Note: Descriptions are shown in the official language in which they were submitted.
llO~Q37
Background of the Invention
ffepatitis B surface antigen (HBsAg) in blood serum
has been a consistent marker for the presence of an infectious
agent, as reported by Alter et al, Am. J. Med. Sci., 270:329-
334, 1975 or Goldfield et al, Am. J. Med. Sci., 270:335-342,
1975. The H~ Ag resembles a virus morpholo~ically, but there
has been no consistent evidence for infectivity or nucleic acid
associated directly with this particle (See Gerin et al, J.
Virol 7:569-576, 1971). The H~SAg is immunogenic since anti-
HBS (antibody) is normally found in patients and animals during
recovery and convalescence. The presence of anti-~Bs is
associated with protective immunity and this sugqests that
HBsAg is a structural component of the infectious virion.
Thus, the asociation of HBsAg as a marker for infectivity of
blood and blood products, and anti-ff~s as a marker for immunity
has stimulated wide spread diagnostic and epidemiologic studies
of hepatitis type B. Highly sensitive and specific immuno-
logic assay systems for antigen and antibody have been necessary
and useful in such studies. One such assay system is
described in U.S. Patent 3,867,517 issued February 18, 1975.
Awareness of multiple transmission routes and the use
of highly sensitive immunologic test procedures for HBsAg have
been reported to reduce significantly transfusion-associated
hepatitis type B (See Hollinger et al, N. Eng. J. Med., 290:1104-
1109, 1974). However, in careful follow-ups of recipients of
~lood negative for ffBsAg, Goldfield et al, Am. J. Med. Sci.,
270:335-342, 1975 has reported evidence of exposure to the
an~igen in 7 o~ 465 patients. This suggests that further
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110~037
studies of other immunologic systems associated with the
hepatitis B virus could lead to more complete diagnosis
of disease and detection of the infectious agent. The Dane
particle, Dane et al, Lancet, 1:695-698, 1970, with its
associated core, has led to identification o~ core antigen
(HBcAg) and its antibody (anti-core or anti-HBc) as a second
immunoloqic system. The finding that the surface of the Dane
particle contains HBsAg and the internal core particle contains
DNA and DNA polymerase have strengthened the proposal for this
particle as a candidate for the infectious virus. However,
until tissue culture or convenient laboratory animal models
are developed, immunochemical, biochemical or electron micro-
scopic techniques are necessary for assessing the presence and
possible infectivity of Dane particles.
lS It has been reported in the literature that the
presence of antibodies to the core of Dane particles (anti-HBc)
in human blood could be an indication of Hepatitis B virus
infection or of a carrier state of this virus. Methods for
detecting anti-HBc as reported in the literature include
complement fixation, immune adherence hemagglutination and
radioimmunoassay. Hoofnagle et al, N. Eng. J. Med. 290:1336-1340
(1974); Tsuda et al, J. Immunol. 115:834-838 (1976); Greenman
et al, Vox Sang. 29:77-80 (1975); Moritsugu et al, J. Immunol.
114:17921798 (1975) Purcell et al, Intervirol. 2:231-243
(1973/74); ~yas et al, Transfusion 16:536-~37 (1976).
--3--
110~037
It seems clear that analysis of anti-HBc can have
epidemiologic and diagnostic significance. Some possible inter-
pretations of the presence of anti-HBc and absence of HBsAg or
anti-HB~ are:
l. Early convalescence from disease with
undetectable levels of replicating virus
and before the appearance of detectable
anti-HB8
2. A carrier state of HB virus in which the
presence of HBsAg is below detecta~le levels
while anti-HBc is continuously stimulated.
3. Restimulation of the immune system in
previously immune individuals where anti-
surface has decreased to undetectable levels.
4. Persistence of anti-HBc, in some cases, for
periods where anti-HBs has decreased to
undetectable level.
Briefly, the present invention provides improved
reagents and methods for the presence of anti-HBc (antibody).
