U.S. patent application number 12/469568 was filed with the patent office on 2010-11-25 for reagents for hcv antigen-antibody combination assays.
Invention is credited to Jianping Yang, Jian Zheng.
Application Number | 20100297607 12/469568 |
Document ID | / |
Family ID | 42558582 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297607 |
Kind Code |
A1 |
Zheng; Jian ; et
al. |
November 25, 2010 |
Reagents For HCV Antigen-Antibody Combination Assays
Abstract
The present invention is directed to combination immunoassays,
reagents and kits for simultaneous detection of HCV antigens and
anti-HCV antibodies in a sample. The combination immunoassays of
the present invention employ a non-ionic detergent that effectively
exposes or releases the HCV core antigen from virions in a sample
without interfering with the performance of other reagents such as
the capture of anti-HCV antibodies by recombinant HCV antigens.
Inventors: |
Zheng; Jian; (Raritan,
NJ) ; Yang; Jianping; (Phillipsburg, NJ) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
42558582 |
Appl. No.: |
12/469568 |
Filed: |
May 20, 2009 |
Current U.S.
Class: |
435/5 |
Current CPC
Class: |
G01N 33/5767 20130101;
G01N 33/5306 20130101; G01N 2469/10 20130101; G01N 33/56983
20130101; G01N 2333/186 20130101; G01N 2469/20 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Claims
1. An immunoassay for simultaneously detecting HCV antigens and
antibodies in a sample, comprising: providing a non-ionic detergent
comprising an N-alkyl-N,N-dimethyl-amine oxide, a first pair of a
capture antigen and a detection antigen, a first pair of a capture
antibody and a conjugate antibody, wherein said capture antigen and
said detection antigen both comprise a first peptide fragment of a
first HCV protein, said capture antibody and said conjugate
antibody specifically bind to a second HCV protein, and said
detection antigen and said conjugate antibody comprise one and same
signal generating means; contacting said sample in the presence of
said detergent with said capture antigen, said detection antigen,
said capture antibody and said conjugate antibody, to form a
sandwich complex between said capture antigen, said detection
antigen, and an anti-HCV antibody present in said sample, and a
complex between said capture antibody, said conjugate antibody, and
an HCV antigen present in said sample; and measuring a signal
generated from said signal-generating means as a result of the
formation of said complexes, thereby simultaneously detecting HCV
antigens and antibodies in said sample.
2. The immunoassay of claim 1, wherein said
N-alkyl-N,N-dimethyl-amine oxide is characterized by the formula,
CH.sub.3--(CH.sub.2)n-N.sup.+--(CH.sub.3).sub.2O.sup.-, wherein n
falls in the range of 9 to 13.
3. The immunoassay of claim 2, wherein said
N-alkyl-N,N-dimethyl-amine oxide is Lauryldimethylamine N-oxide
(LDAO).
4. The immunoassay of claim 1, wherein said first HCV protein and
second HCV protein are independently selected from the group
consisting of the core antigen, E1, E2, NS2, NS3, NS4, and NS5.
5. The immunoassay of claim 1, wherein said first HCV protein and
said second HCV protein are the same, and said capture antibody and
said conjugate antibody bind to a region of said second HCV protein
outside of said first peptide fragment.
6. The immunoassay of claim 5, wherein said first HCV protein and
said second HCV protein are both the HCV core antigen.
7. The immunoassay of claim 1, wherein a second pair of a capture
antigen and a detection antigen is provided, wherein said capture
antigen and said detection antigen of the second pair both comprise
a second peptide fragment of an HCV protein, wherein said second
peptide fragment is different from said first peptide fragment.
8. The immunoassay of claim 7, wherein said first peptide fragment
and said second peptide fragment are derived from different HCV
proteins.
9. The immunoassay of claim 8, wherein at least one of said first
peptide fragment or said second peptide fragment is a fragment of
the HCV core antigen.
10. The immunoassay of claim 1, wherein said capture antibody in
said first pair comprises two or more antibodies.
11. The immunoassay of claim 1, wherein a second pair of a capture
antibody and a conjugate antibody is provided, wherein said capture
antibody and said conjugate antibody in said second pair
specifically bind to said second HCV protein or a different HCV
protein.
12. The immunoassay of claim 1, wherein said capture antigen and
said capture antibody are attached to a solid phase.
13. A kit for simultaneously detecting HCV antigens and antibodies
in a sample, comprising a non-ionic detergent comprising an
N-alkyl-N,N-dimethyl-amine oxide, a first pair of a capture antigen
and a detection antigen, a first pair of a capture antibody and a
conjugate antibody, wherein said capture antigen and said detection
antigen comprise a first peptide fragment of a first HCV protein,
said capture antibody and said conjugate antibody specifically bind
to a second HCV protein, and said detection antigen and said
conjugate antibody comprise one and same signal generating
means.
14. The kit of claim 13, wherein said N-alkyl-N,N-dimethyl-amine
oxide is characterized by the formula,
CH.sub.3--(CH.sub.2)n-N.sup.+--(CH.sub.3).sub.2O.sup.-, wherein n
falls in the range of 9 to 13.
15. The kit of claim 14, wherein said N-alkyl-N,N-dimethyl-amine
oxide is Lauryldimethylamine N-oxide (LDAO).
16. The kit of claim 13, wherein said first HCV protein and said
second HCV protein are independently selected from the group
consisting of the core antigen, E1, E2, NS2, NS3, NS4, and NS5.
17. The kit of claim 13, wherein said first HCV protein and said
second HCV protein are the same, and said capture antibody and said
conjugate antibody bind to a region of said second HCV protein
outside of said first peptide fragment.
18. The kit of claim 17, wherein said first HCV protein and said
second HCV protein are both the HCV core antigen.
19. The kit of claim 13, further comprising a second pair of a
capture antigen and a detection antigen is provided, wherein said
capture antigen and said detection antigen of the second pair
comprise a second peptide fragment of an HCV protein, wherein said
second peptide fragment is different from said first peptide
fragment.
20. The kit of claim 19, wherein said first peptide fragment and
said second peptide fragment are derived from different HCV
proteins.
21. The kit of claim 20, wherein at least one of said first peptide
fragment or said second peptide fragment is a fragment of the HCV
core antigen.
22. The kit of claim 13, wherein said capture antibody in said
first pair comprises two or more antibodies.
23. The kit of claim 13, further comprising a second pair of a
capture antibody and a conjugate antibody, wherein said capture
antibody and said conjugate antibody in said second pair
specifically bind to said second HCV protein or a different HCV
protein.
24. The kit of claim 13, wherein said capture antigen and said
capture antibody are attached to a solid phase.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to immunoassays for
detection and diagnosis of HCV infection. In particular, the
present invention relates to combination immunoassays, reagents and
kits based on use of a non-ionic detergent for simultaneous
detection of HCV antigens and anti-HCV antibodies in a sample.
BACKGROUND OF THE INVENTION
[0002] The hepatitis C virus (HCV), a single-stranded RNA virus, is
the etiological agent of blood-borne non-A, non-B hepatitis.
Chronic active infection with HCV often progresses to liver
cirrhosis and hepatocellular carcinoma. Epidemiological studies
indicate that HCV infects more than 170 million people worldwide
with a high incidence of chronic disease ultimately progressing
(more than 50% of cases) to death. However, since it is mainly a
blood-borne disease, it is possible to identify the pathogen in
blood samples and to eliminate the transmission of disease through
blood transfusion. After exposure to the HCV pathogen, there is
initially no evidence of viral presence, i.e. no detectable viral
RNA or serology markers. This is referred to as the "window period"
(WP). Generally, after 10 days following exposure to HCV, viral RNA
can be detected while anti-HCV antibodies become detectable
approximately 70 days later (Busch M P and Dodd R Y, Transfusion
40(10): 1157-1160, 2000). To prevent spread of HCV infection, it is
extremely important to take this observable fact into consideration
and to establish a reliable blood-screening test, which would
narrow the detection window. Since the commercialization of the
nucleic acid amplification testing (NAT) which detects HCV RNA, the
post-transfusion HVC infection rate has been dramatically reduced.
