EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
lmmunohistochemical of Hepatitis
29,
156-169
( 1978)
and Electron Microscopic Detection B Surface and Core Antigens
G. A. CABRAL,*~ 1 F. GYORKEY,“’ f P. GYORKEY,-/- J. L. MELNICK,” AND G. K. DREESMAN* * Department f Department
of Virology
of Pathology, Receiz;ed
December
and Epidemiology, Houston, Texas 77030; Veterans Administration 19,
1977,
and
in
Baylor College of Medicine, and Hospital, Houston, Texas
revised
form
hlarch
77211
6, 1978
Light, immunofluorescent, and electron microscopic examination of biopsied and of paraffin-embedded hepatitis B virus (HBV)-infected liver was undertaken. Electron microscopy allowed for the specific identification of HBs and HBc particles within hepatocyte cytoplasm and nuclei, respectively. Direct and indirect immunofluorescence proved to be simple, reliable, and specific for detecting HBsAg and HBcAg in biopsy material, but less sensitive in elucidating these antigens in formal’infixed, paraffin-embedded tissue. Immunoperoxidase procedures, employing either conjugated antibody or Fab’, on the other hand, proved extremely sensitive in detecting HBV-associated antigens both in biopsied liver and in formalin-preserved, paraffin-embedded liver which had been stored at room temperature for 10 years.
INTRODUCTION Two distinct antigenic reactivities have been reported in the circulation of individuals infected with hepatitis B virus ( HBV). The first represents excess virus coat material (Brzosko et al., 1973; Gyorkey et al., 1974; Krugman et al., 1974) which is associated with the surface of the intact HBV foriginaIIy referred to as the Dane particle (Dane et al., 1970)] and with spheres 22 nm in diameter or filaments of various lengths (Bayer et al., 1968). This antigen is collectively referred to as hepatitis B surface antigen ( HBsAg). The second distinct antigenic component is associated with the nucleocapsid of the intact HBV and has been termed hepatitis B core antigen ( HBcAg ) ( Almeida et al., 1971). Subsequent studies have demonstrated that HBsAg is principally localized in the cytoplasm of hepatocytes (Edgington and Ritt, 1971; Hadziyannis et al., 1972; Krawczynski et al., 1972; Huang, 1975; Nayak and Sachdeva, 1975), whereas core antigen, although generally associated with the nucleus (Hadziyannis et al., 1973; Huang, 1975; Lamothe et al., 1976), also has been observed in differential distribution patterns within the cytoplasm following application of immunofluorescence or immunoperoxidase methods (Gudat and Bianchi, 1977; Yamada and Nakane, 1977). Standard ultrastructural examination of infected liver cells has confirmed 1 To
whom
requests
for
reprints
should
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0 1978 by Academic Press, of reproduction in any form
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be sent.
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that surface particles, i.e., spheres and filaments, are present within the cytoplasm (Gerber et al., 1974; Sun et al., 1974) and that 26-nm HBcAg structures, observed chiefly in the nucleus (Huang, 1971; Hadziyanms and Gerber, 1974), have also been localized clustered in submembranous spaces of the cytoplasm and in close association with the cell membrane (Gudat and Bianchi, 1977). The biosynthetic origin of each of these antigens within infected cells and the association of HBV with various diseases (i.e., Down’s syndrome, chronic renal disease, and primary hepatic carcinoma) is of prime interest. However, to date studies have been undertaken principally with fluoresceinated antibodies of respective specificities on fresh frozen sections of infected liver. These techniques have proven less than satisfactory for the identification of HBV-associated antigens in formalin-preserved, paraffin-embedded tissues, particularly those which have been stored for extended periods of time. Thus, retrospective examination of samples has been diflicult, and results obtained have been, in general, inconclusive. Several approaches, particularly orcein and immunoperoxidase, have been employed recently in attempts to enhance the staining of tissues stored in paraffin. Orcein staining ( Shikata et al., 1974), although valuable for detecting HBsAg in paraffin-embedded liver tissue, does not react with the particles associated with HBcAg. Furthermore, staining with orcein is less specific than that of immunofluorescence. Horseradish peroxidase (HRP)-anti-HBs or HRP-anti-HBc conjugates, on the other hand, present several attractive features: (i) they are at least as sensitive as those employed in immunofluorescence on frozen sections, (ii) ordinary light microscopes, rather than expensive ultraviolet (UV) scopes, may be utilized for examination of samples, (iii) preparations may be counterstained or treated by various cytochemical or histochemical procedures following development of peroxidatic activity, (iv) samples may be stored for indefinite periods of time without loss of staining intensity, (v) problems peculiar to immunofluorescence procedures, such as autofluorescence, fading, and nonspecificity due ‘to negative charge imparted to conjugates by fluorescein, are minimal, and (vi) conjugates are stable and may be stored for considerable periods of time in the lyophilized state without loss of enzymatic or antibody activities. However, realization of maximum sensitivity with the immunoperoxidase procedure is predicated on the utilization of conjugates which are free of unconjugated antibody that would compete for available intracellular hepatitis-associated antigen sites. Therefore, results concerning application ‘of this technique to paraffinembedded liver containing HBV-associated antigens have been less than ideal or have been successful only after exposure of sections to pronase digestion followed by extended incubation periods with conjugates. In the present communication, we report on the development of highly specific and sensitive HRP conjugates, their purification, and their subsequent employment for the identification of HBs and HBc antigens in acetone-fixed cryostat sections or formalin-preserved, paraffin-embedded tissues. In addition, we have compared the relative efficacy of direct .and indirect immunoperoxidase procedures to that of fluoresceinated antibodies in the detection of HBV-associated antigens in sections of the same material.
158
CABRAL
AL\TERIALS Clinical
El’
AND
AL.
METHODS
Material
Two liver biopsy samples were studied by using fresh frozen cryostat and paraffinized sections for the presence of HBs and HBc antigens, In addition, one autopsy liver specimen was investigated using paraffinized sections. The two patients from whom biopsies were obtained were clinically suspected of having periarteritis nodosa, which was proven by histologic examination. The other patient was suspected of having chronic persistent hepatitis and expired from renal failure. Each patient showed significantly elevated liver function test results. In addition, sera from all 3 patients were positive for HBsAg as determined by solid-phase radioimmunossay (Ausria-‘?“I, Abbott Laboratories, North Chicago, Ill. ) . Preparation
of Antisera
Suspensions of 22 -C 2 nm particles associated with HBsAg were prepared from HBsAg-positive plasma kindly supplied by Dr. M. J. Simons (World Health Organization Immunology Centre, Singapore) and were then employed to produce anti-HBs in rabbits and in goats using methods previously described (Dreesman et al., 1972). The isolation of HBV core particles and the production of anti-HBc in rabbits has been reported (Fields et al., 1976, 1977). Briefly, particles were obtained from infected human livers by a series of freeze/thaw cycles and homogenization in the presence of Triton X-100. Supernatant fluids were extracted with ether and were subsequently purified by centrifugation in a linear sucrose gradient followed by gel filtration in Sepharose 4B. The preparation thus obtained contained approximately 5 x 101’ particles/ml and was emulsified in Freund’s complete adjuvant and injected directly into surgically exposed popliteal lymph nodes. Rabbits were boostered by intramuscular injections at 6-week periods. Serum employed in this study was obtained 10 days following the fourth inoculation and had a titer of 1.2 x 10” as measured by micro-solid immunoradiometric assay (micro-SPIRA) (Fields et al., 1977). Antiserum to rabbit IgG was prepared by hyperimmunization of goats with rabbit IgG purified by DEAE chromatography (Peterson and Sober, 1956). A single IgG precipitin arc was observed when this antiserum was reacted with normal rabbit serum and evaluated by microimmunoelectrophoresis. Goat IgG was prepared from a goat anti-rabbit IgG serum by precipitation with NaZSO1 at final wt/vol concentrations of 18 and 14% followed by anion-exchange chromatography on a DEAE-cellulose column equilibrated wtih 0.01 M phosphate buffer, pH 8.0. The peak protein fractions eluted with the equilibrating buffer were concentrated to 10 mg/ml using an Amicon PM-30 filter (Amicon Corp., Lesington, Mass. ). Analytical ultracentrifugation of this preparation demonstrated the existence of a sedimentation coefficient, SZO,,v, of 6.3 with no detectable contamination. Preparation
of Goat Anti-Rabbit
Fab’
Goat anti-rabbit IgG antibody was digested in acetate buffer at pH 3.9 with 2% pepsin for a period of 4 hr according to the procedure of Fanger et al. ( 1970).
