Note: Descriptions are shown in the official language in which they were submitted.
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BUFFER COMPOSITION FOR REAGENTS FOR IMMUNOASSAY
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to buffering compositions, and more particularly, to
buffering compositions useful for immunological and immunochemical assay
components, such as antibodies and antigens.
2. Discussion of the art
Many types of immunoassays can be run on an apparatus of the type
described in U. S. Patent No. 5,358,691. In general, immunoassays can be
classified into two major categories--homogeneous and heterogeneous.
Homogeneous and heterogeneous immunoassays depend upon the ability of a first
binding member of a binding member pair, e.g., an antigen, to specifically
bind to
a second binding member of a binding member pair, e.g., an antibody. A
conjugate, comprising one of such binding members labeled with a detectable
moiety, is employed to determine the extent of such binding. For example, such
binding member pairs can be an antigen and an antibody to such antigen. The
conjugate, which can comprise the antigen, either participates in a binding
reaction
with the antibody or does not participate in such a reaction. The amount of
detectable moiety detected and measured after the reaction can be correlated
to the
amount of antibody present in the test sample.
Heterogeneous assays can be performed in a competitive immunoassay
format or in a sandwich immunoassay format. In the competitive immunoassay
format, an antigen can be immobilized to a solid phase material whereby the
amount of detectable moiety that is bound to a solid phase material can be
detected, measured, and correlated to the amount of antibody present in the
test
sample. Examples of solid phase materials include beads, particles,
microparticles, and the like. In the sandwich immunoassay format, a test
sample
containing, for example, an antibody is contacted with a protein such as an
antigen. The antigen is immobilized on a solid phase material. Examples of
solid
phase materials include beads, particles, microparticles, and the like. The
solid
phase material is typically treated with a second antigen or antibody that has
been
labeled with a detectable moiety. The second antigen or antibody then becomes
bound to the corresponding antigen or antibody on the solid phase material
and,
after one or more washing steps to remove any unbound material, an indicator
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material, such as a chromogenic substance, is introduced to react with the
detectable moiety to produce a detectable signal, e.g., a color change. The
color
change is then detected, measured, and correlated to the amount of antibody
present in the test sample. It should also be noted that various diluents and
buffers
are also required to optimize the operation of the microparticles, antigens,
conjugates, and other components of the assay that participate in chemical
reactions.
A heterogeneous immunoassay that can be performed with the apparatus of
U. S. Patent No. 5,358,691 in either a competitive or sandwich immunoassay
format is a microparticle capture enzyme immunoassay, such as that described
in
Clinical Chemistry, Volume 34, No. 9, pages 1726-1732 (1988), employing
microparticles as the solid phase material.
A step-by-step description of a microparticle capture enzyme immunoassay
procedure is set forth at col. 35, line 60 through col. 44, line 22 of U. S.
Patent No.
5,358,691.
Various components of kits used in immunoassays run on the apparatus
described in U. S. Patent No. 5,358,691 must fulfill certain functional
requirements. The microparticles must provide a solid phase for antibody
capture,
serving as a support for the antigens used for said capture. These particles
must
themselves be captured, e.g., by a matrix cell filter, to permit detection of
bound
antibody by the conjugate/substrate combination. The microparticle diluent
must
provide a medium that maintains the antigens' ability to be recognized by
complementary antibodies in patient specimens (stability) while not inhibiting
that
recognition (signal generation). The conjugate must provide a means to
specifically recognize antibodies bound to antigens on the microparticle and a
means to generate a signal to indicate the presence of conjugate on the
microparticle. The conjugate diluent must provide a medium that preserves and
optimizes the ability of one binding pair member on the conjugate to recognize
its
complementary binding pair member, while simultaneously preserving and
optimizing the ability of the enzyme portion, e.g., alkaline phosphatase
portion, to
hydrolyze the substrate into a detectable entity. In addition, the conjugate
diluent
must contain components that will minimize, if not eliminate, nonspecific
binding
of the conjugate to the matrix or microparticle, thus preventing the
generation of a
false signal. The sample dilution buffer must provide a medium that optimizes
the
ability of antibodies in a specimen to bind to antigen on the microparticles
while
preventing nonspecific interactions that might lead to the generation of a
false
signal.
