan microscope. Fluorescence staining for M A
Small aliquots of P3HR-1 or Raji cells were gently pelleted, washed twice with PBS, and resuspended in 5 vol (16 units of B1, NPC, 1M or a 1 :10 dilution of " normal ") of serum for 30 min. Again, the cells were washed (PBS) and resuspended in saline glycerol and wet mounts were made for microscopic observation. Cellular fluorescence was determined as indicated in the preceding paragraph. Peroxidase staining for VCA and MA
Ten-ml aliquots of cell suspension were pelletized in a clinical centrifuge (600 g) and the supernatant fluids were discarded. The cell pellets were resuspended in paraformaldehyde for lh, and then pelletized (600 g ) and washed twice in cacodylate buffer by gently overlaying the buffer for 15 min (handling of the cells from this point became critical for preservation of cellular morphology). The buffer was carefully removed by pipetting and the cells were resuspended in a 1 :4 dilution of the serumperoxidase complex for 16-24 h at 4" C with gentle agitation. The cells were allowed to settle for 15 min, then were centrifuged (1250) yielded bright whole-cell fluorescence in both the cytoplasm and nucleus (Fig. 1 ~ ) Moderately . strong and weak sera showed predominantly cytoplasmic reactions. Peroxidase reactions were observed on organelles and inner membranes of cells, on nuclear virions, on cytoplasmic virions, and on extracellular virions. These reactions were readily observable by the increased size, density and irregularity of the positive structures. Figure 4 shows coating of virions in the nucleus of the cells, whereas Figure 5 contains an example
313
of mixed nuclear and cytoplasmic virion reactions. In each of these Figures certain organelles of both the nucleus and the cytoplasm have strong positive reactions. Figure 6a, B shows control and positive extracellular virus. Observation of EA
Glutaraldehyde fixation, used for VCA and MA, resulted in consistently negative reactions for EA by both IF and IP techniques. In contrast, acetonefixed cell smears were positive for observation of both EAD and EAR by IF (Fig. l c , ~ ) .Use of
FIGURE 7 Reaction of IP-labelled anti-EAD positive serum with 42-h-old, EBV-superinfected Raji cells. Enlarged areas show reactions observed on the ribosomes resulting in enlargement and irregularities and also reactions on inner membranes.
3 14
STEPHENS ET AL.
the standard acetone fixation technique proved highly destructive to cells for processing by the IP method. The alternative and more gentle paraformaldehyde/acetone fixation procedure afforded preservation of morphology and EAD was readily observed by IP (Fig. 7). Reactions were found at the level both of the free ribosomes and of the inner membranes throughout the cytoplasm. Here, as with VCA reactions, a positive reaction was recognized by the increased size and irregularity of the structures as compared with those of the control cells (Fig. 8).
We were unable t o observe consistent reactions by IP which correlated with the IF observations of EAR. EAR positive and EA, negative antisera tested on EAR positive cells (by IF) gave negative results. Several combinations of paraformaldehyde and/or acetone as fixative conditions were tried without success. DISCUSSION
We have compared the utility of immuno-electron microscopy and immunofluorescence procedures (Henle et al., 1971; Klein et al., 1968a, b) to localize
FIGURE 8 Observations with IP-labelled anti EAD positive serum and control Raji cells; in contrast to positive reactions demonstrated in Figure 7, these enlarged areas show unreacted ribosomes and inner membranes.
