An Immunoperoxidase Technic for the
Demonstration of the Hepatitis
B Surface Antigen
in Human Livers
ANGELOS P. AFROUDAKIS, M.D.,
CHOONG-TSEK LIEW, B.S., AND ROBERT L. PETERS,
M.D.
Department of Pathology, John Wesley Hospital-University of Southern
California School of Medicine, Los Angeles, California
Afroudakis, Angelos P., Liew, Choong-Tsek, and Peters, Robert L.: An immunoperoxidase technic for the demonstration of the hepatitis B surface antigen in human livers. Am J Clin Pathol 65: 533-539, 1976. A sensitive method
for demonstrating the site of hepatitis B surface antigen (HBsAg) in fixed
tissues embedded in either paraffin or araldite is described. The method
employs the peroxidase-rabbitantiperoxidase linkage through goatantirabbit
to rabbit anti-HBsAg. In staining hepatitis antigen in agar, comparison of fixation (using three common fixatives) with unfixed precipitation arcs revealed
no recognizable differences in antigenicity induced by fixation. The method
allows confirmation of positive reaction by appropriate blocking controls. The
technic is compared with the orcein stain of Shikata and found to be somewhat
more sensitive but slightly more time-consuming. (Key words: Hepatitis;
Hepatitis B antigen; Peroxidase; Horseradish peroxidase; Immunoperoxidase.)
of the cellular localization of the
two major antigenic components of hepatitis B virus (HBV) in patients with acute
and chronic hepatitis B viral disease and in
ostensibly healthy hepatitis antigen carriers
have provided opportunities for re-evaluation of the pathogenetic concepts of these
hepatic conditions. Many studies by the immunofluorescent technic and immunoelectron microscopy have established that the
STUDIES
Received J u n e 2, 1975; accepted for publication
June 18, 1975.
Supported by grant of John Wesley Hospital Attending Staff Association (6001).
Address reprint requests to Dr. Peters: Department
ofPathology.John Wesley Hospital, 2825 South Hope
Street, Los Angeles, California 90007.
antigenic material that makes up the
20-nm. particles found in sera of infected
patients may be found in hepatocyte cytoplasm of certain infected individuals.4-10-21
The cytoplasmic antigen that is called
"hepatitis B surface antigen" (HBsAg) contains negligible, if any, nucleoprotein, and
is generally believed to represent excess
coat material for the virus. The complete
virus, usually considered to be the Dane
particle, has as its outer coat HBsAg,
whereas its nucleoprotein-containing core
is antigenically distinct and is known as
hepatitis B core antigen (HBcAg). HBcAg
is found principally in selected hepatocyte
nuclei of certain patients who have chronic
varieties of hepatitis B infection. 6,7
533
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ABSTRACT
534
AFROUDAKIS, LIEW, AND PETERS
A.J.C.P. —Vol. 65
1 for studies by light and electron microscopy (EM) was pointed out almost ten years
ago. 15 Horseradish peroxidase (HRP) was
found to be the most suitable enzyme for
H Rabbit Immunoglobulin G
Anti-Rabbit Immunoglobulin G produced in goats the intracellular localization of antigens
at an ultrastructural level, having small
HRPI stained by 3,3' Diaminobenzidine
molecular weight and thus increased peneFie. 1. Schematic pattern of the mechanism of PAP
trating
properties. 14
stain for HBsAg.
Applications of the HRP-antiHRP (PAP)
system in demonstration of tissue antigenic
22
Despite the wide acceptance of fluores- factors such as spirochetal antigens,
12
16
fetoprotein
and
cent technics for identification of various pituitary hormones,
tissue antigens as well as for demonstrating a-1-antitrypsin 17 in liver cells have been
circulating antibodies in patients with auto- described. Similarly, indirect identification
immune diseases, routine use of immuno- of circulating antibodies such as antinufluorescence has major disadvantages. 13 clear factors, smooth muscle antibodies,
One is the transitory character of fluores- and mitochondrial antibodies using PAP
cence, which does not permit a review of labeling has been employed. 2,13 T h e sensithe sections at a later time, nor does it allow tivity and advantages of this histochemical
review of tissue already fixed or embedded. system have-been emphasized by many inA capacity to compare tissue from ongoing vestigators.
