U.S. patent application number 13/478377 was filed with the patent office on 2012-10-25 for synthetic antigens for the detection of antibodies to hepatitis c virus.
This patent application is currently assigned to INNOGENETICS, S.A.. Invention is credited to Robert J. DELEYS, Geert Maertens, Dirk Pollet, Hugo Van Heuverswjn.
Application Number | 20120270208 13/478377 |
Document ID | / |
Family ID | 46252886 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120270208 |
Kind Code |
A1 |
DELEYS; Robert J. ; et
al. |
October 25, 2012 |
SYNTHETIC ANTIGENS FOR THE DETECTION OF ANTIBODIES TO HEPATITIS C
VIRUS
Abstract
Peptide sequences are provided which are capable of mimicking
proteins encoded by HCV for use as reagents for screening of blood
and blood products for prior exposure to HCV. The peptides are at
least 5 amino acids long and can be used in various specific assays
for the detection of antibodies to HCV, for the detection of HCV
antigens, or as immunogens.
Inventors: |
DELEYS; Robert J.;
(Grimbergen, BE) ; Pollet; Dirk; (Schilde, BE)
; Maertens; Geert; (Brugge, BE) ; Van Heuverswjn;
Hugo; (Laarne, BE) |
Assignee: |
INNOGENETICS, S.A.
Ghent
BE
|
Family ID: |
46252886 |
Appl. No.: |
13/478377 |
Filed: |
May 23, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10822871 |
Apr 13, 2004 |
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13478377 |
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10044995 |
Jan 15, 2002 |
6872520 |
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10822871 |
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09941611 |
Aug 30, 2001 |
6576417 |
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10044995 |
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09275265 |
Mar 23, 1999 |
6287761 |
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09941611 |
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08391671 |
Feb 21, 1995 |
5922532 |
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09275265 |
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07920286 |
Oct 14, 1992 |
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08391671 |
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Current U.S.
Class: |
435/5 |
Current CPC
Class: |
Y10S 530/826 20130101;
A61K 39/00 20130101; C12N 2770/24222 20130101; C07K 14/005
20130101; Y10S 436/82 20130101 |
Class at
Publication: |
435/5 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 1990 |
EP |
90124241.2 |
Claims
1. A combination of distinct molecules, consisting essentially of
(a) a first molecule which is capable of providing for
immunological competition with at least one strain of HCV,
comprising an amino acid sequence selected from the group
consisting of at least 5 amino acids of an HCV polyprotein located
in the region corresponding to amino acids 1-20 of SEQ ID NO:23, at
least 5 amino acids located in the region corresponding to amino
acids 7-26 of SEQ ID NO:23, at least 5 amino acids located in the
region corresponding to amino acids 8-18 of SEQ ID NO:23, at least
5 amino acids located in the region corresponding to amino acids
13-32 of SEQ ID NO:23, at least 5 amino acids located in the region
corresponding to amino acids 37-56 of SEQ ID NO:23, at least 5
amino acids located in the region corresponding to amino acids
49-68 of SEQ ID NO:23, at least 5 amino acids located in the region
corresponding to amino acids 61-80 of SEQ ID NO:23, at least 5
amino acids located in the region corresponding to amino acids
73-92 of SEQ ID NO:23, at least 5 amino acids of SEQ ID NO: 1, at
least 5 amino acids of SEQ ID NO: 2, at least 5 amino acids of SEQ
ID NO: 3, at least 5 amino acids of SEQ ID NO: 4, at least 5 amino
acids of SEQ ID NO: 5, at least 5 amino acids of SEQ ID NO: 6, at
least 5 amino acids of SEQ ID NO: 7, and at least 5 amino acids of
SEQ ID NO: 8; and (b) a second molecule which is capable of
providing for immunological competition with at least one strain of
HCV, comprising an amino acid sequence selected from the group
consisting of at least 5 amino acids located in the region
corresponding to amino acids 1688-1707 of SEQ ID NO:23, at least 5
amino acids located in the region corresponding to amino acids
1694-1713 of SEQ ID NO:23, at least 5 amino acids located in the
region corresponding to amino acids 1706-1725 of SEQ ID NO:23, at
least 5 amino acids located in the region corresponding to amino
acids 1712-1731 of SEQ ID NO:23, at least 5 amino acids located in
the region corresponding to amino acids 1718-1737 of SEQ ID NO:23,
at least 5 amino acids located in the region corresponding to amino