315
VISUALIZATION OF EBV ANTIGENS
and further characterize the EBV antigens: VCA, MA, EA, (Stephens et al., 1975). The IP procedure is longer and more complicated than IF (Henle, 1966) and could not be recommended as a replacement for routine IF studies. However, IP allows a great extension of the observations that can be made by IF, primarily in the identification of the cellular or virion site of the immune reaction. Peroxidase labelling offers an advantage over labelling with ferritin in that the latter, being a larger molecule, presents more difficulty in penetration of a washout from the cell. We found no difficulty in obtaining good penetration or washout of peroxidase-labelled material. From these observations we conclude that VCA can be identified not only on the intact virion, but also at various sites within the cells (including the ribosomal sites of synthesis). Using the IP technique, we found VCA-positive cells (in the P3HR-1 cell line) in areas which contained no detectable intact virions. Therefore a VCA-positive cell is not necessarily one that is also virion-positive. MA appears as a constituent of the membrane and not on or attached to the membrane. Were the antigen “ on ”, rather than part of the membrane, the spacing between the membrane and peroxidase reaction product might be greater and more irregular. While no difficulty was encountered in differentiating and identifying EAD and EAR by IF, only EAD could be consistently observed by IP. Probably EAR is sensitive to the fixation procedures or is
BTUDES
removed from the cells during the extensive washing procedures which are used. We believe that a technical problem exists at the level of fixation because similar difficulty was found in observing EAD before we developed a different fixation method. Our preliminary observations indicate that EAR is associated with ribosomes of the rough endoplasmic reticulum. While the results are not shown here, we have also observed the development of EAD in IdUrdactivated NC37 cells. Cells activated to EA production in this manner were much more fragile and difficult to handle, even though we used the gentle sedimentation procedure described above. Comparisons of reactivity in the VCA, MA or EAD examinations showed consistently good correlation between IP and IF results. However, use of the IP technique resulted in a higher percentage of positive cells, indicating a somewhat greater sensitivity than that obtained with the IF technique.
ACKNOWLEDGEMENTS
This study was conducted under Contract N01-CP-33234 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS. We thank Drs. J. V. Schlosser and R. C. Chang for their generous supply of sera and Ms. D. Woolf and Ms. G . Lowry for their excellent technical assistance in these studies.
COMPARATIVES DES ANTIGENES DE L’EBV PAR LES TECHNIQUES D’IMM UNOFLUORESCENCE ET D’IMM UNO PER O XY DASE
-
-
Les auteurs ont compar6 trois grouper d’antigines de I’EBV VCA, M A et EA I’aide des techniques d’immunofluorescence (IF) et d’immunoperoxydase (IP) en microscopie Clectronique. Des cellules P3HR-1 et des cellules Raji surinfect6es par I’EBV ont servi de cibles aux serums marqu6s de malades atteints de lymphome de Burkitt, de cancer du nasopharynx e t de mononuclOose infectieuse ou de donneurs sains. Des anticorps marques i la peroxydase I t a s ont 6galement 6th utilises pour mesurer, par autoradiographie, la p6n6tration du complexe dans le systime cellulaire e t pour contraler I’efficacit6 des proc6d6s d’incubation et de ringage. La technique de s6dimentation qui a 6t6 employ6e s’est r6v616e critique du point de vue de la manipulation des cellules-cibles,qui sont fragiles. Le VCA et le M A ont 6t6 facilement identifies et localis6r par les deux proc6dis sans modification sensible des techniques de base. Pour identifier I’antighe pr6coce avec le proc6d6 IP, iI a fallu modifier la m6thode de fixation et pr6voir un bref traitement i I’ac6tone. O n a constat6 que I’antighe pr6coce diffus (EAD) est associ6 aux ribosomes cellulaires.
REFERENCES
BURKITT,D. P., and BURCHENAL, J. H., Treatment of Burkitt’s tumor. Springer-Verlag, New York (1967). BURKITTD. P., and WRIGHT,D. H., Burkitt’s lymphoma. E. & S . Livingstone, Edinburgh and London (1970). . . CARO,L. G., VAN TUBERGEN, R. P., and KOLB,J. A., High resolution autoradiography. I. Methods. J . Cell Biol., 15, 173-188 (1962).
EPSTEIN,M. A., ACHONG,B. G., and BARR,Y . M., Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet, i, 702-703 (1964).
ERNBERG, I., KLEIN, G., KOURILSKY, F. M., and SILD., Differentiation between early and late membrane antigen on human lymphoblastoid cell lines infected with Epstein-Barr virus. I. Immunofluorescence. J . nat. Cancer Inst., 53, 61-65 (1974). VESTRE,
HAMPAR, B., GERBER, P., Hsu, K . , MARTOS, L., WALKER, J., SIQUENZA, R., and WELLS,G., Irnmunoferritin and immunofluorescence studies with Epstein-Barr virus and Herpes simplex virus by use of human sera and hyperimmune rabbit sera. J . nat. Cancer Inst., 45, 75-81 (1970).
316
STEPHENS ET AL.