HBV
ANTIGENS
IN
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150
The reaction mixture was then neutralized with 0.1 N NaOH and precipitated by the addition of 25% NaZS04 to a final concentration of 18% (Nisonoff, 1964). The material thus obtained on ultracentrifugal analysis had an S value of 5.0, characteristic of F( ab’)Y fragments. Fab’ was prepared by reduction with 0.007 M dithiothreitol at pH 7.0 for 2 hr at 37°C. Dry iodoacetamide was then added to a final concentration of 0.015 M and the protein solution was incubated for an additional 2 hr at 37°C. The mixture was fractionated by gel filtration in Sephadex G-100, and the single symmetrical peak eluted with 0.15 A4 NaCl was concentrated to 8 pg/ml by Amicon filtration using a UMlO filter. These Fab’ fragments had an S value of 3.2 as observed in the analytical centrifuge, and no longer formed a precipitin line with rabbit IgG in agar gel diffusion. Preparation
of Conjugates
(i) lmmunofluorescence. Globulin preparations of goat anti-HBs and of goat anti-rabbit IgG were dialyzed overnight in 0.85% saline, pH 8.0, at 4°C and were then conjugated to FITC, isomer I (Sigma Chemical Co., St. Louis, MO.) using procedures previously described (Hebert et al., 1972). Free dye was then separated from the FITC conjugate by filtration through Sephadex G-75 in 0.1 hl NaCl containing 0.005 M phosphate buffer, pH 7.0. Material eluted in the void volume fractions, containing labeled protein free of unreacted dye, was concentrated using a UMlO Diaflo membrane (Amicon Corp., Lexington, Mass.) and was then dialyzed against 0.005 M phosphate buffer, pH 8.1. The crude conjugate was applied to a DEAE-cellulose column equilibrated with the same buffer (McDevitt et al., 1963), and the initial fluorescent eluate was discarded. Purified, labeled antibody of minimal nonspecificity (i.e., relatively low negative charge) was then obtained by eluting with 0.05 M phosphate buffer, pH 6.3 (McDevitt et al., 1963). Following concentration with a UMlO Diaflo membrane, the conjugates were absorbed with acetone-prepared mouse liver powder and were stored in OS-ml aliquots at -70°C. The FITC conjugates were assessed for purity by microimmunoelectrophoresis. In addition, appropriate dilutions in 0.1 N NaOH, which yielded absorbancy readings at 495 nm of 0.05 to 0.6, were prepared. The protein-bound FITC in pg/ml was established by determining the absorbancy of the conjugate at this wavelength and comparing it to that of fluorescein diacetate (Eastman Kodak Co., Rochester, N. Y.) on a standard curve (Hebert et al., 1972). Protein concentrations were determined by the Biuret reaction employing spectrophotometric readings at 560 nm in order to ehminate altered values due to the presence of FITC. Conjugates with fluorescein to protein (F/P) ratios (i.e., pg FITC per mg protein) of 1 to 1.5 were employed for examination of liver sections. Anti-HBs antibody titers as measured by the passive hemagglutination procedure (Vyas and Shulman, 1970) were 1:200,000 and 1:800,000 to HBsAg,/adw and HBsAg/ayw, respectively. Rabbit anti-HBc was conjugated to FITC essentially as described above with the exception that purified labeled antibody fractions were obtained following elution at 4°C from the DEAE-cellulose column with 0.2 M phosphate buffer, pH 6.3. Anti-HBc antibody titer, as measured by micro-SPIRA, was 1:16,000. Anti-HBs antibody was not detected following examination of the conjugate by the Ausab test (Abbott Laboratories, North Chicago, Ill.).
1GO
ET AL.
CABRAL
Fraction
No.
0 0,
30
40 Fraction
50
I 00
No. 15 ml&r 1
FIG. 1. Fractionation of purified goat IgG, containing anti-rabbit IgG antibody, conjugated with horseradish peroxidase by gel filtr-ation using LKB Ultrogel AcA 44. The reactants were eluted with PBS, pH 7.2, from a 2.6 X 100 cm column, pooled as designated, and concentrated to a volume of 5 ml by Amicon ultrafiltration. The concentrated pool was recycled through the same column. The fractions designed in the inset of the figure were pooled and used in this study.
(ii) Immurzoperoxidnse. Goat Fab’ antibody fragments or IgG specific for rabbit IgG and goat anti-HBs IgG were conjugated to HRP with glutaraldehyde using the two-step procedure of Avrameas and Ternynck (1971), with slight modifications. Briefly, 90 mg of HRP (RZ3.0, Sigma Chemical Co., St. Louis, MO.) was dissolved in 1.2 ml of 0.1 M phosphate buffer, pH 6.8, containing 1.25% glutaraldehyde ( Polysciences, Warrington, Pa. ) . The reaction vial was wrapped in tin foil, and the contents were gently stirred for 18 hr at room temperature. Free glutaraldehyde was separated from the activated HRP by gel filtration in a 0.9 x 60 cm column of Sephades G-25 equilibrated with 0.15 M NaCl. The brown-colored fractions were pooled and concentrated to a volume of 6 ml with a UMlO Amicon filter. The activated HRP solution was added to an equal volume (6 ml) of Fab’ or IgG containing 2.5 mg of protein/ml or 5 mg of pruteinml, respectively. Following the addition of 0.6 ml of 1 h/f carbonate buffer, pH 9.5, the mixture was allowed to react in the dark at 4°C for 24 hr with gentle stirring. At the end of this incubation period, 0.6 ml of 0.2 M lysine was added and the contents were mixed for an additional 2 hr. The mixture was subsequently dialyzed against several changes of phosphate-buffered saline (PBS), pH 7.2, and the resultant small precipitate was removed by centrifugation at 20,OOOgfor 20 min at 4°C. The resulting mixture of complexes and unreacted reagents was placed on a 2.6 x 90 cm column of Ultrogel type AcA 44 (LKB Produkter, Bromma, Sweden) equilibrated with PBS at 4°C and 3-ml fractions were collected. The elution pattern for the goat anti-rabbit IgG preparation is shown in the lower part of Fig. 1 following determination of absorbance at 280 nm ancl at 403 nm for protein and peroxidase, respectively. The fractions in the second peak, containing material absorbing at 280 and 403 nm, were concentrated on a UMlO
EIBV
ANTIGENS
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161
FIG. 2. Hepatitis Electron rows ) . cryostat
B surface particles and associated HBsAg in HBV-infected liver cells. (A) micrograph exhibiting intracytoplasmic spherical and filamentous HBs particles (ar~31,400. (B) A direct immunofluorescence staining for HBsAg on an acetone-fixed section showing strong cytoplasmic fluorescence. X510.
filter and were recycled through the same column. The elution pattern is illustrated in the upper part of Fig. 1. Fractions 34 through 36 were pooled, concentrated by ultrafiltration, and analyzed by microimmunoelectrophoresis utilizing both goat anti-peroxidase and rabbit anti-goat IgG. A single precipitin line was observed upon staining with amido-blue-black. The same reaction was observed folIowing treatment with 3,3’-diaminobenzidine tetrahydrochloride (DAB), as indicated by a red-brown precipitin line. Similar procedures and
Amicon
162
CAISHAL
El’ AI,.