Other methods are based on serological screening of the blood for
detecting the presence of HCV core antigen (Ortho HCV Core Antigen
ELISA, Ortho-Clinical Diagnostics, Inc., Raritan, N.J.) or
antibodies against HCV polypeptides (Ortho HCV 3.0 ELISA,
Ortho-Clinical Diagnostics, Inc., Raritan, N.J.) in patient serum
or plasma.
[0003] According to a survey conducted by Seme et al. (J. Clin.
Virol. 32(2): 92-101, 2005), the first generation HCV core antigen
assay detects HCV infection with comparable sensitivity and
detection limits to the nucleic acid techniques (NAT). These assays
detect HCV infection between 40 to 50 days earlier than the current
third generation HCV antibody screening assays. Although the first
generation HCV core antigen assay, designed for blood screening,
has significantly reduced the window period, it only detects core
antigen at pre-seroconversion or early post-seroconversion phase.
Furthermore, the first generation HCV core antigen assay is unable
to detect core antigen when the antigen forms immune-complexes with
anti-core antibodies in the late seroconversion phase. Clearly, it
is desirable to have a combined serology assay that can detect HCV
core antigen in the pre-seroconversion phase as well as anti-HCV
antibodies in the seroconversion phase, thus narrowing the WP
significantly. This combined serology test can especially be a
valuable method of blood screening in settings where the NAT test
can not be carried out due to lack of equipment or competency.
[0004] Such an HCV antigen and antibody combined assay will be a
significant improvement over the current third generation serology
blood screening method (Ortho HCV 3.0 ELISA, Ortho-Clinical
Diagnostics, Inc., Raritan, N.J.) with regards to narrowing the WP.
However, one of the challenges to the successful antigen antibody
combined assay is to select an appropriate detergent to disrupt HCV
virions and release antigen without interfering with the capture of
anti-HCV antibodies by recombinant HCV antigens.
SUMMARY OF THE INVENTION
[0005] The present invention is directed to combination
immunoassays, reagents and kits for simultaneous detection of HCV
antigens and anti-HCV antibodies in a sample.
[0006] The uniqueness of the combination immunoassays of the
present invention resides principally in the use of a non-ionic
detergent that effectively exposes or releases the HCV core antigen
from virions in a sample without any pre-processing of the sample,
yet does not interfere with the capture of anti-HCV antibodies by
recombinant HCV antigens, thereby permitting simultaneous
measurement of the HCV core antigen and anti-HCV antibodies.
[0007] Non-ionic detergents suitable for use in the combination
assays of the present invention are members of the
N-alkyl-N,N-dimethyl-amine oxide family. In a preferred embodiment,
the combination assay employs Lauryldimethylamine N-oxide (LDAO).
In other embodiments, a derivative or functional or chemical
equivalent of LDAO is employed.
[0008] Accordingly, the present invention provides combination
immunoassays in various formats, related reagents and kits for
simultaneous detection of HCV antigens and anti-HCV antibodies in a
sample.
DETAILED DESCRIPTION OF THE INVENTION
[0009] The present invention is directed to HCV antigen-antibody
combination immunoassays, and related methods, reagents and
kits.
[0010] HCV antigen-antibody combination immunoassays or "combo"
assays refer to immunoassays that simultaneously detect both HCV
antigens and anti-HCV antibodies in a sample in a single assay. The
antigen/antibody combination assay methods rely on the
identification and use of antigenic and immunogenic HCV antibodies
and antigens that are present during the early stages of HCV
seroconversion, thereby increasing detection accuracy and reducing
the incidence of false results during the window period.
[0011] The present invention is predicated, at least in part, on
the identification of nonionic detergents that are suitable and
effective for use in a combination immunoassay for sensitively and
accurately detecting early HCV infection. Such detergents
effectively disrupt HCV viral particles in a test sample and as a
result, the released core antigen is effectively captured and
detected by monoclonal antibody probes. In the meantime, the
detergents do not impact negatively the antibody detection either
by leaching the recombinant antigens coated on the solid-phase or
by interfering with the capturing of anti-HCV antibodies during the
combination assay. Therefore, the combination assays provided by
the present invention permit simultaneous detection of both HCV
antigens (including the core antigen) and anti-HCV antibodies in a
sample in a single assay. Compared to the existing assays,
including assays directed to detecting either HCV antigens or
anti-HCV antibodies separately, the combination assays of the
present invention circumvent the need to run two separate assays
and provide improved sensitivity and specificity for HCV
detection.
[0012] In accordance with the present invention, nonionic
detergents suitable for use in a combination immunoassay include
members of the N-alkyl-N-alkyl'-N-methyl-amine oxide family, which
are also known as zwitter-ionic or zwitterionic detergents. The
alkyl groups generally contain not more than 15 carbon atoms
each.
[0013] In one embodiment, the detergent is an
N-alkyl-N-alkyl'-N-methyl-amine oxide wherein the alkyl group is a
mono-chain or branched alkyl group containing 9 to 13 carbon atoms,
and the other alkyl group ("the alkyl" group) is a mono-chain or
branched alkyl group containing 1 to 13 carbon atoms. In another
embodiment, one or both the alkyl groups of
N-alkyl-N-alkyl'-N-methyl-amine oxide are substituted alkyl
groups.
[0014] In a preferred embodiment, the detergent is
N-alkyl-N,N-dimethyl-amine oxide characterized by the formula:
CH.sub.3--(CH.sub.2)n-N.sup.+--(CH.sub.3).sub.2O.sup.-
wherein the length of the alkyl chain is defined by the number n,
which can be in the range of 9 to 13, or preferably 10 to 12, more
preferably n is 11. The number "n" is at least 9; preferably at
least 10; more preferably at least 11.
[0015] In an especially preferred embodiment of the present
invention, the nonionic detergent employed in an HCV combination
immunoassay is Lauryldimethylamine N-oxide (LDAO), having the
formula of
CH.sub.3--(CH.sub.2).sub.11--N.sup.+--(CH.sub.3).sub.2O.sup.-,
which is graphically depicted as:
##STR00001##
[0016] A non-ionic detergent can be provided to a combination assay
in any appropriate manner. For example, the detergent can be added
to a test sample before the sample is placed in contact with
detecting reagents such as capture antigens and antibodies.
Alternatively, the detergent can be mixed with a test sample
simultaneously with the addition of other reagents for detection.
In a preferred embodiment, the detergent is provided in a solution
(or "diluent"), which is used to dilute or suspend a biological
test sample prior to contacting the sample with capture antigens
and antibodies. Regardless of how a non-ionic detergent is provided
to the assay, the concentration of the detergent in the solution
mixture when the contact with the test sample occurs should be in
the range of 0.1%-2% (w/v), preferably at least 0.3% (w/v) but not
more than 1% (w/v), more preferably in about 0.5% (w/v).
[0017] Biological samples that can be tested for HCV using the
combination assays of the present invention include any sample
suspected to contain HCV virions, antigens or antibodies. The
sample can be a biological fluid or tissue, including body fluids,
such as whole blood, dried whole blood, serum, plasma, or other
blood components including red blood cells, white blood cells and
platelets; urine, saliva, cerebrospinal fluid, liver tissue, among
others. The samples can be treated in any appropriate manner prior
to being used in the assay.
[0018] In accordance with the present invention, the anti-HCV
antibody detection part of the combination assay generally employs
at least one (i.e., one or more) capture antigen that binds,
therefore "captures" anti-HCV antibodies in the sample. The capture
antigens are generally antigenic peptides (containing one or more
epitopes) derived from an HCV protein encoded by the HCV genome.
The sequence of the entire HCV genome and the encoded HCV
polyprotein sequence are documented in GenBank (accession #M62321
and #AAA45676, respectively) and available to those skilled in the
art.
[0019] It should be noted that the term "HCV protein" as used
herein includes both a native, full-length protein encoded by the
HCV genome (e.g., a structural or non-structural protein, or a
precursor of a structural or non-structural protein), and an
artificial fusion polypeptide of two native HCV proteins or
fragments thereof (e.g., a fusion of NS3 and partial NS4, also
referred herein as "NS3/NS4").
[0020] The term "HCV antigen" or "HCV antigens", as used herein in
reference to antigens present in a sample from an HCV-infected
individual, can be full-length HCV proteins or antigenic fragments
thereof.