HAMPAR, B., and MARTOS,L., Immunological relationships. In: A. Kaplan (ed.), The herpesviruses, p. 248-252, Academic Press, New York (1973). HENLE,W. and HENLE,G., Immunofluorescence in cells derived from Burkitt’s lymphoma. J . Bact., 91, 1248-1256 (1966). HENLE,W., and HENLE,G., Host responses to herpesviruses -a review. I n : G. de-ThB, M. A. Epstein and H . zur Hausen (ed.), Oncogenesis and herpesviruses, II, pp. 21 5224, International Agency for Research on Cancer Scientific Publication No. 11, Part 2, IARC, Lyons (1975). HENLE,G., HENLE,W., and DIEHL,V., Relation of Burkitt’s tumor-associated herpes-type virus to infectious mononucleosis. Proc. nut. Acad. Sci. (Wash.), 59, 94-101 (1968). HENLE,G., HENLE,W., and KLEIN,G., Demonstration of two distinct components in the early antigen complex of Epstein-Barr virus-infected cells. Int. J . Cancer, 8, 272-282 (1971). W. M., The preparation of radioiodinated proteins HUNTER, of high activity, their reaction with antibody in virro: the radioimmunoassay. In: D. M. Weir (ed.), Handbook of experimental immunology, pp. 608-654, F. A. Davis Co., Philadelphia (1 967). INOUE,M., and KLEIN, G., Reactivity of radioiodinated serum antibody from Burkitt lymphoma nasopharyngeal carcinoma patients against culture lines derived from Burkitt lymphoma. Clin. exp. Immunol., I, 39-49 (1970). KLEIN, G., The Epstein-Barr virus. In: A. Kaplan (ed.), The herpesviruses, pp. 521-550, Academic Press, New York (1973). P., KLEIN,E., and STJERNSWARD, J., KLEIN,G., CLIPFORD, Search for tumor specific immune reactions i n Burkitt lymphoma patients by the membrane immunofluorescence
reaction. Proc. nut. Acad. Sci. (Wash.), 55, 1628-1635 (I 966). G., HENLE,G., HENLE,W., DIEHL,V., KLEIN,G., PEARSON, and NIEDERMAN, J. C., Relation between Epstein-Barr viral and cell membrane immunofluorescence in Burkitt tumor cell. 11. Comparison of cells and sera from patients with Burkitt’s lymphoma and infectious mononucleosis. J . exp. Med., 128, 1021-1030 (1968~). KLEIN,G., PEARSON, G., NADKARNI, J. S., NADKARNI, J. J., KLEIN,E., HENLE,G., HENLE, W., and CLIFFOKD, P., Relation between Epstein-Barr viral and cell membrane immunofluorescence of Burkitt tumor cells. I. Dependence of cell membrane immunofluorescence on presence of EB virus. J . exp. Med., 128, 1011-1019 (1968h). KURSTAK, E., The immunoperoxidase technique: localization of viral antigens in cells. In: K . Maramorosch and H . Koprowski (ed.), Methods in virology, Vol. V, pp. 423-444, Academic Press, New York (1971). 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, 11, 499-520 (1973). J. C., and KIRSCHSTEIN, R., SHABO,A. L., PETRICCIANI, Immunoperoxidase localization of Herpes zoster virus and simian virus 40 in cell culture. Appl. Mioobiol., 23, 10011009 (1972). P., STEPHENS, R., TRAWL,K., WOOLF,D., and GAUDREAU, Studies of Epstein-Barr virus antigens using immunoperoxidase and immunoiluorescence techniques. In: G. W. Baily (ed.), 33rd Ann. Proc. EIectron Microscopy SOC.Amer., Las Vegas, Nevada (1975). M., LYLES,J., LARSON,D., and MAYYASI, S., YEH,J., AHMED, Competition radioimmunoassay for Mason-Pfizer monkey virus: comparison with recent isolates. Inr. J. Cancer, 15, 632-639 (1975).
COMPARATIVE STUDIES ON EBV ANTIGENS BY IMMUNOFLUORESCENCE A N D IMMUNOPEROXIDASE TECHNIQUES R. STEPHENS, K. TRAUL,P. GAUDREAU, J. YEH, L. FISHER and S . A. MAYYASI The John L. Smith Memorial for Cancer Research, Pfizer Inc., Maywood, New Jersey 07607, USA
Three groups of EBV antigens, VCA, M A and EA, were compared by the techniques of electron microscopic immunoperoxidase (IP) and immunofluorescence (IF). P3HR-1 and EBV suparinfected Raji cells served as targets for labelled sera from patients with BL, N P C and IM or from healthy donors. 1251 peroxidase-labelled antibodies were also prepared to determine, autoradiographically, the penetration of the complex into the cell system, and to monitor the incubation and washing procedures. The development of a gentle sedimentation technique proved critical in handling the fragile target cells. VCA and M A were readily identified and localized by both procedures without significant modification of the basic techniques. lndentlfication of early antigens by IP required modification of the fixation method to include a brief treatment in acetone. The diffuse early antigen (EAD) was found to be associated with cellular ribosomes.