work with that stored from previous exBasically, the procedure, as modified,
periments is necessary for obtaining in- provides specificity limited only by the
creased knowledge of pathogenesis of dis- singularity of rabbit antibody used, alloweases related to the hepatitis B virus.
ing demonstration of any antigen to which
Shikata and associates20 recently re- a rabbit antibody is available without the
ported staining methods for HBsAg in tedium of chemically linking peroxidase to
paraffin sections using orcein and aldehyde each. T h e sandwich reaction is demonfuchsin stains, both of which apparently strated schematically in Figure 1.
depict the disulfide bonding of HBsAg.
T h e purpose of the present communicaShikata's technic has been quite an advance tion is to introduce use of the PAP technic
in obtaining permanent preparations by in the demonstration of the HBsAg in
simple procedures demonstrating mate- hepatocytes of fixed human livers, in comrial presumed to be HBAg. Since sub- parison with the orcein stain introduced
stances other than HBsAg may give positive by Shikata and associates. 20
reactions to orcein, and since confirmation
According to the requirements of this
of orcein-positive reaction by serially sec- technic the following reagents were used.
tioned immunofluorescent studies is difficult, a more precise cell-by-cell comparison
Reagents
of a specific reaction with the Shikata
orcein technic is desirable.
Antiserum to HBsAg Made in Rabbits
T o overcome the disadvantages in other
Unlabeled rabbit anti-HBs (R-andHBs)
immunohistologic studies, an immunoobtained commercially from Ortho Diagperoxidase technic has been adopted by
nostics (Hapindex*) was used in a dilumany investigators as an alternative
tion of 1:4 in pH 7.2 phosphate-buffered
method to immunofluorescence in histosaline solution (PBS).
chemistry. T h e usefulness of histochemically demonstrable enzymes in labeling tissue antigens and circulating antibodies
* Ortho Diagnostics, Raritan, New Jersey.
i
V^IBMM^V'V'-JMPWB^
HBsAg^^ntm^GAR.^^RAP^^HRPJ:
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April 1976
IMMUNOPEROXIDASE STAIN OF HBsAg
535
in formalin, and one in Bovin's fixative.
From the unfixed slides, one was stained by
Unlabeled antirabbit IgG produced in orcein, and one by the immunoperoxidase
goats (goat anti-rabbit, GAR) was obtained technic. T h e third unfixed and the three
from Behring Diagnostics! and was used in fixed slides were incubated for 25 minutes
a dilution of 1:4 in PBS.
in 0.1% pronase to destroy the bound antibody. All pronase-treated slides were then
Rabbit Antiperoxidase (RAP)
stained by the immunoperoxidase method
This antiserum was prepared by a series and strengths of reaction compared.
of injections of HRP into albino rabbits
according to a method introduced by
Liver Sections
Mason and colleagues. 12 For the first sensiSpecimens of human liver tissue obtization 4 mg. of type IV HRP (Sigma
Chemical Company:):) were dissolved in 1 tained by needle biopsy or at autopsy
ml. sterile saline solution and 1 ml. com- were fixed overnight in acetate-buffered
plete Freund's adjuvant and injected sub- 10% formalin (pH 7.0). After dehydration
cutaneously. Four weeks and again six through graded alcohol they were embedweeks after the first injection, 2 mg. type ded in paraffin and sectioned into 5-/um.
II HRP (Sigma Chemical Company) were thick sections; an immunoperoxidase indissolved in physiologic saline solution and direct technic was performed.
Other specimens from the same livers
injected intravenously. Seven days after
the last injection, blood was drawn from were fixed in a "B-5" fixative containing
6 Gm. mercuric chloride, 2.074 Cm. hythe rabbits.
After Ouchterlony double diffusion of drated sodium acetate dissolved in 90 ml.