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Because certain antigens used in immunoassays are unstable after
prolonged exposure to heat, it would be desirable to increase the stability of
antigens to heat stress. Because antigens are characterized by certain
epitopes, it
would be desirable to increase the stability of the antigens by increasing the
stability of the characteristic epitopes.
SUMMARY OF THE INVENTION
In accordance with the invention, there is provided an aqueous composition
comprising:
at least one biological buffer in a concentration ranging from about 10 mM
to about 500 mM;
dithiothreitol in a concentration ranging from about 2 mM to about 10 mM;
and
ethylene glycol present in a concentration ranging from about 4% to about
8%, wherein % means g/100 ml,
wherein said composition has a pH of below about 7.2.
The present invention provides an aqueous composition suitable for use as
a buffer, which composition comprises at least one biological buffer,
dithiothreitol
(alternatively referred to herein as "DTT"), and ethylene glycol. The medium
of
the composition is water. The composition can also include at least one
biological
detergent, at least one source of positive and negative counterions, e.g.,
salt, and at
least one viscosity modifier, e.g., sugar. The buffer also can include at
least one
preservative, e.g., sodium azide. The pH of the composition of the present
invention can range from about 6.4 to about 7.2, preferably from about 6.4 to
about 6.8, more preferably from about 6.5 to about 6.7. Most preferably, the
pH
of the composition is about 6.6.
The composition is particularly useful for stabilizing the immunoreactivity
of antigens, e.g., hepatitis C virus (HCV) antigens of the NS3 region of the
viral
genome, under heat stress conditions, in particular, temperatures ranging from
31
to 37 C. The composition of this invention has been found to be useful as a
diluent for microparticles for diagnostic assays in which antigens are
passively
coated onto polymeric microparticles. It has been found that the use of
ethylene
glycol significantly enhances the long-term ability of antigens to detect
complementary antibodies that bind to the antigens disposed on the surface of
a
microparticle.
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The composition of this invention provides immunoreactive stability to
antigens and antibodies at elevated temperatures, e.g., 37 C, for relatively
long
periods of time, e.g., 20 days.
In another aspect of the invention, a kit containing the composition as a
component is also provided.
BRIEF DESCRIPTION OF THE DRAWING
FIG. 1 is a graph illustrating the advantageous effect of ethylene glycol
upon the stability of 33c antigen, as evidenced by the ability to detect
binding of
anti-HCV antibodies to microparticles having 33c antigen coated thereon. The
33c antigen is a segment of the recombinant HCV protein HC43.
DETAILED DESCRIPTION OF THE INVENTION
This invention involves an aqueous composition that is particularly useful
for improving stability of antigens and antibodies under heat stress
conditions.
The composition comprises the following ingredients: at least one a biological
buffer, dithiothreitol [HSCH2(CHOH)2CH2SH], and ethylene glycol
[HOCH2CH2OH]. The medium of the composition is water.
In the preferred embodiments of the invention, the composition further
includes at least one biological detergent, at least one source of positive
and
negative counterions, e.g., salt, at least one viscosity modifier, e.g.,
sugar. A
preservative, e.g., sodium azide, can also be added to the composition to
reduce microbial growth. The composition has a pH ranging from about 6.4 to
about 7.2, more preferably from about 6.5 to about 6.7. If necessary, the pH
of
the composition can be adjusted with acid or base.
The function of the biological buffer is to maintain the pH of the medium
at a constant level. It has been found that optimum results are obtained when
3 0 the pKa of the buffer is within 1.0 pH unit of pH 6.6. In other words, the
pKa of
the buffer preferably ranges from 5.6 to 7.6.