[0021] Generally speaking, a peptide fragment of an HCV protein is
antigenic and capable of binding to and capturing the respective
antibodies when the fragment is at least 6 or 7 amino acids in
length, preferably, at least 8, or at least 9, or at least 10 amino
acids; more preferably, at least 12, 15 or 20 amino acids. Capture
HCV antigens can contain more than one epitope. The term "epitope"
is well understood in the art and refers to a molecular region or
structural determinant on the surface of an antigen capable of
binding to an antibody and eliciting an immune response. Capture
antigens can be made recombinantly, or by conventional chemical
synthesis.
[0022] In certain embodiments, the capture antigen is an antigenic
fragment of an HCV protein selected from core antigen, E1, E2, NS2,
NS3, NS4, or NS5. In a preferred embodiment, the capture antigen is
derived from core antigen, or an NS3/NS4 fusion protein as
illustrated in the examples below. In another embodiment, two or
more peptides are used as capture antigens, which can be derived
from the same or different HCV proteins.
[0023] A capture antigen can be coated on a solid phase prior to
performing the assay. Alternatively, the capture antigen is
conjugated with an appropriate reagent, e.g., biotin, which can
mediate attachment to a solid phase after the capture antigen has
formed a complex with (i.e., has captured) anti-HCV antibodies in a
liquid phase, e.g., in a cocktail ELISA format.
[0024] Examples of solid phases suitable for use in the
immunnassays of the present invention include both porous and
non-porous materials, such as, for example, latex particles,
magnetic particles, beads, membranes, and microtiter wells. The
choice of solid phase material can be determined based upon desired
assay format.
[0025] When a capture antigen is contacted with a test sample, if
anti-HCV antibodies are present in the sample, such antibodies bind
to the capture antigen. The antibodies captured by the capture
antigen, i.e., the captured antibodies, can be detected using a
number of approaches.
[0026] One approach utilizes a second antibody, which recognizes
and binds the captured HCV antibodies. The second antibody, e.g.,
anti-human IgG, is conjugated to a signal-generating means. When
the second antibody binds to a captured (first) antibody, the
signal-generating means generates a measurable signal, which
indicates the presence of the first antibody in the test
sample.
[0027] Another approach utilizes a second antigen, which can be
conjugated to a signal-generating means, and is also referred to as
a "detection antigen". Like capture antigens, a detection antigen
can also contain more than one epitope, as long as one common
epitope is present on both the capture antigen and the detection
antigen. Additionally, one or more detection antigens can be used.
When a detection antigen binds to a captured antibody thereby
forming a capture antigen-antibody-detection antigen sandwich, the
signal-generating means in the detection antigen can generate a
measurable signal, which indicates the presence of the antibody in
the test sample. This "Ag sandwich" approach is premised on the
ability of an anti-HCV antibody to simultaneously bind two
identical epitopes in two separate antigen molecules. This
Ag-sandwich approach permits highly specific detection of the
anti-HCV antibodies; and because of this high specificity, this
approach allows for the use of a larger volume of a test sample,
which in turn permits a more sensitive detection of HCV antigens
present in the sample.
[0028] This signal-generating means is either itself detectable or
may be reacted with one or more compounds to generate a detectable
signal. Examples of signal-generating means include chromogens,
radioisotopes, chemiluminescent compounds, enzymes (e.g., alkaline
phosphatase, acid phosphatase, horseradish peroxidase,
beta-galactosidase and ribonuclease), or one partner of a binding
pair (such as biotin or strepavidin). Where an enzyme is used as a
signal-generating means (e.g., alkaline phosphatase or horseradish
peroxidase), addition of a chromo-, fluro-, or lumo-genic substrate
results in generation of a detectable signal.
[0029] In accordance with the present invention, the HCV antigen
detection part of the combination assay is generally achieved using
one or more pairs of a capture antibody and a conjugate antibody in
an antibody sandwich format.
[0030] The conjugate antibody is attached with any one of the
signal-generating means described hereinabove. The capture antibody
can be coated on a solid phase, i.e., the same solid phase as the
capture antigen(s) described above, prior to performing the assay.
Alternatively, the capture antibody is conjugated with an
appropriate reagent, e.g., biotin, which can mediate attachment to
a solid phase after the capture antibody has formed a complex with
(i.e., has captured) an HCV antigen in a liquid phase which is
either already or subsequently bound with a conjugate antibody. In
either approach, once a capture antibody-antigen-conjugate antibody
sandwich is formed and captured onto a solid phase, the
signal-generating means can generate a measurable signal, which
indicates the presence of the antigen in the test sample.
[0031] Within each pair, there can be one or more capture
antibodies each recognizing different epitopes, and one or more
conjugate antibodies each recognizing different epitopes. In order
to permit simultaneous binding of the capture and conjugate
antibodies to the same antigen molecule and formation of a sandwich
complex, the capture antibody or antibodies should recognize
different epitopes from those recognized by the conjugate antibody
or antibodies in the same pair, yet the epitopes recognized by the
capture and conjugate antibodies, respectively, should be within
the same HCV antigen in order to form an antibody-antigen-antibody
sandwich. For example, the capture and conjugate antibodies can be
directed to epitopes in any HCV protein, structural or
non-structural HCV polypeptide, encoded by the HCV genome.
Preferably, the antibodies are directed to epitopes within an HCV
protein selected from the core antigen, E1, E2, NS2, NS3, NS4, or
NS5. In an especially preferred embodiment, the capture and
conjugate antibodies in one pair are directed to, i.e.,
specifically bind to, epitopes within the HCV core antigen. In
another embodiment, two or more pairs of capture and conjugate
antibodies are used, and at least one of the pairs includes a
capture and a conjugate antibody directed to epitopes within the
HCV core antigen.
[0032] The capture and conjugate antibodies can be monoclonal
antibodies or polyclonal antibodies or combinations thereof.
Preferably, monoclonal antibodies are employed.
[0033] To perform a combination assay in accordance with the
present invention, one is provided with a capture antigen and
either a conjugate antibody or a detection antigen for detecting
anti-HCV antibodies; a capture antibody and an HCV-specific
antibody conjugate for detecting HCV antigens; and an appropriate
non-ionic detergent. A test sample is contacted with the detergent,
the capture and conjugate reagents, sequentially or simultaneously,
and the signal generated from the conjugate reagents can be
correlated with a diagnosis of HCV infection.
[0034] In a specific embodiment, one is provided with a capture
antigen, a detection antigen; a capture antibody and a conjugate
antibody; and an appropriate non-ionic detergent. In a preferred
embodiment, the capture antigen and the capture antibody are both
coated on (covalently or non-covalently attached to) a solid phase.
A test sample is brought into contact with the capture reagents
attached to the solid phase in the presence of the detergent.
Simultaneously or preferably subsequently, the conjugate reagents
(i.e., the detection antigen and conjugate antibody) are added to
the reaction mixture. The signal generated as a result of the
binding of the conjugate reagents also to the solid phase can be
correlated with a diagnosis of HCV infection.
[0035] It should be noted that the capture and conjugate reagents
should be chosen to avoid cross-reactivity and false-positive
results. For example, the HCV antigens employed for detecting
anti-HCV antibodies are derived from an HCV polypeptide different
from the polypeptide against which the capture and conjugate
antibodies are directed. Alternatively, when the antigens employed
for antibody detection are derived from the same HCV protein (e.g.,
the core antigen) as the protein against which the antibody
reagents are raised, the antigens used for antibody detection
should present epitopes that are not recognized by the antibody
reagents used for antigen detection. Essentially, the epitopes to
which antibodies bind in the antigen detection part of the assay
should not be present in the antigens used for HCV antibody
detection.
[0036] The present invention also provides kits containing the
various reagents described above for performing a combination assay
in order to simultaneously detect HCV antigens and anti-HCV
antibodies in support of a diagnosis of HCV infection.
EXAMPLES
[0037] The following examples describe in detail the advantages and
importance of the invention by examining the detergent
Lauryldimethylamine N-oxide (LDAO) and its efficiency in achieving
release of the HCV core antigen in an antigen assay and its
compatibility with an HCV antibody assay. The use of detergent
Lauryldimethylamine N-oxide (LDAO) in simultaneous measurement of
HCV core antigen and HCV antibodies is also described.