Molecular and serologic studies t o define the association of Epstein-Barr virus (EBV) (Epstein et al., 1964; Burkitt and Burchenal, 1967; Burkitt and Wright, 1970) with Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and infectious mononucleosis (IM), have led to many interesting observations regarding the associated antigens (Henle and Henle, 1975; Klein, 1973; Hampar and Martos, 1973). While the role of EBV in all of these diseases is not absolutely defined, it continues to be the prime subject for etiological investigations. Immunofluorescence (IF) has proved t o be an invaluable tool in identifying and locating several of the groups of EBV-associated antigens (Fig. l), as well as in seroepidemiologic analyses. (Henle et al., 1968, 1971; Klein et al., 1966; Reedman and Klein, 1973; Ernberg et al., 1974). The more sensitive immunoelectron microscopic (IEM) techniques of immunoferritin (Hampar et al., 1970) and autoradiography (Inoue and Klein, 1970) have also been employed, but to a lesser extent. Generally the latter studies have been limited to antigens associated with larger structures such as intact virions or cell membranes. We initiated immunoperoxidase (IP) studies of several EBV antigens-virus capsid antigens (VCA), early antigens (EA) and membrane antigens (MA) -with the goal of refining knowledge about the localization of these antigens. Use of the IP techniques has been shown to have significant advan-
tages over IF methods in identifying the various antigenic sites because the IP procedure is more sensitive than IF and provides greater resolution. MATERIAL AND METHODS
Cell cultures
Burkitt-lymphoma-derived P3HR-1 and Raji cells were grown in RPMI 1640 medium supplemented with 8 % fetal calf serum. Changing of the cell culture medium took place every 5-7 days. Cell counts were made using the conventional trypan blue exclusion method and viability of the cells was usually maintained around 85-90 %. Preparation of EBV
EBV was prepared from tissue culture supernatant fluids of P3HR-1 cultures. Six days after the cultures had been set up, the fluids were pooled and the cells removed by centrifugation at 800 g for 15 rnin. The virus-containing supernatant was then centrifuged at 70,000 g for 90 min t o pellet the virus. The pellets were resuspended in a mixture of 50% fresh plus 50 % spent medium which had been previously filtered through a 0.22 pm pore size filter. All virus suspensions were stored at -95" C prior t o use. Induction of early antigen
Twenty-four-hour cultures of Raji cells were harvested, counted and inoculated into 2-oz plastic flasks. Each flask received 4 ml of medium (50% fresh, 50% spent as above) containing 6 x 1 0 5 (viable) cells per ml. One ml of appropriately diluted virus suspension was added t o each flask. The cultures were mixed by shaking and then incubated at 35" C for 42 h. The cells were harvested and washed gently with buffered saline. A small aliquot was used immediately to prepare acetone-fixed smears for immunofluorescence analysis while the remainder of the cell pellets were fixed as described below in the paragraphs dealing with immunoperoxidase procedures. Received: October 21, 1976 and in revised from December 9, 1976.
306
STEPHENS ET AL.
FIGURE 1 Observations of immunofluorescence reactions showing EB viral MA complete and segmented EAD ( c )and EAR (D) in P3HR-1 cells (A, B) and EBV superinfected Raji cells (c, D) x 320.