Biological buffers that are suitable for the composition of the invention
include, but are not limited to, the following acids or bases:
3 5 benzene- 1,2,4,5-tetracarboxylic (pyromellitic)
benzene-1,2,3-tricarboxylic (hemimellitic)
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dimethyimalonic
histidine
hydroxylamine
carbonic (H2C03+002)
malonic
2-(N -morpholino)-ethane sulfonic acid "MES"
glycerophosphoric
propane- 1, 2,3-tricarboxylic (tricarballylic)
benzenepentacarboxylic
maleic
2,2-dimethylsuccinic
ethylenediaminetetraacetic acid "EDTA"
3,3-dimethylglutaric
bis(2-hydoxyethyi)imino-tris(hydroxymethyl)methane "BIS-TRIS"
benzenehexacarboxylic (mellitic)
N -(2-acetamido)imino-diacetic acid "ADA"
butane-1,2,3,4-tetracarboxylic
pyrophosphoric
1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid)
1,4-piperazinebis-(ethanesulfonic acid) "PIPES"
N -(2-acetamido)-2-aminoethanesulfonic acid "ACES"
1,1 -cyclohexanediacetic
3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid "EMTA"
("ENDCA")
imidazole
2-(aminoethyl)trimethylammonium chloride "CHOLAMINE"
N,N -bis(2-hydroxyethyl)-2-aminoethanesulfonic acid "BES"
2-methylpropane-1,2,3-triscarboxylic ( )~-methyltricarballylic)
2-(N -morpholino)propane-sulfonic acid "MOPS"
3 0 phosphoric
N -tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid "TES"
N -2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid "HEPES"
In the foregoing list, the name of the compound in parentheses is the common
name of the compound that precedes the name of the compound in
parentheses. The acronym set off by quotation marks is the name of the
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compound commonly used when ordering the compound that precedes the
acronym set off by quotation marks.
The buffer should be present in an amount sufficient to carry out its
intended function, i. e., maintenance of the desired pH. Effective
concentrations
of the biological buffer can range from about 10 millimoles per liter to about
500
millimoles per liter (mM), more preferably from about 10 mM to about 100 mM.
The preferred buffer is the buffer that goes by the acronym "MES".
The function of the dithiothreitol is to protect sulfhydryl groups in a
protein. By protecting sulfhydryl groupsin a protein, dithiothreitol allows
the
antigens, e.g., antigens of HCV, to retain their characteristic structure,
thereby
allowing recognition of antigens by their complementary antibodies.
Dithiothreitol is described in detail in the brochure entitled "Cleland's
Reagent",
published by Calbiochem-Novabiochem Corporation, San Diego, California,
incorporated herein by reference. As used herein, the term "dithiothreitol"
includes both dithiothreitol and its isomer dithioerythritol. The
dithiothreitol
should be present in an amount sufficient to carry out its intended function,
i. e.,
to protect sulfhydryl groups in a protein. As used herein, "protect sulfhydryl
groups" means to repress oxidation of sulfhydryl groups in a protein to
disulfide
groups while not affecting disulfide groups already present in the protein
groups. Effective concentrations of the dithiothreitol can range from about 2
millimoles per liter to about 10 millimoles per liter (mM), preferably from
about
5 mM to about 10 mM, and more preferably about 10 mM.
The function of ethylene glycol is to prevent dithiothreitol from oxidation
by oxidizing agents. For example, in the absence of ethylene glycol,
2 5 dithiothreitol will be oxidized, thereby rendering dithiothreitol less
effective for
protecting sulfhydryl groups. Ethylene glycol is a 1,2-diol. Ethylene glycol
was
found to be superior to similar polyhydic alcohols or polymers, such as
glycerol,
polyethylene glycol, and polyvinyl acetate, with respect to the ability to
preserve
the binding capacity of antibodies, e.g., anti-HCV antibodies, to antigens.
e.g.,
3 0 HCV antigens. Ethylene glycol should be present in an amount sufficient to
protect dithiothreitol from oxidation. Effective concentrations of the
ethylene
glycol can range from about 4% to about 8% , preferably from about 4% to
about 5%, and more preferably about 4%, based on weight per unit volume
(g/100 mi).
3 5 It is highly desirable to include at least one biological detergent, at
least
one source of positive and negative counterions, e.g., a salt, and at least
one
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viscosity modifier, e.g., a sugar in the composition of this invention in
order to
carry out effective immunoassays.