acids 1724-1743 of SEQ ID NO:23, at least 5 amino acids located in
the region corresponding to amino acids 1730-1749 of SEQ ID NO:23,
at least 5 amino acids of SEQ ID NO: 9, at least 5 amino acids of
SEQ ID NO: 10, at least 5 amino acids of SEQ ID NO: 11, at least 5
amino acids of SEQ ID NO: 12, at least 5 amino acids of SEQ ID NO:
13, at least 5 amino acids of SEQ ID NO: 14 and at least 5 amino
acids of SEQ ID NO: 15, and (c) a third molecule which is capable
of providing for immunological competition with at least one strain
of HCV, comprising an amino acid sequence selected from the group
consisting of at least 5 amino acids located in the region
corresponding to amino acids 2263-2282 of SEQ ID NO:23, at least 5
amino acids located in the region corresponding to amino acids
2275-2294 of SEQ ID NO:23, at least 5 amino acids located in the
region corresponding to amino acids 2287-2306 of SEQ ID NO:23, at
least 5 amino acids located in the region corresponding to amino
acids 2299-2318 of SEQ ID NO:23, at lease 5 amino acids located in
the region corresponding to amino acids 2311-2330 of SEQ ID NO:23,
at least 5 amino acids of SEQ ID NO: 16, at least 5 amino acids of
SEQ ID NO: 17, at least 5 amino acids of SEQ ID NO: 18, at least 5
amino acids of SEQ ID NO: 19, and at least 5 amino acids of SEQ ID
NO: 20, wherein said molecules of (a), (b) and (c) are each
separate molecules from one another, wherein said molecules of (a),
(b) and (c) are each different from one another and wherein said
molecules are selected from the group consisting of peptides and
polypeptides, and said combination further including at least one
molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ
ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11,
SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID
NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20,
a peptide consisting of at least 5 amino acids of the HCV
polyprotein of an HCV isolate which is capable of providing for
immunological competition with at least one strain of HCV located
in the region corresponding to amino acids 7 to 26 of SEQ ID NO:23,
a peptide consisting of at least 5 amino acids of the HCV
polyprotein of an HCV isolate which is capable of providing for
immunological competition with at least one strain of HCV located
in the region corresponding to amino acids 13 to 32 of SEQ ID
NO:23, a peptide of the HCV polyprotein of an HCV isolate which is
capable of providing for immunological competition with at least
one strain of HCV corresponding to the region represented by amino
acids 37 to 56 of SEQ ID NO:23, a peptide consisting of at least 5
amino acids of the HCV polyprotein of an HCV isolate which is
capable of providing for immunological competition with at least
one strain of HCV located in the region corresponding to amino
acids 49 to 68 of SEQ ID NO:23, a peptide of the HCV polyprotein of
an HCV isolate which is capable of providing for immunological
competition with at least one strain of HCV corresponding to the
region represented by amino acids 61 to 80 of SEQ ID NO:23, a
peptide of the HCV polyprotein of an HCV isolate which is capable
of providing for immunological competition with at least one strain
of HCV corresponding to the region represented by amino acids 73 to
92 of SEQ ID NO:23, a peptide consisting of at least 5 amino acids
of the HCV polyprotein of an HCV isolate which is capable of
providing for immunological competition with at least one strain of
HCV located in the region corresponding to amino acids 1688 to 1707
of SEQ ID NO:23, a peptide consisting of at least 5 amino acids of
the HCV polyprotein of an HCV isolate which is capable of providing
for immunological competition with at least one strain of HCV
located in the region corresponding to amino acids 1694 to 1713 of
SEQ ID NO:23, a peptide consisting of at least 5 amino acids of the
HCV polyprotein of an HCV isolate which is capable of providing for
immunological competition with at least one strain of HCV located
in the region corresponding to amino acids 1706 to 1725 of SEQ ID
NO:23, a peptide consisting of at least 5 amino acids located of
the HCV polyprotein of an HCV isolate which is capable of providing
for immunological competition with at least one strain of HCV in
the region corresponding to amino acids 1712 to 1731 of SEQ ID
NO:23, a peptide consisting of at least 5 to at most 12 amino acids
of the HCV polyprotein of an HCV isolate which is capable of
providing for immunological competition with at least one strain of
HCV located in the region corresponding to amino acids 1718 to 1737
of SEQ ID NO:23, a peptide consisting of at least 5 amino acids of