[0038] It is to be understood that the examples presented
hereinbelow are for illustration and are not intended to limit the
scope of the present invention.
General Reagents for HCV Core Antigen and Anti-HCV Antibody
Detection
Ortho HCV Core Antigen ELISA Test System
[0039] Ortho HCV Core Antigen ELISA (Ortho-Clinical Diagnostics,
Inc., Raritan, N.J.) was used for the detection of hepatitis C
nucleocapsid core antigen (HCV core antigen) in human serum or
plasma. The monoclonal antibodies coated onto the microwell solid
phase were used to capture the antigen and the secondary monoclonal
antibodies conjugated to HRP were used to detect the captured
antigen. A detergent-containing specimen diluent was used to expose
the HCV core antigen for capture and detection without a
pretreatment step.
Ortho HCV 3.0 ELISA Test System
[0040] Ortho HCV 3.0 ELISA (Ortho-Clinical Diagnostics, Inc.,
Raritan, N.J.) was for the detection of antibody to hepatitis C
virus (anti-HCV) in human serum or plasma. The recombinant HCV
antigens c22 (amino acid 2-120, core region), c200 (amino acid
1192-1931, NS3/NS4 region) and NS5 (amino acid 2054-2995) coated on
to the microwell solid phase were used to capture the anti-HCV
antibodies. Murine monoclonal anti-human IgG antibody conjugated to
HRP was used to detect the captured human anti-HCV. This format
utilizes detergent Tween 20 in the specimen diluent.
Chiron RIBA HCV 3.0 SIA
[0041] Chiron RIBA HCV 3.0 SIA (Novartis Vaccines &
Diagnostics, Emeryville, Calif.) is a strip immunoblot assay used
for the detection of antibody to hepatitis C virus (anti-HCV) in
human serum or plasma. The HCV antigens or peptides c22p (amino
acid 10-53, core region), c33c (amino acid 1192-1457, NS3 region),
5-1-1 and c100 peptides (amino acid 1694-1735 and 1920-1935
respectively, NS4 region) and NS5 (amino acid 2054-2995) were
immobilized as individual bands onto test strips to capture the
anti-HCV antibodies. Murine monoclonal anti-human IgG antibody
conjugated to HRP then detected the captured human anti-HCV. The
assay provides additional information on antibody specific
reactivity to individual antigen of an Ortho HCV 3.0 ELISA reactive
specimen.
Antigens
[0042] Two peptide derivatives were synthesized to cover the HCV
core protein sequence from amino acid 10 to amino acid 45. For
HCC5N, the sequence is set forth in SEQ ID NO:1 (the N-terminus has
a free amino group, as opposed to an amino group blocked by
acetylation). HCC5N has two residues at the C-terminus, NorLeucine
and Cys, which do not belong to the HCV core protein. For HCC5-b,
the sequence is set forth in SEQ ID NO:2 (the N-terminus has a free
amino group). HCC5-b has two residues at the C-terminus, Gly and
Lys(Biotin), which do not belong to the HCV core protein. HCC5N was
further conjugated to SMCC activated BSA through the C-terminal Cys
of HCC5N under reducing conditions.
[0043] Recombinant HCV NS3 helicase (rNS3(h)), covering residues
P1208 to T1657, was cloned and expressed in E. coli as described
previously (Jin, Arch Biochem Biophys. 323(1): 47-53, 1995). A
6.times.His tag was fused to the N-terminus of the rNS3(h) to
purify protein (resulting in SEQ ID NO:3, representing a fusion
protein of the 6.times.His tag and rNS3(h)). A biotin conjugated
rNS3(h) was made using EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Cat
#21335). The conjugated protein was determined to have 3 biotins
per rNS3(h) molecule.
Monoclonal Anti-HCV Core Antibodies
[0044] Four monoclonal antibodies were used in the study. The
production and epitope recognition sites of monoclonal antibody
C11-3, C11-7 and C11-14 have been described in European Patent
Publication EP 0 967 484 A1. None of the epitope recognition sites
of these monoclonal antibodies fall within amino acids 10-45 of
HCV. The monoclonal anti-HCV antibody 12F11 was raised against
recombinant c22. The recognition site of 12F1 1 is around amino
acids 58-72 of HCV (core antigen). The antibodies C11-14 and 12F11
were conjugated to horseradish peroxidase (HRP) using standard
procedures.
HCV Specimens
[0045] (1) anti-c22 and Anti-c33c Depleted Anti-HCV Specimens
[0046] Specimens WHO Hu-a-c33c, 41530822, 41530832 and 41530883
were originally tested anti-HCV strong reactive by Ortho HCV 3.0
ELISA and non-reactive by Ortho HCV Core Antigen ELISA. The
anti-c22 portion (anti-HCV core) of the antibodies was depleted by
passing the specimen through an affinity column in which
chromatography resins were conjugated with recombinant HCV core
antigen c22, the same antigen as one of the coating antigens used
in the Ortho HCV 3.0 ELISA.
[0047] Specimen WHO Hu-a-c22, I-20 and 360650 were originally
tested anti-HCV strong reactive by Ortho HCV 3.0 ELISA and
non-reactive by Ortho HCV Core Antigen ELISA. The anti-c33c portion
(anti-partial NS3) of the antibodies was depleted by passing the
specimen through an affinity column in which chromatography resins
were conjugated with recombinant HCV core antigen c33, the same
antigen (c33c) immobilized in the Chiron RIBA 3.0 SIA and a shorter
version of one of the coating antigens (c200) used in the Ortho HCV
3.0 ELISA.
(2) HCV Core Antigen Reactive Specimen
[0048] HCV core antigen reactive sample Lot 16 was a plasma pool,
made of specimens that were tested non-reactive of anti-HCV by
Ortho HCV 3.0 ELISA, reactive of HCV RNA by PCR, and reactive of
HCV core antigen by Ortho HCV Core Antigen ELISA. HCV core antigen
sample 9160834 is a plasma specimen from Boston Biomedica Inc.
(BBI). The specimen was tested non-reactive to anti-HCV by Ortho
HCV 3.0 ELISA, reactive to HCV RNA at about 36,379,940 IU/mL or
189,175,500 copies/mL by Versant bDNA (Bayer), and reactive to
Ortho HCV Core Antigen ELISA.
(3) HCV Seroconversion Panels
[0049] HCV Seroconversion Panels tested in the present studies were
all commercially available, purchased from SeraCare Life Sciences,
Inc. USA (BBI Diagnostics) and ZeptoMetrix Corporation, USA (former
Impath/BioClinical Partners, Inc.). A HCV Seroconversion Panel was
composed of serial bleeds of human blood specimens taken from an
individual who was infected with HCV. Specimens covered a period
from anti-HCV non-reactive to reactive.
Example 1
Anti-HCV Antibody Detection (an Indirect Assay Format)
[0050] The assays were performed using reagents from an Ortho HCV
3.0 ELISA kit. 150 .mu.L specimen diluents composed of a phosphate
based buffer containing detergent Tween-20, BSA, casein, yeast
extracts and other proteins, plus 50 .mu.L specimen were added to
each well. The plates were incubated at 37.degree. C. for 60
minutes, and were then washed 5 times with a PBS/Tween20-containing
wash buffer. Afterwards 200 .mu.L HRP conjugated murine monoclonal
anti-human IgG antibody was added to each well. The plates were
incubated at 37.degree. C. for 60 minutes and subsequently washed 6
times. 200 .mu.L OPD/substrate (one OPD tablet (Sigma, Cat #P-8287)
in 6 mL of ELISA substrate buffer) was added to each well and the
plates were incubated in the dark at room temperature for 30
minutes. A 4N H.sub.2SO.sub.4 stop solution was added at 50
.mu.L/well. ODs were recorded at 493 nm using a plate
spectrophotometry reader. Assay cut-off setting was adapted from
HCV 3.0 ELISA that is OD of average negatives plus 0.6. The assay
exceptions were: [0051] (1) In "ELISA-1", specimen diluent was from
the kit, containing detergent Tween 20; while in "ELISA-2", the
detergent was replaced with N-Lauryl sarcosine (NLS), and in
"ELISA-3", the detergent was replaced with Lauryldimethylamine
N-oxide (LDAO). [0052] (2) In "ELISA-1", the HRP-conjugated murine
monoclonal anti-human IgG antibody, provided in the kit, was
diluted at 1:3 prior to use; and in "ELISA-2" and "ELISA-3", the
conjugated antibody provided in the kit was diluted at 1:2 prior to
use.