Detection of anti-HBc (antibody) using the present invention
provides a significant increase in detectability for poten-
tially infective blood in comparison to the detection of
surface antigen (HBsAg)~ using the methods of U.S. Patent
3,867,517. For example, samples have been examined wherein
no surface antigen (HBSAg) is detected using the method of
the 3,867,517 patent but anti-HBc was found present using
the method of this invention.
--4--
11~00:~7
SUMMARY OF THE INVENTION
In one particular aspect the present invention provides
a reagent suitable for use in immunoassay methods for the
detection of antibodies evidencing exposure to heptatitis B
virus, said reagent comprising a solid phase coated with
HBCAg-
In another particular aspect the present inventionprovides in an immunoassay for the detection in blood or
blood constituents of antibodies evidencing exposure to
hepatitis B virus, the improvement comprising the use of a
binding reagent comprising a solid phase coated with HBCAg.
Detailed Description of Reagents
Purification of Dane Particles
Plasmas positive for HBSAg and containing Dane particles
were pooled and used as the source of Dane particles. The
purification procedure for Dane particles consisted of
isopycnic banding in sucrose gradients and a single isopycnic
banding in cesium chloride. DNA polymerase (E.C.2.7.7.7), an
enzyme associated with HBCAg was used as an indicator of
Dane particles throughout the purification procedure. Two
populations of Dane particles with different buoyant densities
were usually present in HBSAg posi~ive plasmas; one banded in
sucrose at a density of about 1.28 gm/cm, and another at about
1.25 gm/cm;
jl/ ; -5-
ll~QQ3~
the latter band was the major band of the two, but relative
quantities of the two populations in terms of DNA polymerase
activities varied f~om batch to batch of plasma pools. The
light and heavy peaks were selected and rebanded in cesium
chloride. In electron micrographs, no morphological diff-
erence was observed between these two populations of Dane
particles with different densities.
Purification of Dane Particle Cores
Highly purified Dane particles from either heavy
or light fraction were treated with a non-ionic detergent
designated Nonidet P-40 and 2-mercaptoethanol to remove
surface components. The preparations were then mixed with
equal volu~es of genetron and shaken vigorously to liberate
the free cores. DNA polymerase remained in the aqueous phase.
This genetron treatment step is not essential and may be
omitted. However, its use contributes to a purer product.
Further purification of the aqueous DNA polymerase activity in
linear sucrose gradients gave a homogeneous band with a
sedimentation rate of approximately llOS. Electron microscopy
showed that 27 nm core particles co-sedimented with the poly-
merase. Only trace amounts of B5Ag were detected in the
sucrose gradient by radioimmunoassay.
ilO0~37
~mmunogenicity and Antigenicity of
Dane Particle Cores
Guinea pigs and rabbits were immunizied by injection
with the sucrose gradient purified core preparation mixed with
equal volume of Freund's adjuvant. The serums from these
~mmunized animals were reactive with purified Dane cores and
were routinely examined for anti-HBc titres by a direct radio-
immunoassay described hereinafter. The hyper-immune anti-HBc
serums were passed through immunoabsorbent columns of purified
H~ Ag and normal human serum proteins to remove possible
antibodies induced by these components. Animals immunized
with highly purified Dane particle cores produced a high titer
of anti-HBc, while anti-HBs titer was comparatively low or
undetectable by solid phase radioimmunoassay, as described
in U.S. Patent 3,867,S17 and as measured by the product sold
under the trademark AUSAB. When the anti-HBcanimal serum was
incubated with highly purified 22 nanometers (nm) HBsAg particles
the anti-H~c antibody formed a complex with the HBsAg particles
but when the HBsAg particles were first digested by pepsin,
the H~ Ag particles lost the binding ability to anti-HBc but
retained full H~ Ag activity, i.e., binding ability to anti~
It has therefore been concluded that on the 22 nm H~ Ag particle
there are antigenic sites similar to or the same as the Dane
particle core. This unexpected property of ~ Ag particles
can be used in various forms of immunoassays for the detection
of anti-H~ .