the HCV polyprotein of an HCV isolate which is capable of providing
for immunological competition with at least one strain of HCV
located in the region corresponding to amino acids 1724 to 1743 of
SEQ ID NO:23, a peptide consisting of at least 5 to at most 12
amino acids of the HCV polyprotein of an HCV isolate which is
capable of providing for immunological competition with at least
one strain of HCV located in the region corresponding to amino
acids 1730 to 1749 of SEQ ID NO:23, a peptide consisting of at
least 5 amino acids of the HCV polyprotein of an HCV isolate which
is capable of providing for immunological competition with at least
one strain of HCV located in the region corresponding to amino
acids 2287 to 2306 of SEQ ID NO:23, a peptide consisting of at
least 5 amino acids of the HCV polyprotein of an HCV isolate which
is capable of providing for immunological competition with at least
one strain of HCV located in the region corresponding to amino
acids 2299 to 2318 of SEQ ID NO:23, a peptide consisting of at
least 5 amino acids of the HCV polyprotein of an HCV isolate which
is capable of providing for immunological competition with at least
one strain of HCV located in the region corresponding to amino
acids 2311 to 2330 of SEQ ID NO:23, a peptide consisting of at
least 5 amino acids of SEQ ID NO:2, a peptide consisting of at
least 5 amino acids of SEQ ID NO:4, a peptide consisting of at
least 5, 6, 8, 12 or 20 amino acids of amino acids of the HCV
polyprotein of an HCV isolate which is capable of providing for
immunological competition with at least one strain of HCV
corresponding to the region represented by 37 to 56 of SEQ ID
NO:23, a peptide consisting of at least 5 amino acids of SEQ ID
NO:6, a peptide consisting of at least 5, 6, 8, 12 or 20 amino
acids of the HCV polyprotein of an HCV isolate which is capable of
providing for immunological competition with at least one strain of
HCV corresponding to the region represented by amino acids 61 to 80
of SEQ ID NO:23, a peptide consisting of at least 5, 6, 8, 12 or 20
amino acids of amino acids of the HCV polyprotein of an HCV isolate
which is capable of providing for immunological competition with at
least one strain of HCV corresponding to the region represented by
73 to 92 of SEQ ID NO:23, a peptide consisting of at least 5 amino
acids of SEQ ID NO:9, a peptide consisting of at least 5 amino
acids of SEQ ID NO:10, a peptide consisting of at least 5 amino
acids of SEQ ID NO:11, a peptide consisting of at least 5 amino
acids of SEQ ID NO:12, a peptide consisting of at least 5 to at
most 12 amino acids of SEQ ID NO:13, a peptide consisting of at
least 5 amino acids of SEQ ID NO:14, a peptide consisting of at
least 5 to at most 12 amino acids of SEQ ID NO:15, a peptide
consisting of at least 5 amino acids of SEQ ID NO: 18, a peptide
consisting of at least 5 amino acids of SEQ ID NO: 19, and a
peptide consisting of at least 5 amino acids of SEQ ID NO: 20.
2. A combination according to claim 1, wherein said molecules
comprising at least 5 amino acids are individually produced by
recombinant expression or chemical synthesis.
3. A combination according to claim 1, wherein one or more of said
first, second, or third molecules comprises a fusion
polypeptide.
4. A combination according to claim 1, wherein the combination is
packaged into a kit further comprising control reagents for
detecting antibodies to hepatitis C virus (HCV) in a human body
component suspected of containing said antibodies.
5. A combination of claim 1, wherein at least one of said molecules
comprises synthetic peptides or polypeptides.
6. A combination of claim 5, wherein at least two of said molecules
comprises synthetic peptides or polypeptides.
7. A combination of claim 6, wherein at least three of said
molecules comprises synthetic peptides or polypeptides.
8. A combination of molecules according to claim 1, wherein at
least one of said first, second and third molecules is selected
from the group consisting of: a first molecule consisting of amino
acids 1-92 of SEQ ID NO: 23 or consisting of amino acids of an HCV
polyprotein corresponding to the region represented by amino acids
1-92 of SEQ ID NO:23; a second molecule consisting of amino acids
1688-1749 of SEQ ID NO: 23 or consisting of amino acids of an HCV
polyprotein corresponding to the region represented by amino acids
1688-1749 of SEQ ID NO: 23; and a third molecule consisting of
amino acids only 2263-2330 SEQ ID NO: 23 or consisting of amino
acids of an HCV polyprotein corresponding to the region represented
by amino acids 2263-2330 of SEQ ID NO: 23.