One function of the biological detergent (surfactant) is to reduce non-
specific binding of antibodies other than the analyte antibodies, e.g., anti-
HCV
antibodies, to the microparticies. In other words, antibodies other than the
analyte antibodies may adhere to the solid phase in the immunoassay for
reasons other than the specific recognition of their complementary antigen.
This non-specific binding is undesirable as it leads to false positive
results.
Biological detergents reduce the incidence of such binding caused by nonpolar
or hydrophobic interactions.
Biological detergents (surfactants) that are useful in the composition
include non-ionic surfactants, anionic surfactants, zwitterionic surfactants,
and
cationic surfactants. Non-ionic detergents include polyoxyethylene sorbitan
monolaurate ("TWEEN 20"), polyoxyethylene sorbitan monooleate ("TWEEN
80"), polyoxyethylene ethers ("TRITON ", "BRIJ "), and octylphenol-ethylene
oxide ("NONIDET(D"). Anionic surfactants include caprylic acid, cholic acid,
deoxycholic acid, glycocholic acid and sodium dodecyl sulfate. Zwitterionic
surfactants include "CHAPS " (3-[3-Cholamidopropyl)-dimethylammonio]-1-
propanesulfonate). Cationic detergents include cetylpyridinium chloride. It is
preferred that non-ionic detergents be used for the reason that they are known
to reduce non-specific binding while simultaneously not inhibiting specific
binding. Effective concentrations of the biological detergent can vary from
detergent to detergent, but can typically range from about 0.01 % to about 1%,
preferably about 0.01 %, based on weight per unit volume (g/100 ml). The
preferred detergent is Triton X-1 00.
The function of the source of positive and negative counterions (cations
and anions) is to reduce non-specific binding of antibodies other than the
analyte antibodies, e.g., anti-HCV antibodies, to the microparticies. In other
words, antibodies other than the analyte antibodies may adhere to the solid
I (1 nhaca in thP immi innaccav fnr reacr}nc nthgr than tl}e sncrrifirr
rernnnitinn of
.,~............ .,y~ ...
their complementary antigen. This non-specific binding is undesirable as it
leads to false positive results. Positive and negative counterions reduce the
incidence of such binding caused by ionic interactions.
Suitable sources of positive and negative counterions include salts.
3 5 Salts that are useful in the composition include such salts as NaCi and
KCI.
= Effective concentrations of the salt can range from about 0.05 moles per
liter to
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about 0.5 moles per liter (M), preferably from about 0.1 M to about 0.3 M, and
most preferably from about 0.15 M to about 0.25 M.
The function of the viscosity modifier is to increase the viscosity of a
solution containing microparticles so that the microparticles more easily
remain
suspended in the solution and can be inhibited from settling. The use of a
viscosity modifier in a diluent for microparticles has been found to achieve
neutral density of the microparticles. Achievement of neutral density entails
the
determination of the optimum concentration of viscosity modifier that will
eliminate settling of the microparticles. The concentration of viscosity
modifier
required to achieve neutral density is assay specific and microparticle lot
specific. The principle involves dissolving viscosity modifier in solution to
increase the density of the diluent. When the density of the diluent and
microparticles are equivalent, the microparticles will be in a suspended
state.
Suitable viscosity modifiers include sugars, thickeners, and other agents,
such
as, for example, metrizamide and metrizoic acid. Sugars that are useful in the
composition include such sugars as sucrose, glucose, and mannitol. Effective
concentrations of the sugar can range from about 5% to about 25%, preferably
from about 7% to about 20%, and most preferably from about 10% to about
15%, based on weight per unit volume (g/100 ml).
The function of the preservative is to reduce microbial growth in the
composition. Preservatives that are useful in the composition include sodium
azide. Effective concentrations of the preservative can range from about 0.1%
to about 1%, preferably from about 0.1 % to about 0.5%, and most preferably
from about 0.1 % to about 0.2%, based on weight per unit volume (g/100 ml).
2 5 It is preferred that the pH of the composition be below 7.2, more
preferably below 7.0, and most preferably from about 6.5 to about 6.7. It is
preferred that the pH of the composition be below 7.2 because experimental
evidence shows that the ability to maintain disulfide bonds in their reduced
state
rapidly disappears as the pH increases above 7.2, as evidenced by reduction of
3.0 antigen stability.