[0053] As shown in Table 1, for both anti-c22 and anti-c33c
depleted specimens, Tween20 (commonly used in anti-HCV antibody
detection) and LDAO each provided better assay sensitivity, as
reflected by the signal vs. cut-off ratios ("S/C"), than NLS
(typically used in HCV antigen detection).
Example 2
Anti-HCV Antibody Detection (Ag Sandwich Assay Format)
[0054] In the Ag sandwich assay format, antigens labeled with
biotin were used as conjugate. The biotin-antigen conjugates bound
to human anti-HCV antibodies that were captured on solid phase to
form an Ag-Ab-Ag sandwich. The sandwiched complexes were then
detected by HRP conjugated streptavidin in a subsequent
incubation.
[0055] COSTA.TM. high-binding microtiter plates were coated with
200 .mu.L/well of premixed monoclonal antibodies C11-3 and C11-7 at
2.2 .mu.g/mL of each and HCC5N-BSA at 0.1 .mu.g/mL in 20 mM
phosphate buffer (pH 7.0). The plates were incubated at 25.degree.
C. overnight. The coating solutions were aspirated and 200
.mu.l/well of rNS3(h) at 2 .mu.g/mL in PB (pH7.0) was added. Plates
were incubated at 25.degree. C. overnight. The plates were then
washed one time with PBS/Tween wash buffer, followed by addition of
300 .mu.L/well PBS/BSA blocking solution (1% bovine serum albumin
and 30% sucrose in PBS) for 1 hour at 25.degree. C. The plates were
aspirated and dried overnight at 25.degree. C. with 10% humidity
and pouched with desiccant.
[0056] Specimen diluent composition was the same as used in Example
1, except for containing detergent Tween 20 in "ELISA-4", detergent
N- Lauryl sarcosine (NLS) in "ELISA-5", and detergent
Lauryldimethylamine N-oxide (LDAO) in "ELISA-6". 100 .mu.L specimen
diluent and 100 .mu.L specimen were added to each well. The
specimen incubation was at 37.degree. C. for 1 hour with shake. The
plates were then washed 5 times with PBS/Tween20. 200 .mu.L
biotin-rNS3(h) (or "rNS3(h)-b") was added at 60 ng/mL or
biotin-HCC5 (HCC5-b) was added at 5 ng/mL to each well.
Biotin-antigen was diluted in conjugate diluent CD-1 (Casein
Blocker in PBS, Pierce Cat #37528, supplemented with Tween 20 to
0.05% and EDTA to 2 mM). The biotin-antigen incubation was kept at
37.degree. C. for 60 minutes with shake. The plates were washed 5
times and HRP-Streptavidin (Jackson ImmunoResearch, Cat
#016-030-084) 1:8,000 diluted in CD-1 was added at 200 .mu.L/well.
The HRP-Streptavidin incubation was kept at room temperature for 30
minutes. The plates were then washed 6 times, after which 200 .mu.L
OPD/substrate was added to each well. The plates were incubated in
the dark at room temperature for 30 minutes, and an aliquot of 50
.mu.L 4N H.sub.2SO.sub.4 stop solution was added to each well. ODs
were recorded at 493 nm using a plate spectrophotometry reader.
Assay cut-off was determined as OD of negative average plus 0.300.
Assay results are shown in Table 2. The results show that with the
anti-c33c depleted specimen, Tween20 and LDAO each provided better
assay sensitivity than NLS. The results also demonstrate that the
antigen sandwich format (Ag-Ab-Ag) is better than the indirect
format (Ag-Ab-2.sup.nd Ab).
Example 3
HCV Core Antigen Detection
[0057] The HCV core antigen detection shown in Example 3 was a
monoclonal antibody-HCV core antigen-monoclonal antibody sandwich
ELISA. The exposed HCV core antigen in specimen was captured by two
monoclonal anti-HCV core antibodies coated on plates and detected
by another two HRP labeled monoclonal anti-HCV antibodies.
[0058] Except specimen diluent, ELISA reagents used in Example 3
were basically from the Ortho HCV Core Antigen ELISA kit. "ELISA-7"
used the original Kit specimen diluent that contained 1.0%
detergent N-Lauroylsarcosine (NLS). However, "ELISA-8" used
specimen diluent containing 1.0% detergent Tween 20 and "ELISA-9"
used specimen diluent containing 1.0% detergent Lauryldimethylamine
N-oxide (LDAO).
[0059] ELISA was performed following HCV Core Antigen ELISA's
protocol. 100 .mu.L specimen diluent and 100 .mu.L specimen were
added to each well. The specimen incubation was performed at
37.degree. C. for 90 minutes with shake. The plate was washed 5
times with PBS/Tween20 and conjugate antibodies were added at 200
.mu.l to each well. The conjugate incubation was performed at
37.degree. C. for 30 minutes. The plate was washed 6 times and 200
.mu.L OPD/substrate was added to each well. The plate was then
incubated in the dark at room temperature for 30 minutes. A 4N
H.sub.2SO.sub.4 stop solution was then added at 50 .mu.L/well. ODs
were recorded at 493 nm using a plate spectrophotometer reader.
Assay cut-off setting was similar to HCV Core Antigen ELISA, i.e.,
the OD of negative average plus 0.300.
[0060] Assay results are shown in Table 3. For specimens, NLS and
LDAO each provided better assay sensitivity than Tween20.
Example 4
HCV Core Antigen/Anti-HCV Combination Assay
[0061] HCV core antigen/anti-HCV antibody combination ELISA was
performed on the same plates as used in Example 2. Plates were
coated with monoclonal anti-HCV antibody C11-3 and C11-7, HCC5-BSA
and rNS3(h). Specimen diluent was composed of 20 mM phosphate
buffer (pH 7.3), 0.5M Sodium Chloride, 1 mM EDTA, 1% detergent
LDAO, 1% BSA, 0.03% Yeast extracts and 0.01% denatured Superoxidase
Dismutase (SOD), 200 .mu.g/mL mouse immunoglobulin G (IgG) and 0.1%
2-Chloroacetamide. The antibody/antigen conjugate was composed of
HRP labeled anti-HCV monoclonal antibody C11-14 at 4 .mu.g/mL and
HRP labeled monoclonal antibody 12F11 at 4 .mu.g/mL, HCC5-biotin at
5 ng/mL and rNS3(h)-biotin at 60 ng/mL in a buffer of 10 mM
phosphate buffer (pH 7.3), 142 mM sodium chloride, 3 mM potassium
chloride, 0.1% detergent Tween 20, 1.5% BSA, 20% heat inactivated
Newborn Calf Serum, 0.03% potassium ferricyanide, 100 ug/mL mouse
immunoglobulin G (monoclonal) and 0.1% 2-Chloroacetamide. The HRP
labeled Streptavidin conjugate was composed of HRP-Streptavidin
(Jackson ImmunoResearch, Cat #016-030-084) 1:8,000 diluted in CD-1
(Casein Blocker in PBS, Pierce Cat #37528, added Tween 20 to 0.05%
and EDTA to 2 mM).
[0062] Assay protocol was the same as described in Example 2. 100
uL specimen diluent and 100 uL specimen were added to each well.
Plates were then incubated at 37.degree. C. for 1 hour with shake,
followed by washing 5 times with PBS/Tween20. 200 .mu.L
antibody/antigen conjugate mixture was then added to each well. The
antibody/antigen conjugate incubation was performed at 37.degree.
C. for 60 minutes with shake. The plates were then washed 5 times,
200 .mu.L/well HRP-Streptavidin was subsequently added, and the
plates were incubated at room temperature for 30 minutes.
Afterwards the plates were washed 6 times, 200 .mu.L OPD/substrate
was added to each well, and the plates were incubated in the dark
at room temperature for 30 minutes. 50 .mu.L/well 4N
H.sub.2SO.sub.4 stop solution was added, and ODs were recorded at
493 nm using a plate spectrophotometry reader. Assay cut-off was
set at 0.300.