HRP against RAP in 1% purified agar at distilled water, and 10 ml. 40 per cent
pH 7.6, rabbit antiperoxidase was used in formaldehyde (pH 5.8-5.9). After dehydration through graded alcohol, the
a dilution of 1:8 in PBS.
specimens were embedded in araldite embedding mixture" and sectioned serially at
Pronase
5 fim. T h e Epoxy resin was removed subA nonspecific protease isolated from sequently by treatment with equal parts of
Streptomyces griseus and available commerci- saturated sodium E-thoxide (Mallinckrodt
ally from Sigma Chemical Co. was used in Chemical Works) and Benzene (Allied
a concentration of 0.1% for treatment of Chemical). Before staining, the sections
the sections prior to testing.
were incubated with 0.1% pronase at 37 C.
for 2 5 - 4 0 minutes and rinsed thoroughly
Methods
with distilled water.
Goal Antirabbit immunoglobulin G
Immune Complexes
T o obtain HBsAg-HBsAb i m m u n e
complexes, six counterelectrophoresis
slides utilizing agarose were made, using
two different HBsAg titers electrophoresed against antisera from human sources.
All slides were dried and three of them
were fixed for one hour, one in "B-5", one
t Behring Diagnostics, Summerville, New Jersey.
t Sigma Chemical Company, St. Louis, Missouri.
Ivimunohistochemistry
We have performed a four-layer indirect
immunoenzyme technic in which the enzyme (HRP) is bound to the antigenic
side (HBsAg) through a bridge of antigenantibody reaction.
Paraffin- and araldite-embedded sections were flooded for 30 minutes with
the R-anti HBs (dilution 1:4) at room
temperature in a moist chamber. Then they
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HBsAg-HBsAb
536
AFROUDAKIS, LIEW, AND PETERS
AJ.C.P. —Vol. 65
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Fie. 2 (upper). Low-power photomicrograph of liver of patient with HBsAg-positive chronic active
hepatitis, now quiescent. T h e dark material is deposited at the site of HBsAg. (Five-jum. paraffin section,
PAP technic. x 100.
FIG. 3 (lower). Peroxidase reaction on pronase-treated 2-/u.m. sections of araldite-embedded liver of HBsAgpositive patient. Note the discrete granular pattern in some cells (G) and the faint membranous distribution
in others. x500.
April 1976
537
IMMUNOPEROXIDASE STAIN OF HBsAg
were washed for 10 minutes in PBS,
changed two times, and treated in succession with GAR (dilution 1:4) for 30 minutes, RAP (dilution 1:8) for 30 minutes,
and HRP, type II, in a solution of 5 mg.
per 100 ml. PBS for another 30 minutes.
Interspersed between incubations, the sections were washed for 10 minutes in PBS,
changed twice.
The visualization of HRP was obtained
finally by incubation of the treated sections for 10-20 minutes in 3,3'diaminobenzidine tetrahydrochloride and hydrogen peroxide, according to the method of
Graham and Karnovsky. 5
After dehydration, the sections were
examined with the light microscope. Other
treated sections from the same specimens
were counterstained with hematoxylin and
eosin.
In addition, paraffin- and araldite-embedded sections were stained with orcein
stain. 20 T o establish the specificity of the
immunoperoxidase procedure, control sections were incubated initially with R anti-
HBs that had been adsorbed previously
by purified HBsAg. T h e remaining incubation steps were the same for control and
test sections.
Results
HBsAg- HBsAb complexes on unfixed
agar slides were stained positively by orcein
but not by peroxidase. Positive stain was
obtained with the immunoperoxidase technic only on fixed and unfixed agar slides
where the bound HBsAb had been removed previously from the precipitating
arcs by exposure to the proteolytic activity
of pronase. None of the three fixatives
used affected the intensity of the immunoperoxidase reaction.
As for the liver sections, a brown cytoplasmic stain of variable intensity in peroxidase-positive hepatocytes was achieved.
The intensity of the brown color was evidently proportional to the amount of the
contained antigenic material. Liver cells
containing large amounts of HBsAg and
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FIG. 4. Five-/im. paraffin-embedded sections of liver of patient with asymptomatic persistent viral
hepatitis, showing the "ground glass" hepatocytes (a) and the corresponding HBsAg distribution (6).
X500.
538
AFROUDAKIS, LIEW, AND PETERS
NH2
NH
HaN
X
-2H
NH 2
Colourless
H2N
NH
Blue
L
NH
Brown
FIG. 5. Diagram depicting the conversion of benzidine
by two-step oxidation to a brown precipitate.