110003~
Coating of solid surface with the cores of Dane particles:
Dane particle preparations of various stages of purity
are treated with 2-mercaptoethanol (0.30-0.75/~) and a non-
ionic detergent, such as Triton~X-lO0 or Nonidet~P-40, in a
concentration of about l.0 to 2.5% at 37 C. for one hour.
The purpose of this treatment is to remove the lipoprotein
coat and to release the core antigen of the Dane particles.
The above conditions for the treatment are preferred, but
they may be varied without adverse effect. After the treatment,
the mixture is appropriately diluted with a buffer solution;
for example, O.OlM Tris-HCl, pH 7.1, in physiological saline
solution containing O.OOlM EDTA. The solution is used immediatel~
to coat the solid surface of objects such as beads, tubes
or wells made of plastic or glass. Dane cores prepared in
this manner are very n sticky", and easily attach to solid
surfaces by absorption. When preparations of Dane particles
are grossly impure and contain high levels of extraneous proteins
it is necessary to pre-coat the solid objects with anti-~Bc
before reacting with Dane cores, as above. However, with our
preparations, when polystyrene beads were incubated with the
Dane core solution for 24 to 72 hours, there was more Dane
core on plain beads than on anti-HBcpre-coated beads, especially
when very low concentrations of Dane core were used. It has
been discovered ~hat in the coating solution, the detergent
concentration should be very low (preferably lower than 0.005~),
if Dane cores are coated directly on solid surfaces. Another
advantage of coating solid objects with Dane core directly
on the surface rather than first pre-coatinq with anti-HBc
-- 8
~l~OQ37
is the adaptability of the reagent for use in a direct
sandwich type RIA for anti-HBc.
Preparation of Radioactive Anti-HBc Immuno-
globulins for Radioimmunoassay
Serum containing anti-HBc activity was fractionated on
DEAE-cellulose columns to obtain the immune globulin fraction.
The fraction was then labeled with I or other marker labels
by conventional techniques.
Radioimmunoassay (RIA) Methodology for Anti-HBc Detection:
Use of Category A Reagents:
Example 1
Solid-phase Competitive RIA - Single-
Antibody System
HBsAg coated beads are incubated with an aliquot of
a serum sample for a length of time to allow the anti-HBc in
the sample, if present, to react with the HBcAg-like sites on
the beads. An aliquot of I-anti-HBc is then added to the
reaction mixture to bind to the available ~BcAg-like sites.
After this reaction time, the bead is washed and the amount of
I-anti-HBc bound to the bead is determined. A normal serum
known to be negative for anti-HBc is run parallel with the
test sample. The presence of anti-HBc in the test sample is
signified by a decrease of I-anti-HBc uptake by the bead,
compared to the controls.
The above procedure may be modified for some specific
purposes. For example, in the above assay procedure, the
sample and I-anti-HBc may be pre-mixed before reacting with
the bead, or a washing step may be incorporated between the two
_g_
1100037
incubation periods of the bead with the sample and with the
125
I-anti-HBc.
Example 2
Solid-phase Competitive RIA Double-anti-
bodY sYstem
_
This procedure is similar to the single antibody
system described above, except that the anti-HBc (guinea piq
for example) used is not radio- or otherwise- labeled. After
washing to remove this excess reagent, another incubation step
with labeled antibody to the anti-HBc immune globulin
(guinea pig, for example) prepared in another animal
species (rabbit, for example) is added to make the anti-HBc
on the bead ~visible" by radioactivity, or other marker.
Use of Cate~ory B Reagents:
Example 3
Solid-phase Competitive RIA
An aliquot of labeled human immune globulin contain-
ing anti-HBc and the solution to be assayed are incubated with
a Dane core coated bead. After thorough washing the bead is
counted for the amount of label marker attached. An anti-8Bc
negative sample is assayed in the same manner as a control.
A sample is positive for anti-HBc when the count rate or other
measure of the sample is significantly lower than that of the
negative control sample. Alternatively, variants of the
above procedure can be used:
----10--
ll~QQ37
(l) The Dane core coated beads are first incubated
with the sample before the addition of labeled anti-
HBC; (2) same as (l), except the bead is washed before
the addition of labeled anti-HBc.