[0063] Assay results of HCV antigen/antibody combination ELISA on
BBI HCV seroconversion panels are shown in Table 4. Assay results
of HCV antigen/antibody combination ELISA on ZeptoMetrix HCV
seroconversion panels are shown in Table 5. It can be seen that at
an early infection stage, detection was negative in the antibody
detection assay, whereas detection was positive using the HCV RNA
assay, the HCV antigen assay, or the HCV antigen-antibody
combination assay. See, for example, Table 4, with "PHV907", days
4, 7 and 13 from first bleed. On the other hand, at a later stage
of infection (e.g., day 164 with "PHV907"), when detection of
antigen became negative and the quantity of HCV RNA was reduced,
anti-HCV antibodies were strongly detected in the antibody
detection assay, and the S/C ratio was also well above 1 in the
antigen-antibody combination assay. These results demonstrate that
the HCV antigen-antibody combination assay provided a wider window
of detection than an assay based on detection of either HCV antigen
or anti-HCV antibody alone.
TABLE-US-00001 TABLE 1 Detergent comparison in the indirect format
anti-HCV assay. ELISA assays and format ELISA-1 ELISA-2 ELISA-3
Indirect Indirect Indirect Detergent in specimen diluent Kit's
conjugate dilution Tween20 NLS LDAO (0.25%) (0.67%) (0.67%) 1:3 1:2
1:2 Negative plasma OD OD OD 0.001 0.001 0.002 RIBA3.0 0.002 0.002
0.001 HCV Specimens Dilution c33c c22p 0.002 S/C 0.001 S/C 0.001
S/C anti-c22 depleted specimens WHO Hu-a- 1:25 2+ - 2.122 3.5 0.745
1.2 1.773 2.9 c33c 1:50 2+ - 1.179 2.0 0.414 0.7 0.985 1.6 1:100 1+
- 0.911 1.5 0.297 0.5 0.707 1.2 1:200 + - 0.608 1.0 0.230 0.4 0.547
0.9 41530822 1:25 +/- - 1.235 2.1 0.306 0.5 1.094 1.8 1:50 +/- -
0.686 1.1 0.170 0.3 0.608 1.0 1:100 +/- - 0.441 0.7 0.089 0.1 0.212
0.4 41530832 1:100 2+ - 0.808 1.3 0.322 0.5 0.765 1.3 1:200 + -
0.648 1.1 0.230 0.4 0.547 0.9 1:400 +/- - 0.325 0.5 0.084 0.1 0.201
0.3 41530883 1:200 + - 0.985 1.6 0.203 0.3 0.590 1.0 1:400 +/- -
0.547 0.9 0.113 0.2 0.268 0.4 1:800 +/- - 0.276 0.5 0.053 0.1 0.127
0.2 anti-c33c depleted specimens WHO Hu-a-c22 1:25 - 4+ 1.346 2.2
1.146 1.9 1.678 2.8 1:50 - 4+ 1.165 1.9 0.895 1.5 1.439 2.4 1:100 -
2+ 0.772 1.3 0.420 0.7 0.675 1.1 I-20 1:100 - 3+ 0.896 1.5 0.538
0.9 0.865 1.4 1:200 - 2+ 0.410 0.7 0.230 0.4 0.370 0.6 360650 1:100
- 3+ 0.637 1.1 0.380 0.6 0.611 1.0 1:200 - 2+ 0.325 0.5 0.197 0.3
0.316 0.5 1:400 - + 0.171 0.3 0.108 0.2 0.173 0.3 cut-off ->
0.602 0.601 0.601
TABLE-US-00002 TABLE 2 Detergent comparison in the Ag sandwich
format anti-HCV assay. ELISA assays and format ELISA-4 ELISA-5
ELISA-6 Ag sandwich Ag sandwich Ag sandwich Detergent in specimen
diluent-> Tween20 NLS LDAO (0.25%) (1%) (1%) Negative plasma
Ag-biotin OD OD OD conjugate 0.044 0.012 0.013 rNS3(h)-b 0.013
0.008 0.010 at 60 ng/mL RIBA3.0 0.018 0.020 0.030 HCC5-b HCV
Specimens Dilution c33c c22p 0.036 S/C 0.017 S/C 0.027 S/C at 5
ng/mL anti-c22 depleted specimens WHO Hu-a-c33c 1:100 1+ - 2.997
>9 2.997 >9 2.997 >9 rNS3(h)-b 1:200 +/- - 2.997 >9
2.997 >9 2.997 >9 at 60 ng/mL 1:400 +/- - 1.567 4.8 1.922 6.4
1.446 4.8 1:800 - - 0.470 1.4 0.754 2.5 0.427 1.4 41530822 1:100
+/- - 0.560 1.9 1.720 5.7 0.580 1.9 1:200 - - 0.284 0.9 0.724 2.4
0.236 0.8 41530832 1:200 +/- - 2.997 >9 2.997 >9 2.997 >9
1:400 +/- - 1.418 4.7 2.538 8.5 1.984 6.6 1:800 +/- - 0.232 0.8
0.720 2.4 0.360 1.2 41530883 1:200 + - 2.800 9.3 2.997 >9 2.997
>9 1:400 +/- - 0.510 1.7 1.560 5.2 0.838 2.8 1:800 - - 0.168 0.6
0.612 2.0 0.294 1.0 Cut-off -> 0.329 0.310 0.312 anti-c33c
depleted specimens WHO Hu-a-c22 1:200 - 2+ 2.119 6.5 0.174 0.5
2.997 >9 HCC5-b 1:400 - 1+ 1.588 4.9 0.117 0.4 2.858 8.7 at 5
ng/mL 1:800 - +/- 1.122 3.4 0.066 0.2 1.909 5.8 I-20 1:200 - 2+
0.637 1.9 0.087 0.3 1.127 3.4 1:400 - 2+ 0.343 1.0 0.050 0.2 0.610
1.9 1:800 - +/- 0.185 0.6 0.046 0.1 0.310 0.9 360650 1:200 - 2+
1.659 5.1 0.137 0.4 2.086 6.4 1:400 - 2+ 0.739 2.3 0.075 0.2 1.156
3.5 1:800 - +/- 0.337 1.0 0.056 0.2 0.592 1.8 Cut-off -> 0.327
0.319 0.329
TABLE-US-00003 TABLE 3 Detergent comparison in HCV Core Ag assay.
ELISA assays ELISA-7 ELISA-8 ELISA-9 Detergent in SD NLS Tween20
LDAO (1.0%) (1.0%) (1.0%) Negative plasma OD OD OD 0.010 0.014
0.011 0.010 0.017 0.008 0.008 S/C 0.013 S/C 0.009 S/C Dilution Lot
16, a pool of neat 0.722 15 0.300 5.5 1.852 38 plasma 1:1.5 1.075
22 0.189 3.5 1.220 25 specimens that 1:2.0 1.115 23 0.149 2.7 0.948
19 were all 1:3.0 0.802 16 0.103 1.9 0.621 13 characterized 1:4.0
0.602 12 0.074 1.3 0.431 8.8 as anti-HCV (-), 1:5.0 0.490 9.9 0.064
1.2 0.335 6.8 HCV RNA (+) 1:6.0 0.379 7.7 0.051 0.9 0.279 5.7 and
HCV core 1:8.0 0.271 5.5 0.041 0.7 0.199 4.1 Ag (+) 1:10 0.214 4.3
0.040 0.7 0.148 3.0 1:16 0.155 3.1 0.026 0.5 0.104 1.0 X million
IU/ml Dilution (copies/ml) 9160834 @ 1:5 7.28 (37.8) 1.390 28 0.178
3.2 1.121 23 36.4 .times. 10.sup.6 IU/ml 1:10 3.64 (18.9) 0.734 15
0.086 1.6 0.527 11 or 189 .times. 10.sup.6 1:20 1.82 (9.46) 0.364
7.4 0.047 0.9 0.251 5.1 copies/ml 1:30 1.21 (6.31) 0.253 5.1 0.036
0.6 0.157 3.0 1:40 0.91 (4.73) 0.181 3.7 0.028 0.5 0.116 2.4
cut-off -> 0.049 0.055 0.049
TABLE-US-00004 TABLE 4 HCV antigen/antibody combination assay on
BBI HCV seroconversion panels anti-HCV Seroconversion Days HCV HCV
Ag HCV Ag/Ab Panels from 1st RIBA 3.0 3.0 HCV RNA (HCV core)
combination ID Bleed bleed c22p c33c S/C testing kit quantity Pos
or assay From Panel Data Sheet (Neg) OD S/C PHV907 1 0 - - 0.0 HCV
RNA >5 .times. 10.sup.5 pos 0.311 1.04 genotype 2 4 - - 0.0
Roche >5 .times. 10.sup.5 pos 0.317 1.06 1b 3 7 - - 0.0 Amplicor
>5 .times. 10.sup.5 pos 0.371 1.24 4 13 1+ - 0.1 PCR (BBI) >5
.times. 10.sup.5 pos 0.437 1.46 5 18 4+ +/- 0.4 copies/ml >5
.times. 10.sup.5 pos 0.877 2.92 6 21 4+ 1+ 1.0 >5 .times.