analyses were undertaken for conjugation of tlic goat anti-11Bs IgG and for Fab’ preparations of goat anti-rabbit IgG antibody. Light Microscopy For routine histological staining, liver blocks were embedded in paraplast, sectioned at 4 to 5 pm and stained with hematoxylin and eosin. Liver sections were also stained with orcein as described by Shikata et al. (1974). lmmunohistochemical Staining (i) Zmmunofluorescence. For the direct immunofluorescence technique, conjugated anti-HBs and anti-HBc were employed at dilutions of 1:20 and 1:40, respectively, on S-pm cryostat sections of acetone-fixed (5 min) tissue or on 3-5 pm paraplast sections of formalin-preserved tissue obtained from the same liver and from liver previously stored in paraffin blocks. Preparations were mounted in Elvanol (Rodriguez and Deinhardt, 1960) or in PBS-glycerol (1:9) and stored at 4°C until examined (within 24 hr of preparation). The staining in hepatocyte cytoplasm was abolished following absorption of fluoresceinated anti-HBs with purified HBsAg. Furthermore, staining of HBcAg or HBsAg with fluoresceinated anti-HBc or anti-HBs, respectively, was blocked by preincubation of sections with unlabeled homologous antibody. For the indirect immunofluorescence method, sections were treated with rabbit anti-HBc antiserum at a dilution of 1:40 and were then incubated with labeled goat anti-rabbit IgG diluted 1:20. Sections were then processed as described for the direct procedure. All sections were examined with a Zeiss UV microscope using an HBO-200 Hg light and a 470 or 540 barrier filter. (ii) Zmmunoperoxidase. Sections were processed by direct procedures using labeled goat anti-HBs and by indirect methods employing unlabeled rabbit antiHBc or rabbit anti-HBs sera in conjunction with either IgG or Fab’ preparations of goat anti-rabbit IgG antibody HRP conjugates essentially as described for immunofluorescence with the following modifications. Following incubation with the appropriate peroxidase-labeled antibody, sections were treated with DAB and with hydrogen peroxide for the histochemical demonstration of peroxidatic activity (Graham and Karnovsky, 1966; Nakane and Pierce, 1967; Palmer et al., 1974), dehydrated in a graded series of ethanol, passed through xylene, and embedded in Permount (Fisher Scientific Co., Fair Lawn, N. J.), Controls similar to those used for immunofluorescence were also examined. Electron Microscopy Selected liver areas, chosen by gross inspection, were cut into small (1 mm?) chunks and were immediately fixed in 2% buffered glutaraldehyde followed by postfixation in osmium tetroxide (Gyorkey et al., 1975). Following dehydration in ethanol containing uranyl acetate, the specimens were embedded in EponAraldite. Formalin-fixed, paraffinized liver material, which had been stored for 10 years at room temperature, was also prepared for electron microscopy by immersing the paraffin blocks initially in xylene, then in chloroform, followed by hydration
HBV
ANTIGENS
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FIG. 3. Hepatitis B core particles and associated HBcAg in HBV-infected hepatocytes. (A) Electron micrograph of a nucleus containing monodispersed hepatitis type B cores. ~14,700. (B) A high magnification micrograph of the intranuclear hepatitis B cores. The particles measure approximately 26 nm in diameter. X89,000. (C) Negatively stained preparation of HBc particIes isolated from nuclei of infected cells. These purified particles were utilized to inoculate rabbits to produce anti-HBc as described in Materiak and Methods. X89,000. (D) Direct immunofluorescent staining for HBcAg on acetone-fixed frozen sections of infected liver. Note the presence of strong nuclear fluorescence. Occasionally whole nuclei were fluorescent (arrow). These sections were obtained from the same liver as those employed for demonstration of HBsAg. X480.
through a graded series of ethanol. Following washing in distilled water, the material was refixed in 2% buffered glutaraldehyde, postfixed in osmium tetroxide, and processed for microscopy as described above, Ultrathin sections were stained with uranyl acetate followed by lead citrate and were examined in a Philips model 301 electron microscope.
164
C,4131~;\1, E’L .\I,.
Frc. 4. Parafbembedded liver sample reprocessed for electron n~i~ro~.copy as tkscril~ed in Materials and Methods. Note the presence of numerous intranuclear care particles. Y 12,800.
RESULTS Light, immunofluorescent and electron microscopic rcbults were compared and correlated for the presence of surface antigen and core antigen. In fresh frozen cryostat sections (Fig. 2B), extensive positive fluorescence was disclosed in the cytoplasm of hepatocytes following treatment with fluoresceinated goat-anti-H& Electron microscopic examination of these cells revealed the presence of typical intracytoplasmic HBsAg particles (Fig. 2A, arrows). The same cryostat sections were studied for the presence of HBcAg and exhibited positive fluorescence in hepatocyte nuclei following incubation with FITC-labeled rabbit anti-HBc (Fig. 3D) or with labeled goat anti-rabbit IgC preceded by rabbit anti-HBc antiserum (not shown). Occasionally, whole nuclei were fluorescent ( arrow), an observation which correlated well with electron microscopic findings of nuclei containing evenly dispersed core particles (Fig. 3A). A comparative assessmentof the IIBc particles is provided on a low-powered electron micrograph (Fig. 3X), a highpowered eIectron micrograph (Fig. 3B), and on 3 micrograph of isolated core particles purified from the necropsy liver (Fig. 3C). In form&n-fixed, paraffinized liver reprocessed for electron microscopy, HBs particles were not readily detected. However, intranuclear core particles were readily seen (Fig. 4). Moderate intracytoplasmic fluorescence, noted in these sections following treatment with fluoresceinated goat anti-HBs (not shown), corresponded quite well with the pattern of “amber” coloration observed in hepatocytes following application of orcein staining (not shown). Identical sections processed for the presence of HBcA g, by either direct or indirect methods, exhibited positive, but weak, intranuclear fluorescence (not shown). Cryostat sections obtained from the same material as above and processed by direct and indirect immunoperoridase procedllres, .+lded y results similar to those
HBV
ANTIGENS
IN
LIVER
1tXI
FIG. 5. Immunoperoxidase staining of HBV-infected river. (A) Indirect staining for HBcAg using rabbit anti-HBc and HRP-conjugated goat anti-rabbit IgG on acetone-fixed frozen sections reveals the presence of strong intranuclear staining. X 195. (B ) Localization of HBcAg in formalin-fixed paraffin sections using the same indirect technique. X800. (C) Formalin-fixed paraffin sections stained for the demonstration of HBsAg using the direct method. X480. (D) Formalin-fixed paraffin section processed by the indirect method. The section was incubated with rabbit anti-HBs followed by goat anti-rabbit Fab’-peroxidase. Note the presence of strong cytoplasmic staining. The nucleus in the cell at the center of the micrograph is unstained, demonstrating the specificity of the anti-HBs antiserum. X480.
obtained by immunofluorescence. Following incubation with goat anti-HBs-HRP, HBsAg was localized in the cytoplasm in a pattern which varied from cell-to-cell, some being negative, other strongly positive (not shown). HBcAg was present within nuclei of most hepatocytes after treatment of sections with rabbit antiHRc followed hy IgC or Fnh’ preparations of goat anti-rabbit IgC antibody7 linked to HRP (Fig. 5A).
166
CABRAL TABLE Comparative Results Human Liver Procedure
Immunofluorescence Direct Indirect Immunoperoxidase Direct Indirect Fab’-conjugate IgG-conjugate
El’ AL. I
of Specific Histochemical Staining for HBc and HBs Antigens” Acetone-fixed cryostat sections
of
Formalin-fixed paraffin sections
HBcAg
HBsAg
HBcAg
4+ 4+
4+ N.D.b
f +
N.D.
N.D.
4+
X.D.