In a preferred embodiment, the buffer of the present invention contains
the following ingredients in the amounts indicated, wherein mM, M and % are
as previously defined:
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,.~o us;
~~~ri redient'~~ " o m ~~'C Y~ :~Arrmn:un
Biological buffer 10 mM to 100 mM
Biological detergent 0.01% to 1%
Source of positive and negative 0.1 M to 0.3 M
counterions (salt)
Viscosity modifier su ar 7 % to 20%
Preservative 0.1 % to 0.5%
Dithiothreitol 5 mM to 10 mM
Ethylene glycol 4% to 5%
The composition of the invention can be prepared simply by mixing the
foregoing components in water in any order and taking care to ensure that the
pH of the composition is adjusted to the proper level. The order of addition
of
ingredients is not critical.
The composition of the invention is useful as a diluent of assay
components. The term "assay components", as used herein, means the solid
phase that contains antigen for antibody capture, e.g., microparticles coated
with antigen. In addition, assay components include commercial assay
reagents such as polyclonal and/or monoclonal antibodies, particularly of IgG
or
IgM class, and fragments thereof; antigens and fragments thereof; antigenic
lysates, recombinant proteins, synthetic peptides, and the like. It also is
contemplated that inert assay materials, such as latex particles, magnetic
beads, microparticles and the like, if provided in a suspension, may be
suspended in the composition of the invention, or if dilution of these
materials in
a suspension is desired, the dilution may be performed with the composition of
the invention.
The composition of the invention may be used to dilute assay
components prior to performing the assay itself. The composition is especially
useful for diluting antigens that are used in determining the presence or
absence of specific antibodies to HCV. An example of such a method of dilution
is described at col. 37, line 20 through col. 41, line 21 of U.S. Patent No.
5,358,691.
2 5 In another aspect ot the invention, the composition can be provided as a
component of a kit. As used herein, the term "kit" means a collection of
reagents
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and associated materials, e.g., diluents, buffers, required to perform an
assay.
In the apparatus described in U.S. Patent No. 5,358,691, a form of a kit is
shown
in FIGS. 4A and 4B. The kit is referred to as a reagent pack. The composition
of
this invention can be packaged in one of the receptacles of the reagent pack.
The following non-limiting examples will further illustrate the invention.
In the examples, all concentrations are based on moles per liter (M),
millimoles
per liter (mM), or per cent (grams per 100 milliliters).
EXAMPLES
EXAMPLE 1 AND COMPARATIVE EXAMPLES A-D
This example illustrates the effect of a composition containing a
biological buffer, dithiothreitol, and ethylene glycol on the stability of
hepatitis C
virus (HCV) antigen under heat stress conditions.
A stock composition containing biological buffer was prepared by
combining the following ingredients to arrive at the indicated concentrations:
Ingredient Concentration
Biological buffer ("MES") 10 mM
Biological detergent ("TRITON X-1 00) 0.01%
Salt (NaCI) 0.2 M
Sugar (sucrose) 11.5%
Preservative (sodium azide) 0.1%
Dithiothreitol 10 mM
Each of the following additives was added to a separate portion of the
stock composition to form four modified stock compositions. Each additive and
the concentration thereof are set forth below:
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Example Additive Concentration
Ethylene glycol 5%
Comp. A Polyethylene glycol 1%
Comp. B Polyvinyl alcohol 1%
Comp. C Glycerol 5%
Comp. D None 0%
Microparticies (polystyrene, 0.8 micrometer average diameter,
commercially available from Seradyn) were coated with HCV antigens. The
antigens were HC43/c100, c200, and NS5. The 33c antigen was present in
both the HC34 antigen and the c200 antigen. One set of microparticles was
coated with the HC43/c100 antigen. A second set of microparticles was coated
with the c200 antigen. A third set of microparticies was coated with the NS5
antigen. Coating of the microparticles was carried out by passive adsorption.