10.sup.5 pos 2.442 8.14 7 164 4+ 4+ 4.4 4 .times. 10.sup.4 neg
>3 >10 PHV909 s1 0 - - 0.0 1 .times. 10.sup.4 pos 0.305 1.02
genotype 3 2 28 1+ - 1.3 4 .times. 10.sup.4 pos 0.701 2.34 3 30 2+
- 1.3 2 .times. 10.sup.4 pos 0.960 3.20 PHV910 1 0 - - 0.0 >5
.times. 10.sup.5 pos 0.599 2.00 genotype 2 4 - - 0.0 >5 .times.
10.sup.5 pos 0.424 1.41 1b 3 8 3+ 1+ 3.0 >5 .times. 10.sup.5 pos
1.497 4.99 4 11 4+ 3+ 6.7 >5 .times. 10.sup.5 pos >3 >10 5
15 4+ 4+ 8.0 >5 .times. 10.sup.5 pos >3 >10 PHV913 1 0 - -
0.0 >5 .times. 10.sup.5 pos 0.310 1.03 genotype 2 2 - - 0.1
>5 .times. 10.sup.5 pos 0.330 1.10 2b 3 7 2+ - 1.5 >5 .times.
10.sup.5 pos 0.867 2.89 4 9 2+ +/- 1.7 >5 .times. 10.sup.5 pos
1.256 4.19 PHV914 1 0 - - 0.0 >5 .times. 10.sup.5 pos 0.292 0.97
genotype 2 5 - - 0.0 >5 .times. 10.sup.5 pos 0.366 1.22 2b 3 9 -
- 0.0 >5 .times. 10.sup.5 pos 0.307 1.02 4 12 - - 0.1 >5
.times. 10.sup.5 pos 0.325 1.08 5 16 2+ - 1.2 >5 .times.
10.sup.5 pos 0.310 1.03 6 19 2+ - 2.1 >5 .times. 10.sup.5 pos
0.323 1.08 7 24 4+ +/- 4.5 >5 .times. 10.sup.5 pos 0.392 1.31 8
30 4+ 3+ 6.8 >5 .times. 10.sup.5 pos 1.452 4.84 9 33 4+ 3+ 7.6
>5 .times. 10.sup.5 pos 2.052 6.84 PHV916 1 0 - - 0.0 3 .times.
10.sup.5 pos 0.127 0.42 genotype 2 2 - - 0.0 >5 .times. 10.sup.5
pos 0.297 0.99 2b 3 7 - - 0.0 >5 .times. 10.sup.5 pos 0.375 1.25
4 9 - - 0.0 >5 .times. 10.sup.5 pos 0.483 1.61 5 16 - +/- 0.3
>5 .times. 10.sup.5 pos 0.314 1.05 6 19 - +/- 1.1 >5 .times.
10.sup.5 pos 0.607 2.02 7 23 - 3+ 2.7 4 .times. 10.sup.5 pos 0.861
2.87 8 28 - 3+ 3.7 2 .times. 10.sup.5 neg 1.051 3.50 PHV918 1 0 - -
0.0 HCV RNA >8 .times. 10.sup.5 pos 0.272 0.91 genotype 2 2 - -
0.0 Roche >8 .times. 10.sup.5 pos 0.544 1.81 1a 3 7 - - 0.0
COBAS >8 .times. 10.sup.5 pos 0.393 1.31 4 9 - - 0.0 Amplicor
>8 .times. 10.sup.5 pos 0.660 2.20 5 14 - - 0.0 PCR (BBI) >8
.times. 10.sup.5 pos 0.441 1.47 6 16 +/- - 0.0 IU/ml >8 .times.
10.sup.5 pos 0.998 3.33 7 24 3+ - 0.6 >8 .times. 10.sup.5 pos
0.368 1.23 8 27 3+ 1+ 0.8 >8 .times. 10.sup.5 pos 0.627 2.09
PHV920 1 0 - - 0.0 >8 .times. 10.sup.5 pos 0.107 0.36 genotype 2
5 - - 0.0 >8 .times. 10.sup.5 pos 0.891 2.97 3 7 - - 0.0 >8
.times. 10.sup.5 pos 0.304 1.01 4 13 +/- 1+ 0.5 >8 .times.
10.sup.5 pos 2.292 7.64 5 16 1+ 3+ 3.1 >8 .times. 10.sup.5 pos
2.840 9.47 6 20 1+ 3+ 3.6 3 .times. 10.sup.4 neg 1.911 6.37 7 26 1+
3+ >5 8 .times. 10.sup.4 neg 1.637 5.46 8 28 2+ 4+ >5 2
.times. 10.sup.3 neg 1.533 5.11 9 33 2+ 4+ >5 BLD neg 2.374 7.91
negative plasma control-1 0.016 0.05 negative plasma control-2
0.019 0.06 negative plasma control-3 0.011 0.04
TABLE-US-00005 TABLE 5 HCV antigen/antibody combination assay on
ZeptoMetrix HCV seroconversion panels Seroconversion Days anti-HCV
HCV Ag HCV Ag/Ab Panels from 1st RIBA 3.0 HCV 3.0 HCV RNA (HCV
core) combination ID Bleed bleed c22p c33c S/C testing kit quantity
Pos or assay From Panel Data Sheet (Neg) OD S/C 6212 1 0 - - 0.00
CHIRON 1.88 .times. 10.sup.6 pos 0.086 0.29 2 12 - +/- 0.15 HCV RNA
2.21 .times. 10.sup.5 neg 1.965 6.55 3 14 - +/- 0.30 Copies/mL
<200,000 neg 2.503 8.34 4 23 - 1+ 1.49 2.27 .times. 10.sup.5 neg
>3 >10 5 26 - 1+ 1.87 2.03 .times. 10.sup.5 neg >3 >10
6 32 - 1+ 2.37 <200,000 neg >3 >10 7 37 - 1+ 2.46 3.91
.times. 10.sup.5 neg >3 >10 8 53 - 4+ 4.13 4.66 .times.