(A),
VCA
(B),
307
VISUALIZATION OF EBV ANTIGENS
Sera and serum conjugation
EBV-positive and -negative sera used for these studies were from healthy persons, or patients with nasopharyngeal carcinoma, Burkitt's lymphoma or infectious mononucleosis. Sera were stored undiluted in 1-ml aliquots at -95" C prior to use. Peroxidase conjugates were formed with 12 parts horse-radish peroxidase and 5 parts serum protein (Kurstak, 1971) after which the complex was purified by the Shabo (1972) sedimentation procedure. Iodination of peroxidase-conjugated globulins was accomplished by the chloramine T method (Hunter, 1967; Yeh et al., 1975). One hundred pl of peroxidase-conjugated immunoglobulins (75 pg/ml) were incubated for 1 min with 1 mCi of lZ5I2and 25 pl of chloramine T (4 mg/ml), after which 100 p1 of sodium metabisulfite (3.5 mglml) and 200 pl KI (2 mg/ml) were added. The mixture was eluted off a BioGel P-60 column (1 cm x10 cm, mesh 100-200) with 0.5 M phosphate buffer, PH 7.4, then 0.5-pI fractions were collected and each fraction was assayed for TCA precipitability. The specific activity of the peak fractions was found to contain 5 x lo4 cpm per ,ug conjugated protein. Fluorescence staining for VCA andlor EA
Following the indirect procedure of Henle (1966), acetone-fixed smears were allowed t o incubate for 30 min with appropriate dilutions (8-16 units of antibody) of either NPC, IM, or BL sera. EBVnegative serum was used at 1:lO dilution. After incubation, the smears were rinsed (three times) in PBS and stained with 16 units of fluorescein-labelled (goat) anti-human IgG serum. After the cell smears
had been rinsed again three times in PBS they were counterstained with Evans' blue dye and allowed to air dry. The smears were mounted in glycerol containing 10% PBS, and the nature and quality of fluorescence was based on the evaluation of 300-500cells using a Reichert Zetopan microscope. Fluorescence staining for M A
Small aliquots of P3HR-1 or Raji cells were gently pelleted, washed twice with PBS, and resuspended in 5 vol (16 units of B1, NPC, 1M or a 1 :10 dilution of " normal ") of serum for 30 min. Again, the cells were washed (PBS) and resuspended in saline glycerol and wet mounts were made for microscopic observation. Cellular fluorescence was determined as indicated in the preceding paragraph. Peroxidase staining for VCA and MA
Ten-ml aliquots of cell suspension were pelletized in a clinical centrifuge (600 g) and the supernatant fluids were discarded. The cell pellets were resuspended in paraformaldehyde for lh, and then pelletized (600 g ) and washed twice in cacodylate buffer by gently overlaying the buffer for 15 min (handling of the cells from this point became critical for preservation of cellular morphology). The buffer was carefully removed by pipetting and the cells were resuspended in a 1 :4 dilution of the serumperoxidase complex for 16-24 h at 4" C with gentle agitation. The cells were allowed to settle for 15 min, then were centrifuged (1250) yielded bright whole-cell fluorescence in both the cytoplasm and nucleus (Fig. 1 ~ ) Moderately . strong and weak sera showed predominantly cytoplasmic reactions. Peroxidase reactions were observed on organelles and inner membranes of cells, on nuclear virions, on cytoplasmic virions, and on extracellular virions. These reactions were readily observable by the increased size, density and irregularity of the positive structures. Figure 4 shows coating of virions in the nucleus of the cells, whereas Figure 5 contains an example
313
of mixed nuclear and cytoplasmic virion reactions. In each of these Figures certain organelles of both the nucleus and the cytoplasm have strong positive reactions. Figure 6a, B shows control and positive extracellular virus. Observation of EA
Glutaraldehyde fixation, used for VCA and MA, resulted in consistently negative reactions for EA by both IF and IP techniques. In contrast, acetonefixed cell smears were positive for observation of both EAD and EAR by IF (Fig. l c , ~ ) .Use of
FIGURE 7 Reaction of IP-labelled anti-EAD positive serum with 42-h-old, EBV-superinfected Raji cells. Enlarged areas show reactions observed on the ribosomes resulting in enlargement and irregularities and also reactions on inner membranes.
3 14
STEPHENS ET AL.
the standard acetone fixation technique proved highly destructive to cells for processing by the IP method. The alternative and more gentle paraformaldehyde/acetone fixation procedure afforded preservation of morphology and EAD was readily observed by IP (Fig. 7). Reactions were found at the level both of the free ribosomes and of the inner membranes throughout the cytoplasm. Here, as with VCA reactions, a positive reaction was recognized by the increased size and irregularity of the structures as compared with those of the control cells (Fig. 8).