9. A combination of molecules according to claim 1, wherein at
least two of said first, second and third molecules is selected
from the group consisting of: a first molecule consisting of amino
acids 1-92 of SEQ ID NO: 23 or consisting of amino acids of an HCV
polyprotein corresponding to the region represented by amino acids
1-92 of SEQ ID NO: 23; a second molecule consisting of amino acids
1688-1749 of SEQ ID NO: 23 or consisting of amino acids of an HCV
polyprotein corresponding to the region represented by amino acids
1688-1749 of SEQ ID NO: 23; and a third molecule consisting of
amino acids 2263-2330 of SEQ ID NO: 23 or consisting of amino acids
of an HCV polyprotein corresponding to the region represented by
amino acids 2263-2330 of SEQ ID NO: 23.
10. A combination of molecules according to claim 1, wherein: said
first molecule consisting of amino acids 1-92 of SEQ ID NO: 23 or
consisting of amino acids of an HCV polyprotein corresponding to
the region represented by amino acids 1-92 of SEQ ID NO: 23; said
second molecule consisting of amino acids 1688-1749 of SEQ ID NO:
23 or consisting of amino acids of an HCV polyprotein corresponding
to the region represented by amino acids 1688-1749 of SEQ ID NO:
23; and said third molecule consisting of amino acids 2263-2330 of
SEQ ID NO: 23 or consisting of amino acids of an HCV polyprotein
corresponding to the region represented by amino acids 2263-2330 of
SEQ ID NO: 23.
11. A method for making a kit for detecting human antibodies that
bind to HCV amino acids, comprising combining the combination of
claim 8 with a support suitable for detecting whether antibodies
bind to the molecules of said combination.
12. A method for making a kit for detecting human antibodies that
bind to HCV amino acids, comprising combining the combination of
claim 9 with a support suitable for detecting whether antibodies
bind to the molecules of said combination.
13. A method for making a kit for detecting human antibodies that
bind to HCV amino acids, comprising combining the combination of
claim 10 with a support suitable for detecting whether antibodies
bind to the molecules of said combination.
14. A combination of claim 1, wherein said at least 5 amino acids
of said first molecule are selected from the amino acids of SEQ ID
NO: 2, of SEQ ID NO: 4, of SEQ ID NO: 6, or from the amino acids
located in the region of an HCV polyprotein corresponding to amino
acids 7-26 of SEQ ID NO:23, located in the region of an HCV
polyprotein corresponding to amino acids 13-32 of SEQ ID NO:23, or
located in the region of an HCV polyprotein corresponding to amino
acids 49-68 of SEQ ID NO:23; said at least 5 amino acids of said
second molecule are selected from the amino acids or SEQ ID NO:10,
or from the amino acids located in the region of an HCV polyprotein
corresponding to amino acids 1694-1713 of SEQ ID NO:23; and wherein
said at least 5 amino acids of said third molecule are selected
from amino acids of SEQ ID NO:19, or from the amino acids located
in the region of an HCV polyprotein corresponding to amino acids
2299-2318 of SEQ ID NO:23.
15. A combination of claim 1, wherein said at least 5 amino acids
of said first molecule are selected from the amino acids of SEQ ID
NO: 1, of SEQ ID NO: 2, of SEQ ID NO: 6, or from the amino acids
located in the region of an HCV polyprotein corresponding to amino
acids 1-20 SEQ ID NO:23, located in the region of an HCV
polyprotein corresponding to amino acids 7-26 of SEQ ID NO:23, or
located in the region of an HCV polyprotein corresponding to amino
acids 49-68 of SEQ ID NO:23; said at least 5 amino acids of said
second molecule are selected from the amino acids of SEQ ID NO: 10,
of SEQ ID NO: 12, or from the amino acids located in the region of
an HCV polyprotein corresponding to amino acids 1694-1713 of SEQ ID
NO:23, or located in the region of an HCV polyprotein corresponding
to amino acids 1712-1731 of SEQ ID NO:23; and wherein said at least
5 amino acids of said third molecule are selected from the amino
acids of SEQ ID NO: 17, of SEQ ID NO: 19, or from the amino acids
located in the region of an HCV polyprotein corresponding to amino
acids 2275-2294 of SEQ ID NO:23, or located in the region of an HCV
polyprotein corresponding to amino acids 2299-2318 of SEQ ID
NO:23.