4+
4+ 4+
4+ 4+
3+ 4f
4+ 4+
5 The intensity of staining was recorded and graded from negative to 4+. recorded samples in which staining was intense at low power and at high power * N.D. = not done.
HBsAg
2+
Designations of 4f using oil immersion.
In formalin-fixed, paraffined material, brown cytoplasmic staining was readily observed in various hepatocytes treated by the direct immunoperoxidase method for the demonstration of HBsAg (Fig. 5C, D). Nuclei were intensely stained for HBcAg and in patterns similar to those described for cryostat sections examined by immunofluorescence. This intranuclear staining generally paralleled the observed distribution of HB core particles in paraffinized tissue reprocessed for electron microscopy (Fig. 5B). A comparative assessmentof the conjugates employed in this study is given in Table I. No liver staining was found with the techniques used in this study in hepatocytes of two patients shown to contain neither HBs nor HBc particles by electron microscopy and to be HBsAg seronegative by radioimmunoassay ( Ausria-IZ51). DISCUSSION Several studies have been undertaken dealing with the demonstration of HBsAg or HBcAg in HBV-infected liver. These have included ultrastructural, immunofluorescence, and immunohistochemical analyses. However, to our knowledge, no studies have been presented which correlate the efficacy of these procedures and of IgG- or Fab’-HRP conjugates in the identification of HBs and HBc antigens in acetone-fixed cryostat or formalin-preserved, paraffinized liver sections. In the present investigation, the direct immuuofluorescence technique proved to be a reliable, sensitive and rapid method for the detection of HBV-associated antigens in acetone-fixed cryostat sections obtained from liver biopsies. This technique also proved reliable and specific in localizing HBsAg in formalin-fixed, paraffin-embedded liver sections stored for as long as 10 years. However, the identification of HBcAg in this latter material proved difficult, even following application of indirect immunofluorescence, due to weak staining. It should be noted that the fluorescence conjugates employed were separated on the basis of
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charge by elution through DEAE-cellulose and had low F/P ratios and, consequently, nonspecificity due to negative charge imparted by fluorescein was minimal. The direct and the indirect immunoperoxidase procedures proved to be as sensitive as fluorescence in the detection of HBsAg or HBcAg in frozen sections. These enzyme probes were particularly reliable for elucidating HBV-associated antigens in formalin-fixed, paraffinized liver. Sections of this tissue treated by indirect methods employing HRP conjugated to IgG or Fab’ for the demonstration of HBsAg were stained with equal intensity. This suggests that the tagged antibody fragments may prove ideal for ultrastructural examination of both paraffinized and fresh, appropriately fixed HBV-infected tissue. In addition, while orcein staining was applicable only to revelation of HBsAg sites, HRP conjugates proved especially useful for identifying HBcAg. This increased sensitivity of the enzyme conjugates could allow for the retrospective examination of stored paraffin-embedded liver samples, particularly those obtained from individuals with diseases having a probable association with HBV infection. In summary, electron microscopic examination allows for the specific identification of HBs and HBc particles within HBV-infected liver, but is tedious, timeconsuming and may not permit adequate diagnosis in those situations where relatively few hepatocytes are infected. Immunofluorescence methods are simple, reliable, specific techniques for detection of HBcAg and HBsAg in liver biopsies, but are less sensitive in elucidating these antigens in formalin-fixed, paraffinembedded material. Immunoperoxidase staining, employing various conjugates, proved equally sensitive in detecting antigens in acetone-fixed biopsy material and in formalin-preserved, paraffin-embedded tissue stored for as long as 10 years. ACKNOWLEDGMENTS We gratefully acknowledge research was supported by Institute, National Institutes Foundation.
the valuable technical assistance of Ms. Becky Clarke. This Research Grant HL-17269 from the National Heart and Lung of Health, and by Grant Q-435 from the Robert A. Welch
REFERENCES ALMEIDA, J. D., RUBENSTEIN, D., and STOTT, E. J. (1971). New antigen-antibody system in Australia-antigen-positive hepatitis. Lancet 2, 1225. AVRAMEAS, S., and TERNYNCK, T. ( 1971). Peroxidase labelled antibody and Fab conjugates with enhanced intracellular penetration. Immunochem. 8, 1175. BAYER, M. E., BLUMBERG, B. S., and WERNER, B. (1968). Particles associated with Australia antigen in the sera of patients with leukaemia, Down’s syndrome and hepatitis. Nature 218, 1057. BRZOSKO, W. J., MADALINSKI, K., KRAWCZYNSU, K., and NOWOSLAWSKI, A. ( 1973). Duality of hepatitis B antigen and its antibody. I. Immunofluorescence studies. J. Infect. Dis. 127, 424. DAIXE, D. S., CAMERON, C. H., and BRIC:GS, M. ( 1970). Virus-like particles in serum of patients with Australia-antigen-associated hepatitis. Lancet 1, 695. DREESMAN, G. R., HOLLINGER, F. B., MCCOTVIBS, R. M., and MELNICK, J. L. (1972). Production of potent anti-Australia antigen sera of high specificity and sensitivity in goats. Infect. Immun. 5, 213. EDGINGTON, T. S., and RITT, D. J. (1971). Intrahepatic expression of serum hepatitis virusassociated antigens. J. Erp. Med. 134, 871.
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168
L’2’ AL.
13. A., WELLS, J. V., and NISONOFF, A. (1970). Requirements for stimulation of transformation of rabbit peripheral lymphocytes by J. Immunol. 105, 1484. FIELDS, H. A., DREES~IAN, G. R., HOLLINGER, F. B., MARCIANO-CAURAL, F., GYORKEY, F., and MELNICK, J. L. (1976). Endogenous DNA polymerase-positive core particles from hepatitisinfected hepatocytes. Intervirology 6, 224. FIELDS, H. A., HOLLINGER, F. B., DESMYTEH, J., MELNICK, J. L., and DWEES~IAS, G. R. (1977). Biochemical and biophysical properties of hepatitis B core particles derived from Dane particles and infected hepatocytes. Interoirology 8, 336. GERBER, M. A., HAUZIYANNIS, S., VISSOULIS, C., SCHAFFNER, F., PAROSETTO, F., and POPPER, H. (1974). Electron microscopy and immunoelectron microscopy of cytoplasmic hepatitis B antigen in hepatocytes. Am. f. Pathol. 75, 489. GRAHAM, R. C., JH., and KARNOVSKY, M. J. ( 1966). The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291. GUDAT, F., and BIANCHI, L. ( 1977). E wence 1 for phasic sequences in nuclear HBcAg formation and cell-membrane directed flow of core particles in chronic hepatitis B. Gastroentcrology 73, 1191. GYORKEY, F., MIN, K. W., GYORKEY, I’., and MEI~NICK, J. L. ( 1974). Cytoplasmic localization of hepatitis B surface (viral coat) antigen (HB,Ag) by electron microscopy. New Engl. J. Med. 290, l-188. GYORKEY, F., MIN, K. W., KRISKO, I., and GYORKEY, P. (1975). Usefulness of electron microscopy in the diagnosis of human tumors. Human Pathol. 