Example 4
Solid-phase Direct Assay: Ho~ologous
Sandwith Type
Dane core coated beads in conjunction with labeled
- HBcAg can be used in direct RIA of the sandwich type. An
aliquot of a sample is incubated with a Dane core coated bead,
tube or object. The object is then washed and further incubated
with a solution of labeled HBcAg. If anti-HBc is present in
the sample, a sandwich structure of HBcAg-anti-HBc-labeled
HBcAg is formed. After washing, the bead will show signifi-
cantly higher count rate or other measurement than control
beads which were assayed with anti-8Bc negative samples.
ExamPle S
Solid-phase Direct Assay - Heterologous
Sandwich Tvpe
Dane core coated beads are first incubated in an
aliquo~ of properly diluted human serum. If the sample
contains anti-~Bc, the anti-~Bc will specifically attach to the
Dane core on the beads. After washing the beads with water or
a buffer solution, a labeled anti-human immunoglobulin solution
is added to the beads to react with the anti-~Bc previously
attached. This method can distinguish anti-HBc being IgG or
IgM, if labeled specific anti-human IgG or IgM is used.
37
Findings Using the Assay Procedures
~requency Distribution of Anti-Core
A collection of serums from consecutive plasma-
pheresis donors (paid donors) were analyzed for anti-HBc using
the methods of the present invention, and for HBSAg (using
I-anti-HBs), and for anti-HBs (using 125I-HBSA~) using the
method of U.S. Patent 3,867,517. Correlation of the findings
with regard to the Hepatitis a surface and core systems using
category A reagents is summarized in Table I. About 81% of the
HBsAg positive specimens also contained anti-HBc. About 15% of
the specimens containing anti-HBs also contained anti-HBc.
This suggests that the persistence of anti-HBc in these donors
may be of shorter duration than persistence of anti-HBs. A
third category of 3105 donors showed no evidence of prior
exposure to the hepatitis B virus, based on negative results
with tests for surface antigen and antibody. However, almost
5% of this group were positive for anti-core. Similar results
were observed with a group of 1049 volunteer blood donors. The
HBsAg positive serums were not available for testing. About
21% of the anti-H~s specimens were also positive for anti-core.
About 2% of the surface antigen and antibody negative donors
were positive for anti-core. In a collection of ~B~Ag positive
serums from carriers, 106 of 113 (94%) were positive for anti-
HBc~
2S In the above studies anti-HBc was found in 29~ (7.3%)
of the 3929 plasmapheresis donors, and 34 (3.2%) of 1049 volun- ~`
teer blood donors. There were 145 and 19 donors, respectively,
where an~i-HBc was the only detectable marker for hepatitis 8
-12-
Q37
virus exposure.
In another population study, anti-HBc was
determined by using the category B reagents, comparable
results were obtained (Table II), except a higher per-
centage of anti-HBs positive samples were also found to
be anti-HBc positive.
These results agree qualitatively with two
other studies of the occurrence of anti-HBc in healthy
populations. Hoofnagle et al, Lancet 2:869-873, 1973 and
N. Eng. J. Med. 290:1336-1340, 1974 analyzed serums from
commercial donors, volunteer donors and H~ Ag carriers
by complement fixation with HBCAg from infected chimpanzee
liver hepatocytes. Anti-HBc was found in 8 of 100 com-
mercial donor serums and 2 of 200 volunteer donor serums.
There were 5 and 1 donors, respectively, where anti-HBc was
the only evidence of exposure to the hepatitis B virus.
Tsuda et al, J. Immunol. 115:834-838 1975 used immune ad-
herence hemagglutination with purified plasma Dane particle
cores for the analyses of the serum from 215 healthy blood
donors. There were 36 positive for anti-H~c These were
composed of 2 of 2 HB Ag positives, 28 of 31 anti-HBs
positives and 6 from the 192 surface-negative group.
~100~37
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