All three sets of the coated microparticies were blended together and a
portion
of the resulting blend was introduced into each of the four modified stock
solutions (Example 1 and Comparative Examples A, B, and C) and into the
unmodified stock solution, which acted as a control (Comparative Example D).
2 5 The microparticles were used to detect antibodies in single marker
specimens used in assay development, specifically, panels containing anti-
HCV antibodies to viral core, c100, or 33c antigens, as well as a negative
control The negative control contained no antibodies. All tests were run in
triplicate and averaged.
3 0 Each solution containing the coated microparticles was incubated for a
period of approximately 12 days at each of the following temperatures:
2-8 C
31 C
35 37 C
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The solution containing glycerol severely reduced signal generation in the
assay. Accordingly, this solution was not evaluated beyond the fourth day. The
solution containing polyvinyl acetate also reduced signal generation in the
assay. Accordingly, this solution was not evaluated beyond the fourth day. The
solutions containing glycol and polyethylene glycol and the control were
maintained at their respective temperatures and tested on the twelfth day.
The assay employed to test the effect of the additives was a microparticle
capture enzyme immunoassay, was similar to that described in U.S. Patent No.
5,358,691, wherein the enzyme was alkaline phosphatase and the enzyme
substrate was 4-methylumbelliferyl phosphate. However, in this assay, the
antigens were coated onto microparticles, the analyte in the test sample was
anti-HCV antibody, and the enzyme was attached to anti-human IgG antibody to
form the conjugate. The assay format was a sandwich assay wherein the
antigen would bind to the anti-HCV antibody, which, in turn, would bind to the
conjugate.
FIG. 1 shows the results for the experiments carried out under the most
extreme heat stress conditions (37 C for 12 days). FIG. 1 shows results for
antibodies to the c100 antigen, the 33c antigen, and the negative control.
The composition containing ethylene glycol showed superiority over the
2 0 control and the composition containing polyethylene glycol with respect to
maintaining detection of 33c activity following subjection to extreme heat
stress
conditions.
The antigens used in the assay are described below. Individual
blood or plasma donors or patients (based on clinical evaluation) may be
tested
2 5 for antibodies to HCV. The presence of these antibodies indicates that the
individual (donor or patient) has been infected with HCV, may harbor
infectious
HCV, and may be capable of transmitting non-A, non-B hepatitis (NANBH).
3 0 HC43
The recombinant HCV protein HC43, expressed in E. coli, contains sequences
of putative HCV core structural protein and HCV nonstructural protein NS3.
HC43 is a fusion protein consisting of amino acids 1 to 150 and 1192 to 1457
of
3 5 the HCV polyprotein. The amino acids 1192 to 1457 of the HCV polyprotein
constitute the 33c antigen.
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c100-3
ow
The recombinant HCV protein c100-3, expressed in Saccharomyces cerevisiae
(yeast), contains sequences of the putative HCV nonstructural proteins NS3
and NS4. c100-3 is a chimeric fusion region of 154 amino acids of human
superoxide dismutase (SOD), five linker amino acids, amino acids 1569 to 1931
of the HCV polyprotein and an additional five linker amino acids at the
carboxyl
terminus.
c200
The recombinant HCV protein c200 expressed in yeast contains HCV amino
acids 1192 to 1931, presumed to encode the NS3 and NS4 regions of the HCV
genome. c200 is a chimeric fusion protein with 154 amino acids of superoxide
dismutase (SOD).
NS5
The recombinant HCV protein NS5, expressed in yeast, contains sequences of
the putative HCV nonstructural protein NS5. NS5 is a chimeric fusion region of
154 amino acids of SOD and amino acids 2054 to 2995 of the HCV polyprotein.
A more detailed discussion of the hepatitis C viral genome can be found
in Selby et al., "Expression, identification and subcellular localization of
the
proteins encoded by the hepatitis C viral genome", Journa/ of General Virology
3 0 (1993), 74, 1103-1113, incorporated herein by reference.
Various modifications and alterations of this invention will become apparent
to
those skilled in the art without departing from the scope and spirit of this
invention, and it should be understood that this invention is not to be unduly
3 5 limited to the illustrative embodiments set forth herein.
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