10.sup.5 neg >3 >10 9 55 - 4+ 4.13 3.57 .times. 10.sup.5 neg
>3 >10 6215 1 0 - - 0.00 CHIRON >120 .times. 10.sup.6 pos
0.898 2.99 2 3 - - 0.01 HCV RNA 104 .times. 10.sup.6 pos 0.952 3.17
3 10 - - 0.02 (bDNA) 58 .times. 10.sup.6 pos 0.456 1.52 4 20 4+ +/-
4.66 (copies/ml) 48 .times. 10.sup.6 pos 2.321 7.74 6225 10 35 - -
0.00 CHIRON <0.2 neg 0.034 0.11 11 39 - - 0.00 bDNA <0.2 neg
0.021 0.07 12 45 - - 0.00 (MEq/mL) 27.02 pos 0.266 0.89 13 47 - -
0.00 98.84 pos 0.678 2.26 14 52 - - 0.00 77.79 pos 0.627 2.09 15 56
- - 0.01 54.71 pos 0.545 1.82 16 60 - - 0.00 >120 pos 1.027 3.42
17 72 - - 0.07 9.37 pos 0.635 2.12 18 77 - 1+ 1.73 4.97 pos 1.380
4.60 19 79 - 1+ 2.11 3.24 pos 1.400 4.67 6229 1 0 - - 0.01 52.19
pos 0.490 1.63 2 3 - - 0.01 55.83 pos 0.528 1.76 3 7 - - 0.01 55.53
pos 0.452 1.51 4 10 - - 0.01 94.76 pos 0.823 2.74 5 18 - - 0.53
31.88 pos 1.346 4.49 6 21 - +/- 1.06 69.85 pos 1.636 5.45 7 25 +/-
1+ 1.71 51.02 pos 1.594 5.31 8 29 +/- 3+ 4.07 43.89 pos 2.590 8.63
9041 1 0 - - 0.00 CHIRON <0.2 neg 0.044 0.15 2 24 - - 0.01 HCV
RNA 15.78 pos 0.125 0.42 3 27 - - 0.01 bDNA 2.0 63.72 pos 0.603
2.01 4 31 - - 0.01 (MEq/mL), >120 pos 1.106 3.69 5 62 +/- 4+
7.30 * one Mega 72.27 pos 2.924 9.75 6 64 +/- 4+ 8.03 Equivalent/
66.27 pos >3 >10 7 69 1+ 4+ 8.20 mL 21.80 pos >3 >10 8
71 3+ 4+ 8.78 (MEq/mL) 11.24 pos >3 >10 9044 1 0 - - 0.00 is
.apprxeq. 1 105.90 pos 0.652 2.17 2 4 - - 0.01 million 52.71 pos
0.496 1.65 3 17 - - 0.00 equivalents 77.18 pos 0.722 2.41 4 21 -
+/- 0.69 of HCV 71.52 pos 1.089 3.63 5 25 - 2+ 3.70 RNA. 72.82 pos
1.147 3.82 6 29 - 3+ 4.51 48.83 pos 0.855 2.85 9045 1 0 - - 0.00
17.73 pos 0.361 1.20 2 2 - - 0.00 16.42 pos 0.321 1.07 3 7 - - 0.00
36.19 pos 0.395 1.32 4 9 - - 0.01 38.07 pos 0.557 1.86 5 26 - -
0.00 44.86 pos 0.389 1.30 6 32 - - 0.04 17.78 pos 0.302 1.01 7 37 -
1+ 2.61 18.36 pos 1.314 4.38 8 41 - 2+ 3.51 2.32 pos 1.043 3.48
9047 1 0 - - 0.10 65.95 pos 0.474 1.58 2 2 - - 0.12 81.76 pos 0.517
1.72 3 10 - - 0.09 54.85 pos 0.368 1.23 4 12 - - 0.05 64.03 pos
0.501 1.67 5 19 - - 0.10 55.45 pos 0.346 1.15 6 21 - - 0.09 42.97
pos 0.334 1.11 7 28 - 2+ 1.51 9.13 pos 2.921 9.74 8 30 - 4+ 3.92
26.58 pos 2.992 9.97 9 35 - 4+ 7.36 22.77 pos >3 >10 10 37
+/- 4+ 6.77 nd pos >3 >10 9054 7 52 - - 0.00 <0.2 neg
0.040 0.13 8 74 +/- - 0.00 86.79 pos 0.555 1.85 9 77 1+ +/- 0.14
86.75 pos 0.532 1.77 10 82 2+ 1+ 3.16 59.14 pos >3 >10 9058 1
0 +/- - 0.07 90.58 pos 0.762 2.54 2 3 +/- - 0.08 85.34 pos 0.764
2.55 3 7 + +/- 0.15 37.64 pos 0.487 1.62 4 10 1+ 1+ 0.66 48.33 pos
0.930 3.10 5 14 2+ 1+ 3.16 29.46 pos 1.209 4.03 negative plasma
control-1 0.016 0.05 negative plasma control-2 0.019 0.06 negative
plasma control-3 0.011 0.04
Sequence CWU 1
1
3138PRTArtificialSynthetic HCV antigenic peptide "HCC5N" 1Lys Thr
Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro1 5 10 15Gly
Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly 20 25
30Pro Arg Leu Gly Xaa Cys 35238PRTArtificialSynthetic HCV antigenic
peptide "HCC5-b" 2Lys Thr Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp
Val Lys Phe Pro1 5 10 15Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu
Leu Pro Arg Arg Gly 20 25 30Pro Arg Leu Gly Gly Xaa
353462PRTArtificialRecombinant fusion protein between 6xHis tag and
HCV rNS3(h) 3Met Arg Gly Ser His His His His His His Gly Ser Pro
Val Phe Thr1 5 10 15Asp Asn Ser Ser Pro Pro Val Val Pro Gln Ser Phe
Gln Val Ala His 20 25 30Leu His Ala Pro Thr Gly Ser Gly Lys Ser Thr
Lys Val Pro Ala Ala 35 40 45Tyr Ala Ala Gln Gly Tyr Lys Val Leu Val
Leu Asn Pro Ser Val Ala 50 55 60Ala Thr Leu Gly Phe Gly Ala Tyr Met
Ser Lys Ala His Gly Val Asp65 70 75 80Pro Asn Ile Arg Thr Gly Val
Arg Thr Ile Thr Thr Gly Ser Pro Ile 85 90 95Thr Tyr Ser Thr Tyr Gly
Lys Phe Leu Ala Asp Gly Gly Cys Ser Gly 100 105 110Gly Ala Tyr Asp
Ile Ile Ile Cys Asp Glu Cys His Ser Thr Asp Ala 115 120 125Thr Ser
Ile Leu Gly Ile Gly Thr Val Leu Asp Gln Ala Glu Thr Ala 130 135
140Gly Ala Arg Leu Val Val Leu Ala Thr Ala Thr Pro Pro Gly Ser
Val145 150 155 160Thr Val Pro His Pro Asn Ile Glu Glu Val Ala Leu
Ser Thr Thr Gly 165 170 175Glu Ile Pro Phe Tyr Gly Lys Ala Ile Pro
Leu Glu Ala Ile Lys Gly 180 185 190Gly Arg His Leu Ile Phe Cys His
Ser Lys Lys Lys Cys Asp Glu Leu 195 200 205Ala Thr Lys Leu Val Ala
Leu Gly Ile Asn Ala Val Ala Tyr Tyr Arg 210 215 220Gly Leu Asp Val
Ser Val Ile Pro Ser Ser Gly Asp Val Val Val Val225 230 235 240Ala
Thr Asp Ala Leu Met Thr Gly Phe Thr Gly Asp Phe Asp Ser Val 245 250
255Ile Asp Cys Asn Thr Cys Val Thr Gln Thr Val Asp Phe Ser Leu Asp
260 265 270Pro Thr Phe Thr Ile Glu Thr Thr Thr Leu Pro Gln Asp Ala
Val Ser 275 280 285Arg Thr Gln Arg Arg Gly Arg Thr Gly Arg Gly Lys
Pro Gly Ile Tyr 290 295 300Arg Phe Val Ala Pro Gly Glu Arg Pro Ser
Gly Met Phe Asp Ser Ser305 310 315 320Val Leu Cys Glu Cys Tyr Asp
Ala Gly Cys Ala Trp Tyr Glu Leu Thr 325 330 335Pro Ala Glu Thr Thr
Val Arg Leu Arg Ala Tyr Met Asn Thr Pro Gly 340 345 350Leu Pro Val
Cys Gln Asp His Leu Glu Phe Trp Glu Gly Val Phe Thr 355 360 365Gly
Leu Thr His Ile Asp Ala His Phe Leu Ser Gln Thr Lys Gln Ser 370 375
380Gly Glu Asn Phe Pro Tyr Leu Val Ala Tyr Gln Ala Thr Val Cys
Ala385 390 395 400Arg Ala Gln Ala Pro Pro Pro Ser Trp Asp Gln Met
Trp Lys Cys Leu 405 410 415Ile Arg Leu Lys Pro Thr Leu His Gly Pro
Thr Pro Leu Leu Tyr Arg 420 425 430Leu Gly Ala Val Gln Asn Glu Ile
Thr Leu Thr His Pro Ile Thr Lys 435 440 445Tyr Ile Met Thr Cys Met
Ser Ala Asp Leu Glu Val Val Thr 450 455 460
* * * * *