6, 421. HADZIYANNIS, S., and GERFIER, M. A. ( 1974). and electron microscopic I mmunofluorescence study of the hepatitis B “core” antigen in the liver. 25th rlnniv. Mtg. Am. Assoc. Study of Liver Diseases, Chicago. HADZIYANNIS, S., MOUSSOUROS, A., GUISTO~I, A., and AL~ZIEIL)A, J. ( 1973). Immunofluorescence study of the “core” antigen in the liver (abstract). Dige.don 8, 437. HADZIYANXS, S., MOUSS~U~~OS, A., VISSOULIS, C., and AFIIOUDAKIS, A. ( 1972). Cytoplasmic Iocalisation of Australia antigen in the liver. Luncet 1, 976. HERERT, G., PITTXAN, B., MCKINNEY, R., and CHERR’I’, W. ( 1972). The preparation and physicochemical characterization of fluorescent antibody reagents. U. S. DHEW Pub]. AD9603372, Center for Disease Control, Atlanta, Ca., 41 pp. HUANG, S. ( 1971). Hepatitis-associated antigen hepatitis. An electron microscopic study of virus-like particles in liver cells. Am. J. Pathol. 64, 18’3. HUANG, S. ( 1975). Immunohistochemical demonstrution of hepatitis B core and surface antigens in paraffin sections. Lab. Inuest. 33, 88. KRAWCZYNSKI, K., NAZAREWICZ, T., Bnzos~;o, W. J., and NOWOSLATVSKI, A. (1972). Cellular localization of hepatitis-associated antigen in livers of patients with different forms of hepatitis. J. Infect. Dis. 126, 373. KRUG~IAN, S., HOOFNAGLE, J. H., GERETY, R. J., KAPLAX, P. M., alld GEIIIN, J. L. ( 1974). Viral hepatitis, type B: DNA polymerase activity and aniillody to hepatitis B core antigen. New Engl. J. Al&. 290, 1331. LAlrfoTHE, F., LAUHENCIX-PICH~?, J., Ciurf, J., G&SIX, R., VIALLET, A., and RICHER, G. ( 1976). Detection of surface and core antigens of hepatitis B virus in the iiver of 164 human subjects. A study of immunoperoxidase and orcein staining. Gu.str.ocnterology 71, 10.2. MCDEVITT, H. O., PETERS, J. H., POLLARD, L. W., HARTER, J, G., and COONS, A. H. ( 1963). Purification and analysis of fluorescein-labeled antisera by column chromatography. J. Immunol. 90, 634. NAKANE, P. K., and PIERCE, G. B. (1967). Enzyme-labelecl antiboclies for the light and elcctron microscopic localization of tissue antigens. J. Cell Biol. 33, 307. NAYAK, N. C., and SACHDEVA, R. (1975). Localization of hepatitis B surface antigen in conventional paraffin sections of the liver. Am. J. Puthol. 81, 479. NISONOFF, A. (1964). Enzymatic digestion of rabbit gamma globulin and antibody and rhromatographv of digestion products. In “Methods in hledifal Resr-atch” (FT. E&t,, Eel. i, \‘ol. IO, p. 13k. \‘c*ar 11ook Rl(~l. I’ul~l., Cllicayo, Ill. FANGER,
M.
cross-linkage antiglobulin
W.,
HART,
in the reagents.
HBV
ANTIGENS
IN
LIVER
16’3
PALMER, P. E., DE LELLIS, R. A., and WOLFE, H. J. (1974). Immunohistochemistry of liver in alpha 1-antitrypsin deficiency. A comparative study. Am. J. Clin. P&o/. 62, 350. PETERSON, E. A., and SOBER, H. A. ( 1956). Fractionation of serum protein on anion-exchange cellulose. J. Am. Chem. Sot. 78, 751. RODRIGUEZ, J., and DEINHARIYT, F. (19GO). Preparation of semipermanent mounting medium for fluorescent antibody studies. Virology 12, 316. SHIKATA, T., UZAWA, T., YOSHIWARA, N., OKATSUICA, T., and YAMAZAKI, S. (1974). Staining methods of Australia antigen in paraffin sections. Jap. J. Erp. Med. 44, 25. SUN, S. C., ANDERSON, K. E., Hsu, C. P., and KAU, S. L. (1974). Hepatocellular ultrastructure in asymptomatic hepatitis B antigenemia. Arch. Pathol. 97, 373. VYAS, G. N., and SHULMAN, N. R. (1970). H emagglutination assay for antigen and antibody associated with viral hepatitis. Science 170, 332. YAMADA, G., and NAKANE, P. K. ( 1977). Hepatitis B core and surface antigens in liver tissue. Light and electron microscopic localization by the peroxidase-labeled antibody method. Lab. Invest. 36, 649.
AND
MOLECULAR
PATHOLOGY
lmmunohistochemical of Hepatitis
29,
156-169
( 1978)
and Electron Microscopic Detection B Surface and Core Antigens
G. A. CABRAL,*~ 1 F. GYORKEY,“’ f P. GYORKEY,-/- J. L. MELNICK,” AND G. K. DREESMAN* * Department f Department
of Virology
of Pathology, Receiz;ed
December
and Epidemiology, Houston, Texas 77030; Veterans Administration 19,
1977,
and
in
Baylor College of Medicine, and Hospital, Houston, Texas
revised
form
hlarch
77211
6, 1978
Light, immunofluorescent, and electron microscopic examination of biopsied and of paraffin-embedded hepatitis B virus (HBV)-infected liver was undertaken. Electron microscopy allowed for the specific identification of HBs and HBc particles within hepatocyte cytoplasm and nuclei, respectively. Direct and indirect immunofluorescence proved to be simple, reliable, and specific for detecting HBsAg and HBcAg in biopsy material, but less sensitive in elucidating these antigens in formal’infixed, paraffin-embedded tissue. Immunoperoxidase procedures, employing either conjugated antibody or Fab’, on the other hand, proved extremely sensitive in detecting HBV-associated antigens both in biopsied liver and in formalin-preserved, paraffin-embedded liver which had been stored at room temperature for 10 years.
INTRODUCTION Two distinct antigenic reactivities have been reported in the circulation of individuals infected with hepatitis B virus ( HBV). The first represents excess virus coat material (Brzosko et al., 1973; Gyorkey et al., 1974; Krugman et al., 1974) which is associated with the surface of the intact HBV foriginaIIy referred to as the Dane particle (Dane et al., 1970)] and with spheres 22 nm in diameter or filaments of various lengths (Bayer et al., 1968). This antigen is collectively referred to as hepatitis B surface antigen ( HBsAg). The second distinct antigenic component is associated with the nucleocapsid of the intact HBV and has been termed hepatitis B core antigen ( HBcAg ) ( Almeida et al., 1971). Subsequent studies have demonstrated that HBsAg is principally localized in the cytoplasm of hepatocytes (Edgington and Ritt, 1971; Hadziyannis et al., 1972; Krawczynski et al., 1972; Huang, 1975; Nayak and Sachdeva, 1975), whereas core antigen, although generally associated with the nucleus (Hadziyannis et al., 1973; Huang, 1975; Lamothe et al., 1976), also has been observed in differential distribution patterns within the cytoplasm following application of immunofluorescence or immunoperoxidase methods (Gudat and Bianchi, 1977; Yamada and Nakane, 1977). Standard ultrastructural examination of infected liver cells has confirmed 1 To
whom
requests
for
reprints
should
0014~4800/78/0292-OIsfi$02.00/0 Copyrkht All rights
0 1978 by Academic Press, of reproduction in any form
Inc. reserved.
be sent.