16. A combination of claim 1, wherein said at least 5 amino acids
of said first molecule are selected from the amino acids of SEQ ID
NO: 2, of SEQ ID NO: 4, of SEQ ID NO: 5, of SEQ ID NO: 6, or from
the amino acids located in the region of an HCV polyprotein
corresponding to amino acids 7-26 of SEQ ID NO:23, located in the
region of an HCV polyprotein corresponding to amino acids 13-32 of
SEQ ID NO:23, located in the region of an HCV polyprotein
corresponding to amino acids 37-56 of SEQ ID NO:23, or located in
the region of an HCV polyprotein corresponding to amino acids 49-68
of SEQ ID NO:23; said at least 5 amino acids of said second
molecule are selected from the amino acids of SEQ ID NO: 9, of SEQ
ID NO: 12, or from the amino acids located in the region of an HCV
polyprotein corresponding to amino acids 1688-1707 of SEQ ID NO:23,
or located in the region of an HCV polyprotein corresponding to
amino acids 1712-1731 of SEQ ID NO:23; and wherein said at least 5
amino acids of said third molecule are selected from the amino
acids of SEQ ID NO: 17, of SEQ ID NO: 19, or from the amino acids
located in the region of an HCV polyprotein corresponding to amino
acids 2275-2294 of SEQ ID NO:23, or located in the region of amino
acids 2299-2318 of SEQ ID NO:23.
17. A combination of claim 14, wherein said at least 5 amino acids
of said first molecule are selected from the amino acids of SEQ ID
NO: 2, or from the amino acids located in the region of an HCV
polyprotein corresponding to amino acids 7-26 of SEQ ID NO:23.
18. A combination of claim 1, wherein said combination coats a well
of an immunoassay plate.
19. A combination of claim 1, wherein said combination coats a well
of an immunoassay plate.
20. A combination of claim 14, wherein said combination coats a
well of an immunoassay plate.
21. A combination of claim 15, wherein said combination coats a
well of an immunoassay plate.
22. A combination of claim 16, wherein said combination coats a
well of an immunoassay plate.
23. A combination of claim 1, wherein said combination is bound to
an immunoassay strip.
24. A combination of claim 14, wherein said combination is bound to
an immunoassay strip.
25. A combination of claim 15, wherein said combination is bound to
an immunoassay strip.
26. A combination of claim 16, wherein said combination is bound to
an immunoassay strip.
Description
[0001] The implementation of systematic testing for hepatitis B
virus (HBV) has been instrumental in eliminating this virus from
the blood supply. Nevertheless, a significant number of
post-transfusion hepatitis (PTH) cases still occur. These cases are
generally attributable to non-A, non-B to hepatitis (NANBH)
virus(es), the diagnosis of which is usually made by exclusion of
other viral markers.
[0002] The etiological agent responsible for a large proportion of
these cases has recently been cloned (Choo, Q-L et al. Science
(1988) 244:359-362) and a first-generation antibody test developed
(Kuo, G. et al. Science (1989) 244:362-364). The agent has been
identified as a positive-stranded RNA virus, and the sequence of
its genome has been partially determined. Studies suggest that this
virus, referred to subsequently as hepatitis C virus (HCV), may be
related to flaviviruses and pestiviruses. A portion of the genome
of an HCV isolated from a chimpanzee (HCV.sub.CDC/CHI) is disclosed
in EPO 88310922.5. The coding sequences disclosed in this document
do not include sequences originating from the 5'-end of the viral
genome which code for putative structural-proteins. Recently
however, sequences derived from this region of the HCV genome have
been published (Okamoto, H. et al., Japan J. Exp. Med. 60:167-177,
1990.). The amino acid sequences encoded by the Japanese clone
HC-J1 were combined with the HCV.sub.CDC/CHI sequences in a region
where the two sequences overlap to generate the composite sequence
depicted in FIG. 1. Specifically, the two sequences were joined at
glycine.sub.451 . It should be emphasized that the numbering system
used for the HCV amino acid sequence is not intended to be absolute
since the existence of variant HCV strains harboring deletions or
insertions is highly probable. Sequences corresponding to the 5'
end of the HCV genome have also recently been disclosed in EPO
90302866.0.