We were unable t o observe consistent reactions by IP which correlated with the IF observations of EAR. EAR positive and EA, negative antisera tested on EAR positive cells (by IF) gave negative results. Several combinations of paraformaldehyde and/or acetone as fixative conditions were tried without success. DISCUSSION
We have compared the utility of immuno-electron microscopy and immunofluorescence procedures (Henle et al., 1971; Klein et al., 1968a, b) to localize
FIGURE 8 Observations with IP-labelled anti EAD positive serum and control Raji cells; in contrast to positive reactions demonstrated in Figure 7, these enlarged areas show unreacted ribosomes and inner membranes.
315
VISUALIZATION OF EBV ANTIGENS
and further characterize the EBV antigens: VCA, MA, EA, (Stephens et al., 1975). The IP procedure is longer and more complicated than IF (Henle, 1966) and could not be recommended as a replacement for routine IF studies. However, IP allows a great extension of the observations that can be made by IF, primarily in the identification of the cellular or virion site of the immune reaction. Peroxidase labelling offers an advantage over labelling with ferritin in that the latter, being a larger molecule, presents more difficulty in penetration of a washout from the cell. We found no difficulty in obtaining good penetration or washout of peroxidase-labelled material. From these observations we conclude that VCA can be identified not only on the intact virion, but also at various sites within the cells (including the ribosomal sites of synthesis). Using the IP technique, we found VCA-positive cells (in the P3HR-1 cell line) in areas which contained no detectable intact virions. Therefore a VCA-positive cell is not necessarily one that is also virion-positive. MA appears as a constituent of the membrane and not on or attached to the membrane. Were the antigen “ on ”, rather than part of the membrane, the spacing between the membrane and peroxidase reaction product might be greater and more irregular. While no difficulty was encountered in differentiating and identifying EAD and EAR by IF, only EAD could be consistently observed by IP. Probably EAR is sensitive to the fixation procedures or is
BTUDES
removed from the cells during the extensive washing procedures which are used. We believe that a technical problem exists at the level of fixation because similar difficulty was found in observing EAD before we developed a different fixation method. Our preliminary observations indicate that EAR is associated with ribosomes of the rough endoplasmic reticulum. While the results are not shown here, we have also observed the development of EAD in IdUrdactivated NC37 cells. Cells activated to EA production in this manner were much more fragile and difficult to handle, even though we used the gentle sedimentation procedure described above. Comparisons of reactivity in the VCA, MA or EAD examinations showed consistently good correlation between IP and IF results. However, use of the IP technique resulted in a higher percentage of positive cells, indicating a somewhat greater sensitivity than that obtained with the IF technique.
ACKNOWLEDGEMENTS
This study was conducted under Contract N01-CP-33234 within the Virus Cancer Program of the National Cancer Institute, NIH, PHS. We thank Drs. J. V. Schlosser and R. C. Chang for their generous supply of sera and Ms. D. Woolf and Ms. G . Lowry for their excellent technical assistance in these studies.
COMPARATIVES DES ANTIGENES DE L’EBV PAR LES TECHNIQUES D’IMM UNOFLUORESCENCE ET D’IMM UNO PER O XY DASE
-
-
Les auteurs ont compar6 trois grouper d’antigines de I’EBV VCA, M A et EA I’aide des techniques d’immunofluorescence (IF) et d’immunoperoxydase (IP) en microscopie Clectronique. Des cellules P3HR-1 et des cellules Raji surinfect6es par I’EBV ont servi de cibles aux serums marqu6s de malades atteints de lymphome de Burkitt, de cancer du nasopharynx e t de mononuclOose infectieuse ou de donneurs sains. Des anticorps marques i la peroxydase I t a s ont 6galement 6th utilises pour mesurer, par autoradiographie, la p6n6tration du complexe dans le systime cellulaire e t pour contraler I’efficacit6 des proc6d6s d’incubation et de ringage. La technique de s6dimentation qui a 6t6 employ6e s’est r6v616e critique du point de vue de la manipulation des cellules-cibles,qui sont fragiles. Le VCA et le M A ont 6t6 facilement identifies et localis6r par les deux proc6dis sans modification sensible des techniques de base. Pour identifier I’antighe pr6coce avec le proc6d6 IP, iI a fallu modifier la m6thode de fixation et pr6voir un bref traitement i I’ac6tone. O n a constat6 que I’antighe pr6coce diffus (EAD) est associ6 aux ribosomes cellulaires.
REFERENCES
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