HBV
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157
that surface particles, i.e., spheres and filaments, are present within the cytoplasm (Gerber et al., 1974; Sun et al., 1974) and that 26-nm HBcAg structures, observed chiefly in the nucleus (Huang, 1971; Hadziyanms and Gerber, 1974), have also been localized clustered in submembranous spaces of the cytoplasm and in close association with the cell membrane (Gudat and Bianchi, 1977). The biosynthetic origin of each of these antigens within infected cells and the association of HBV with various diseases (i.e., Down’s syndrome, chronic renal disease, and primary hepatic carcinoma) is of prime interest. However, to date studies have been undertaken principally with fluoresceinated antibodies of respective specificities on fresh frozen sections of infected liver. These techniques have proven less than satisfactory for the identification of HBV-associated antigens in formalin-preserved, paraffin-embedded tissues, particularly those which have been stored for extended periods of time. Thus, retrospective examination of samples has been diflicult, and results obtained have been, in general, inconclusive. Several approaches, particularly orcein and immunoperoxidase, have been employed recently in attempts to enhance the staining of tissues stored in paraffin. Orcein staining ( Shikata et al., 1974), although valuable for detecting HBsAg in paraffin-embedded liver tissue, does not react with the particles associated with HBcAg. Furthermore, staining with orcein is less specific than that of immunofluorescence. Horseradish peroxidase (HRP)-anti-HBs or HRP-anti-HBc conjugates, on the other hand, present several attractive features: (i) they are at least as sensitive as those employed in immunofluorescence on frozen sections, (ii) ordinary light microscopes, rather than expensive ultraviolet (UV) scopes, may be utilized for examination of samples, (iii) preparations may be counterstained or treated by various cytochemical or histochemical procedures following development of peroxidatic activity, (iv) samples may be stored for indefinite periods of time without loss of staining intensity, (v) problems peculiar to immunofluorescence procedures, such as autofluorescence, fading, and nonspecificity due ‘to negative charge imparted to conjugates by fluorescein, are minimal, and (vi) conjugates are stable and may be stored for considerable periods of time in the lyophilized state without loss of enzymatic or antibody activities. However, realization of maximum sensitivity with the immunoperoxidase procedure is predicated on the utilization of conjugates which are free of unconjugated antibody that would compete for available intracellular hepatitis-associated antigen sites. Therefore, results concerning application ‘of this technique to paraffinembedded liver containing HBV-associated antigens have been less than ideal or have been successful only after exposure of sections to pronase digestion followed by extended incubation periods with conjugates. In the present communication, we report on the development of highly specific and sensitive HRP conjugates, their purification, and their subsequent employment for the identification of HBs and HBc antigens in acetone-fixed cryostat sections or formalin-preserved, paraffin-embedded tissues. In addition, we have compared the relative efficacy of direct .and indirect immunoperoxidase procedures to that of fluoresceinated antibodies in the detection of HBV-associated antigens in sections of the same material.
158
CABRAL
AL\TERIALS Clinical
El’
AND
AL.
METHODS
Material
Two liver biopsy samples were studied by using fresh frozen cryostat and paraffinized sections for the presence of HBs and HBc antigens, In addition, one autopsy liver specimen was investigated using paraffinized sections. The two patients from whom biopsies were obtained were clinically suspected of having periarteritis nodosa, which was proven by histologic examination. The other patient was suspected of having chronic persistent hepatitis and expired from renal failure. Each patient showed significantly elevated liver function test results. In addition, sera from all 3 patients were positive for HBsAg as determined by solid-phase radioimmunossay (Ausria-‘?“I, Abbott Laboratories, North Chicago, Ill. ) . Preparation
of Antisera
Suspensions of 22 -C 2 nm particles associated with HBsAg were prepared from HBsAg-positive plasma kindly supplied by Dr. M. J. Simons (World Health Organization Immunology Centre, Singapore) and were then employed to produce anti-HBs in rabbits and in goats using methods previously described (Dreesman et al., 1972). The isolation of HBV core particles and the production of anti-HBc in rabbits has been reported (Fields et al., 1976, 1977). Briefly, particles were obtained from infected human livers by a series of freeze/thaw cycles and homogenization in the presence of Triton X-100. Supernatant fluids were extracted with ether and were subsequently purified by centrifugation in a linear sucrose gradient followed by gel filtration in Sepharose 4B. The preparation thus obtained contained approximately 5 x 101’ particles/ml and was emulsified in Freund’s complete adjuvant and injected directly into surgically exposed popliteal lymph nodes. Rabbits were boostered by intramuscular injections at 6-week periods. Serum employed in this study was obtained 10 days following the fourth inoculation and had a titer of 1.2 x 10” as measured by micro-solid immunoradiometric assay (micro-SPIRA) (Fields et al., 1977). Antiserum to rabbit IgG was prepared by hyperimmunization of goats with rabbit IgG purified by DEAE chromatography (Peterson and Sober, 1956). A single IgG precipitin arc was observed when this antiserum was reacted with normal rabbit serum and evaluated by microimmunoelectrophoresis. Goat IgG was prepared from a goat anti-rabbit IgG serum by precipitation with NaZSO1 at final wt/vol concentrations of 18 and 14% followed by anion-exchange chromatography on a DEAE-cellulose column equilibrated wtih 0.01 M phosphate buffer, pH 8.0. The peak protein fractions eluted with the equilibrating buffer were concentrated to 10 mg/ml using an Amicon PM-30 filter (Amicon Corp., Lesington, Mass. ). Analytical ultracentrifugation of this preparation demonstrated the existence of a sedimentation coefficient, SZO,,v, of 6.3 with no detectable contamination. Preparation
of Goat Anti-Rabbit
Fab’
Goat anti-rabbit IgG antibody was digested in acetate buffer at pH 3.9 with 2% pepsin for a period of 4 hr according to the procedure of Fanger et al. ( 1970).
HBV
ANTIGENS
IN
LIVEK
150
The reaction mixture was then neutralized with 0.1 N NaOH and precipitated by the addition of 25% NaZS04 to a final concentration of 18% (Nisonoff, 1964). The material thus obtained on ultracentrifugal analysis had an S value of 5.0, characteristic of F( ab’)Y fragments. Fab’ was prepared by reduction with 0.007 M dithiothreitol at pH 7.0 for 2 hr at 37°C. Dry iodoacetamide was then added to a final concentration of 0.015 M and the protein solution was incubated for an additional 2 hr at 37°C. The mixture was fractionated by gel filtration in Sephadex G-100, and the single symmetrical peak eluted with 0.15 A4 NaCl was concentrated to 8 pg/ml by Amicon filtration using a UMlO filter. These Fab’ fragments had an S value of 3.2 as observed in the analytical centrifuge, and no longer formed a precipitin line with rabbit IgG in agar gel diffusion. Preparation
of Conjugates
(i) lmmunofluorescence. Globulin preparations of goat anti-HBs and of goat anti-rabbit IgG were dialyzed overnight in 0.85% saline, pH 8.0, at 4°C and were then conjugated to FITC, isomer I (Sigma Chemical Co., St. Louis, MO.) using procedures previously described (Hebert et al., 1972). Free dye was then separated from the FITC conjugate by filtration through Sephadex G-75 in 0.1 hl NaCl containing 0.005 M phosphate buffer, pH 7.0. Material eluted in the void volume fractions, containing labeled protein free of unreacted dye, was concentrated using a UMlO Diaflo membrane (Amicon Corp., Lexington, Mass.) and was then dialyzed against 0.005 M phosphate buffer, pH 8.1. The crude conjugate was applied to a DEAE-cellulose column equilibrated with the same buffer (McDevitt et al., 1963), and the initial fluorescent eluate was discarded. Purified, labeled antibody of minimal nonspecificity (i.e., relatively low negative charge) was then obtained by eluting with 0.05 M phosphate buffer, pH 6.3 (McDevitt et al., 1963). Following concentration with a UMlO Diaflo membrane, the conjugates were absorbed with acetone-prepared mouse liver powder and were stored in OS-ml aliquots at -70°C. The FITC conjugates were assessed for purity by microimmunoelectrophoresis. In addition, appropriate dilutions in 0.1 N NaOH, which yielded absorbancy readings at 495 nm of 0.05 to 0.6, were prepared. The protein-bound FITC in pg/ml was established by determining the absorbancy of the conjugate at this wavelength and comparing it to that of fluorescein diacetate (Eastman Kodak Co., Rochester, N. Y.) on a standard curve (Hebert et al., 1972). Protein concentrations were determined by the Biuret reaction employing spectrophotometric readings at 560 nm in order to ehminate altered values due to the presence of FITC. Conjugates with fluorescein to protein (F/P) ratios (i.e., pg FITC per mg protein) of 1 to 1.5 were employed for examination of liver sections. Anti-HBs antibody titers as measured by the passive hemagglutination procedure (Vyas and Shulman, 1970) were 1:200,000 and 1:800,000 to HBsAg,/adw and HBsAg/ayw, respectively. Rabbit anti-HBc was conjugated to FITC essentially as described above with the exception that purified labeled antibody fractions were obtained following elution at 4°C from the DEAE-cellulose column with 0.2 M phosphate buffer, pH 6.3. Anti-HBc antibody titer, as measured by micro-SPIRA, was 1:16,000. Anti-HBs antibody was not detected following examination of the conjugate by the Ausab test (Abbott Laboratories, North Chicago, Ill.).