Discussion
The performance of the peroxidaseantiperoxidase technic in the demonstration of the HBsAgin liver cells with successful results confirms once again the usefulness of this sytem in the demonstration
of tissue antigens. T h e reproducibility and
advantages of the technic have already
been mentioned by many investigators 12,13
and are not repeated. However, its sensitivity is emphasized, despite the utilization
of whole rabbit antiserum instead of isolated anti-HRP gamma-globulin. 12 After
serial estimations, Petrali and co-workers 19
concluded recently that use of purified
antibody to HRP yields four times higher
sensitivity than antiserum to HRP. We believe that, in spite of this limitation, the
multilayered reactivity of the immunoglobulins contributes to increased sensitivity of the technic.
The fact that the immunoperoxidasepositive cells corresponded in shape,
number, and distribution to the typical
"ground-glass" hepatocytes as described
by Hadziyannis and associates 8 provides
another confirmation, in an indirect manner, of the specificity and sensitivity of the
proposed technic in comparison with immunofluorescence.
In an attempt to abolish any background
stain, we treated the sections prior to testing
with the proteolytic enzyme, pronase. Kim
and Bissel had found five years ago 9 that
incubation of the HBsAg with certain degradative enzymes, which include pronase,
did not affect its antigenicity. They assumed that the antigenic determinant of
HBsAg is protein that is protected against
the digestive activity of proteolytic enzymes
by a lipid covering component. On the
other hand, pronase can digest the protein
component of the haem protein, including
endoplasmic hemoprotein enzyme systems,
cytochrome oxidase, peroxidase, and catalase, and eliminate their peroxidase activity
on Benzidine, which normally gives a blue
or brown product (Fig. 5). 18 Although pronase was successful with araldite sections
in reducing background cytoplasmic
material and is capable of splitting HBsAgantiHBs complexes, which theoretically
may be present, pronase digestion of paraffin-embedded sections resulted in floating
of tissue on the slides. However, paraffin-
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thus increased amounts of binding peroxidase were stained dark brown, and others
with smaller amounts were stained lightly
(Fig. 2). A granular pattern of the histochemically demonstrable antigen was easily
recognizable in high magnifications (Fig.
3). T h e brown stain was in many sections
much more accentuated around the always
definitely negative nuclei, and to a lesser
extent at the margins of the cellular membranes.
The stained cytoplasm corresponded in
shape and distribution to the eosinophilic,
finely granular, "ground-glass"-appearing
cytoplasm of the so-called "ground-glass"
cells in routine histologic sections (Fig. 4,
a and b).8 T h e cellular distribution of the
brown stain was either diffuse, covering
whole cells, or partial, confined to a part
of the cytoplasm, or even marginal along
the sinusoidal border of the cells, according to the location of the antigenic material
(Fig. 3). Brown granular material was identified also in a few mesenchymal cells of
some sections. A nonspecific background
stain of other tissue elements was, in most
sections, unremarkable, especially in the
araldite-embedded, pronase-treated tissues. All control sections were negative
for peroxidase-stained material.
A.J.C.P. —Vol. 65
April 1976
IMMUNOPEROXIDASE STAIN OF HBsAg
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embedded sections contained less material
giving a background stain than did aralditeembedded sections.
The pattern of antigen distribution in the
liver cells was in agreement with that seen
with the orcein stain, and is consistent with
the pattern found by other investigators
using the orcein stain, 1,3,2 ° though in a few
sections, the smaller amounts of antigen
located at the peripheral portions of the
cells were identified more easily by the immunoperoxidase than by the orcein
method. In addition, HBsAg-antiHBs
complexes, or structures other than HBsAg
that have disulfide bonds, are also stained
by orcein and give a stain not specific
for HBsAg and not completely eliminated
by pronase treatment. The background
staining, though unremarkable, is undesirable, and sometimes may give rise to difficulties in interpretation of equivocal results.
In conclusion, the achievement of permanent preparations, histochemically
stained specifically for HBsAg, unaffected
by three common fixatives, and in either
paraffin- or araldite-embedded sections,
as well as the potential for counterstaining
with hematoxylin and eosin, provides in
our hands the opportunities for an accurate
and more reliable retrospective technic for
examination of the hepatitis problem.
539