1GO
ET AL.
CABRAL
Fraction
No.
0 0,
30
40 Fraction
50
I 00
No. 15 ml&r 1
FIG. 1. Fractionation of purified goat IgG, containing anti-rabbit IgG antibody, conjugated with horseradish peroxidase by gel filtr-ation using LKB Ultrogel AcA 44. The reactants were eluted with PBS, pH 7.2, from a 2.6 X 100 cm column, pooled as designated, and concentrated to a volume of 5 ml by Amicon ultrafiltration. The concentrated pool was recycled through the same column. The fractions designed in the inset of the figure were pooled and used in this study.
(ii) Immurzoperoxidnse. Goat Fab’ antibody fragments or IgG specific for rabbit IgG and goat anti-HBs IgG were conjugated to HRP with glutaraldehyde using the two-step procedure of Avrameas and Ternynck (1971), with slight modifications. Briefly, 90 mg of HRP (RZ3.0, Sigma Chemical Co., St. Louis, MO.) was dissolved in 1.2 ml of 0.1 M phosphate buffer, pH 6.8, containing 1.25% glutaraldehyde ( Polysciences, Warrington, Pa. ) . The reaction vial was wrapped in tin foil, and the contents were gently stirred for 18 hr at room temperature. Free glutaraldehyde was separated from the activated HRP by gel filtration in a 0.9 x 60 cm column of Sephades G-25 equilibrated with 0.15 M NaCl. The brown-colored fractions were pooled and concentrated to a volume of 6 ml with a UMlO Amicon filter. The activated HRP solution was added to an equal volume (6 ml) of Fab’ or IgG containing 2.5 mg of protein/ml or 5 mg of pruteinml, respectively. Following the addition of 0.6 ml of 1 h/f carbonate buffer, pH 9.5, the mixture was allowed to react in the dark at 4°C for 24 hr with gentle stirring. At the end of this incubation period, 0.6 ml of 0.2 M lysine was added and the contents were mixed for an additional 2 hr. The mixture was subsequently dialyzed against several changes of phosphate-buffered saline (PBS), pH 7.2, and the resultant small precipitate was removed by centrifugation at 20,OOOgfor 20 min at 4°C. The resulting mixture of complexes and unreacted reagents was placed on a 2.6 x 90 cm column of Ultrogel type AcA 44 (LKB Produkter, Bromma, Sweden) equilibrated with PBS at 4°C and 3-ml fractions were collected. The elution pattern for the goat anti-rabbit IgG preparation is shown in the lower part of Fig. 1 following determination of absorbance at 280 nm ancl at 403 nm for protein and peroxidase, respectively. The fractions in the second peak, containing material absorbing at 280 and 403 nm, were concentrated on a UMlO
EIBV
ANTIGENS
IN
LIVER
161
FIG. 2. Hepatitis Electron rows ) . cryostat
B surface particles and associated HBsAg in HBV-infected liver cells. (A) micrograph exhibiting intracytoplasmic spherical and filamentous HBs particles (ar~31,400. (B) A direct immunofluorescence staining for HBsAg on an acetone-fixed section showing strong cytoplasmic fluorescence. X510.
filter and were recycled through the same column. The elution pattern is illustrated in the upper part of Fig. 1. Fractions 34 through 36 were pooled, concentrated by ultrafiltration, and analyzed by microimmunoelectrophoresis utilizing both goat anti-peroxidase and rabbit anti-goat IgG. A single precipitin line was observed upon staining with amido-blue-black. The same reaction was observed folIowing treatment with 3,3’-diaminobenzidine tetrahydrochloride (DAB), as indicated by a red-brown precipitin line. Similar procedures and
Amicon
162
CAISHAL
El’ AI,.
analyses were undertaken for conjugation of tlic goat anti-11Bs IgG and for Fab’ preparations of goat anti-rabbit IgG antibody. Light Microscopy For routine histological staining, liver blocks were embedded in paraplast, sectioned at 4 to 5 pm and stained with hematoxylin and eosin. Liver sections were also stained with orcein as described by Shikata et al. (1974). lmmunohistochemical Staining (i) Zmmunofluorescence. For the direct immunofluorescence technique, conjugated anti-HBs and anti-HBc were employed at dilutions of 1:20 and 1:40, respectively, on S-pm cryostat sections of acetone-fixed (5 min) tissue or on 3-5 pm paraplast sections of formalin-preserved tissue obtained from the same liver and from liver previously stored in paraffin blocks. Preparations were mounted in Elvanol (Rodriguez and Deinhardt, 1960) or in PBS-glycerol (1:9) and stored at 4°C until examined (within 24 hr of preparation). The staining in hepatocyte cytoplasm was abolished following absorption of fluoresceinated anti-HBs with purified HBsAg. Furthermore, staining of HBcAg or HBsAg with fluoresceinated anti-HBc or anti-HBs, respectively, was blocked by preincubation of sections with unlabeled homologous antibody. For the indirect immunofluorescence method, sections were treated with rabbit anti-HBc antiserum at a dilution of 1:40 and were then incubated with labeled goat anti-rabbit IgG diluted 1:20. Sections were then processed as described for the direct procedure. All sections were examined with a Zeiss UV microscope using an HBO-200 Hg light and a 470 or 540 barrier filter. (ii) Zmmunoperoxidase. Sections were processed by direct procedures using labeled goat anti-HBs and by indirect methods employing unlabeled rabbit antiHBc or rabbit anti-HBs sera in conjunction with either IgG or Fab’ preparations of goat anti-rabbit IgG antibody HRP conjugates essentially as described for immunofluorescence with the following modifications. Following incubation with the appropriate peroxidase-labeled antibody, sections were treated with DAB and with hydrogen peroxide for the histochemical demonstration of peroxidatic activity (Graham and Karnovsky, 1966; Nakane and Pierce, 1967; Palmer et al., 1974), dehydrated in a graded series of ethanol, passed through xylene, and embedded in Permount (Fisher Scientific Co., Fair Lawn, N. J.), Controls similar to those used for immunofluorescence were also examined. Electron Microscopy Selected liver areas, chosen by gross inspection, were cut into small (1 mm?) chunks and were immediately fixed in 2% buffered glutaraldehyde followed by postfixation in osmium tetroxide (Gyorkey et al., 1975). Following dehydration in ethanol containing uranyl acetate, the specimens were embedded in EponAraldite. Formalin-fixed, paraffinized liver material, which had been stored for 10 years at room temperature, was also prepared for electron microscopy by immersing the paraffin blocks initially in xylene, then in chloroform, followed by hydration
HBV
ANTIGENS
IN
LIVER
163
FIG. 3. Hepatitis B core particles and associated HBcAg in HBV-infected hepatocytes. (A) Electron micrograph of a nucleus containing monodispersed hepatitis type B cores. ~14,700. (B) A high magnification micrograph of the intranuclear hepatitis B cores. The particles measure approximately 26 nm in diameter. X89,000. (C) Negatively stained preparation of HBc particIes isolated from nuclei of infected cells. These purified particles were utilized to inoculate rabbits to produce anti-HBc as described in Materiak and Methods. X89,000. (D) Direct immunofluorescent staining for HBcAg on acetone-fixed frozen sections of infected liver. Note the presence of strong nuclear fluorescence. Occasionally whole nuclei were fluorescent (arrow). These sections were obtained from the same liver as those employed for demonstration of HBsAg. X480.
through a graded series of ethanol. Following washing in distilled water, the material was refixed in 2% buffered glutaraldehyde, postfixed in osmium tetroxide, and processed for microscopy as described above, Ultrathin sections were stained with uranyl acetate followed by lead citrate and were examined in a Philips model 301 electron microscope.
164
C,4131~;\1, E’L .\I,.
Frc. 4. Parafbembedded liver sample reprocessed for electron n~i~ro~.copy as tkscril~ed in Materials and Methods. Note the presence of numerous intranuclear care particles. Y 12,800.
RESULTS Light, immunofluorescent and electron microscopic rcbults were compared and correlated for the presence of surface antigen and core antigen. In fresh frozen cryostat sections (Fig. 2B), extensive positive fluorescence was disclosed in the cytoplasm of hepatocytes following treatment with fluoresceinated goat-anti-H& Electron microscopic examination of these cells revealed the presence of typical intracytoplasmic HBsAg particles (Fig. 2A, arrows). The same cryostat sections were studied for the presence of HBcAg and exhibited positive fluorescence in hepatocyte nuclei following incubation with FITC-labeled rabbit anti-HBc (Fig. 3D) or with labeled goat anti-rabbit IgC preceded by rabbit anti-HBc antiserum (not shown). Occasionally, whole nuclei were fluorescent ( arrow), an observation which correlated well with electron microscopic findings of nuclei containing evenly dispersed core particles (Fig. 3A). A comparative assessmentof the IIBc particles is provided on a low-powered electron micrograph (Fig. 3X), a highpowered eIectron micrograph (Fig. 3B), and on 3 micrograph of isolated core particles purified from the necropsy liver (Fig. 3C). In form&n-fixed, paraffinized liver reprocessed for electron microscopy, HBs particles were not readily detected. However, intranuclear core particles were readily seen (Fig. 4). Moderate intracytoplasmic fluorescence, noted in these sections following treatment with fluoresceinated goat anti-HBs (not shown), corresponded quite well with the pattern of “amber” coloration observed in hepatocytes following application of orcein staining (not shown). Identical sections processed for the presence of HBcA g, by either direct or indirect methods, exhibited positive, but weak, intranuclear fluorescence (not shown). Cryostat sections obtained from the same material as above and processed by direct and indirect immunoperoridase procedllres, .+lded y results similar to those
HBV
ANTIGENS
IN
LIVER
1tXI
FIG. 5. Immunoperoxidase staining of HBV-infected river. (A) Indirect staining for HBcAg using rabbit anti-HBc and HRP-conjugated goat anti-rabbit IgG on acetone-fixed frozen sections reveals the presence of strong intranuclear staining. X 195. (B ) Localization of HBcAg in formalin-fixed paraffin sections using the same indirect technique. X800. (C) Formalin-fixed paraffin sections stained for the demonstration of HBsAg using the direct method. X480. (D) Formalin-fixed paraffin section processed by the indirect method. The section was incubated with rabbit anti-HBs followed by goat anti-rabbit Fab’-peroxidase. Note the presence of strong cytoplasmic staining. The nucleus in the cell at the center of the micrograph is unstained, demonstrating the specificity of the anti-HBs antiserum. X480.
obtained by immunofluorescence. Following incubation with goat anti-HBs-HRP, HBsAg was localized in the cytoplasm in a pattern which varied from cell-to-cell, some being negative, other strongly positive (not shown). HBcAg was present within nuclei of most hepatocytes after treatment of sections with rabbit antiHRc followed hy IgC or Fnh’ preparations of goat anti-rabbit IgC antibody7 linked to HRP (Fig. 5A).
166
CABRAL TABLE Comparative Results Human Liver Procedure
Immunofluorescence Direct Indirect Immunoperoxidase Direct Indirect Fab’-conjugate IgG-conjugate
El’ AL. I
of Specific Histochemical Staining for HBc and HBs Antigens” Acetone-fixed cryostat sections
of
Formalin-fixed paraffin sections
HBcAg
HBsAg
HBcAg
4+ 4+
4+ N.D.b
f +
N.D.
N.D.
4+
X.D.
4+
4+ 4+
4+ 4+
3+ 4f
4+ 4+
5 The intensity of staining was recorded and graded from negative to 4+. recorded samples in which staining was intense at low power and at high power * N.D. = not done.
HBsAg
2+
Designations of 4f using oil immersion.
In formalin-fixed, paraffined material, brown cytoplasmic staining was readily observed in various hepatocytes treated by the direct immunoperoxidase method for the demonstration of HBsAg (Fig. 5C, D). Nuclei were intensely stained for HBcAg and in patterns similar to those described for cryostat sections examined by immunofluorescence. This intranuclear staining generally paralleled the observed distribution of HB core particles in paraffinized tissue reprocessed for electron microscopy (Fig. 5B). A comparative assessmentof the conjugates employed in this study is given in Table I. No liver staining was found with the techniques used in this study in hepatocytes of two patients shown to contain neither HBs nor HBc particles by electron microscopy and to be HBsAg seronegative by radioimmunoassay ( Ausria-IZ51). DISCUSSION Several studies have been undertaken dealing with the demonstration of HBsAg or HBcAg in HBV-infected liver. These have included ultrastructural, immunofluorescence, and immunohistochemical analyses. However, to our knowledge, no studies have been presented which correlate the efficacy of these procedures and of IgG- or Fab’-HRP conjugates in the identification of HBs and HBc antigens in acetone-fixed cryostat or formalin-preserved, paraffinized liver sections. In the present investigation, the direct immuuofluorescence technique proved to be a reliable, sensitive and rapid method for the detection of HBV-associated antigens in acetone-fixed cryostat sections obtained from liver biopsies. This technique also proved reliable and specific in localizing HBsAg in formalin-fixed, paraffin-embedded liver sections stored for as long as 10 years. However, the identification of HBcAg in this latter material proved difficult, even following application of indirect immunofluorescence, due to weak staining. It should be noted that the fluorescence conjugates employed were separated on the basis of
HBV
ANTIGENS
IN
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charge by elution through DEAE-cellulose and had low F/P ratios and, consequently, nonspecificity due to negative charge imparted by fluorescein was minimal. The direct and the indirect immunoperoxidase procedures proved to be as sensitive as fluorescence in the detection of HBsAg or HBcAg in frozen sections. These enzyme probes were particularly reliable for elucidating HBV-associated antigens in formalin-fixed, paraffinized liver. Sections of this tissue treated by indirect methods employing HRP conjugated to IgG or Fab’ for the demonstration of HBsAg were stained with equal intensity. This suggests that the tagged antibody fragments may prove ideal for ultrastructural examination of both paraffinized and fresh, appropriately fixed HBV-infected tissue. In addition, while orcein staining was applicable only to revelation of HBsAg sites, HRP conjugates proved especially useful for identifying HBcAg. This increased sensitivity of the enzyme conjugates could allow for the retrospective examination of stored paraffin-embedded liver samples, particularly those obtained from individuals with diseases having a probable association with HBV infection. In summary, electron microscopic examination allows for the specific identification of HBs and HBc particles within HBV-infected liver, but is tedious, timeconsuming and may not permit adequate diagnosis in those situations where relatively few hepatocytes are infected. Immunofluorescence methods are simple, reliable, specific techniques for detection of HBcAg and HBsAg in liver biopsies, but are less sensitive in elucidating these antigens in formalin-fixed, paraffinembedded material. Immunoperoxidase staining, employing various conjugates, proved equally sensitive in detecting antigens in acetone-fixed biopsy material and in formalin-preserved, paraffin-embedded tissue stored for as long as 10 years. ACKNOWLEDGMENTS We gratefully acknowledge research was supported by Institute, National Institutes Foundation.
the valuable technical assistance of Ms. Becky Clarke. This Research Grant HL-17269 from the National Heart and Lung of Health, and by Grant Q-435 from the Robert A. Welch
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