[0003] In order to detect potential carriers of HCV, it is
necessary to have access to large amounts of viral proteins. In the
case of HCV, there is currently no known method for culturing the
virus, which precludes the use of virus-infected cultures as a
source of viral antigens. The current first-generation antibody
test makes use of a fusion protein containing a sequence of 363
amino acids encoded by the HCV genome. It was found that antibodies
to this protein could be detected in 75 to 85% of chronic NANBH
patients. In contrast, only approximately 15% of those patients who
were in the acute phase of the disease, had antibodies which
recognized this fusion protein (Kuo, G. et al. Science (1989)
244:362-364). The absence of suitable confirmatory tests, however,
makes it difficult to verify these statistics. The seeming
similarity between the HCV genome and that of flaviviruses makes it
possible to predict the location of epitopes which are likely to be
of diagnostic value. An analysis of the HCV genome reveals the
presence of a continuous long open reading frame. Viral RNA is
presumably translated into a long polyprotein which is subsequently
cleaved by cellular and/or viral proteases. By analogy with, for
example, Dengue virus, the viral structural proteins are presumed
to be derived from the amino-terminal third of the viral
polyprotein. At the present time, the precise sites at which the
polyprotein is cleaved can only be surmised. Nevertheless, the
structural proteins are likely to contain epitopes which would be
useful for diagnostic purposes, both for the detection of
antibodies as well as for raising antibodies which could
subsequently be used for the detection of viral antigens.
Similarly, domains of nonstructural proteins are also expected to
contain epitopes of diagnostic value, even though these proteins
are not found as structural components of virus particles.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] FIG. 1 shows the amino acid sequence of the composite
HCV.sub.HC-J1/CDC/CHI
[0005] FIG. 2 shows the antibody binding to individual peptides and
various mixtures in an ELISA assay
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0006] It is known that RNA viruses frequently exhibit a high rate
of spontaneous mutation and, as such, it is to be expected that no
two HCV isolates will be completely identical, even when derived
from the same individual. For the purpose of this disclosure, a
virus is considered to be the same or equivalent to HCV if it
exhibits a global homology of 60 percent or more with the
HCV.sub.HC-J1/CDC/CHI composite sequence at the nucleic acid level
and 70 percent at the amino acid level.
[0007] Peptides are described which immunologically mimic proteins
encoded by HCV. In order to accommodate strain-to-strain variations
in sequence, conservative as well as non-Conservative amino acid
substitutions may be made. These will generally account for less
than 35 percent of a specific sequence. It may be desirable in
cases where a peptide corresponds to a region in the HCV
polypeptide which is highly polymorphic, to vary one or more of the
amino acids so as to better mimic the different epitopes of
different viral strains.
[0008] The peptides of interest will include at least five,
sometimes six, sometimes eight, sometimes twelve, usually fewer
than about fifty, more usually fewer than about thirty-five, and
preferably fewer than about twenty-five amino acids included within
the sequence encoded by the HCV genome. In each instance, the
peptide will preferably be as small as possible while still
maintaining substantially all of the sensitivity of the larger
peptide. It may also be desirable in certain instances to join two
or more peptides together in one peptide structure.
[0009] It should be understood that the peptides described need not
be identical to any particular HCV sequence, so long as the subject
compounds are capable of providing for immunological competition
with at least one strain of HCV. The peptides may therefore be
subject to insertions, deletions, and conservative or
non-conservative amino acid substitutions where such changes might
provide for certain advantages in their use.
[0010] Substitutions which are considered conservative are those in
which the chemical nature of the substitute is similar to that of
the original amino acid. Combinations of amino acids which could be
considered conservative are Gly, Ala; Asp, Glu; Asn, Gln; Val, Ile,
Leu; Ser, Thr, Lys, Arg; and Phe, Tyr.
[0011] Furthermore, additional amino acids or chemical groups may
be added to the amino- or carboxyl terminus for the purpose of
creating a "linker arm" by which the peptide can conveniently be
attached to a carrier. The linker arm will be at least one amino
acid and may be as many as 60 amino acids but will most frequently
be 1 to 10 amino acids. Tne nature of the attachment to a solid
phase or carrier need not be covalent.
[0012] Natural amino acids such as cysteine, lysine, tyrosine,
glutamic acid, or .aspartic acid may be added to either the amino-
or carboxyl terminus to provide functional groups for coupling to a
solid phase or a carrier. However, other chemical groups such as,
for example, biotin and thioglycolic acid, may be added to the
termini which will endow the peptides with desired chemical or
physical properties. The termini of the peptides may also be
modified, for example, by N-terminal acetylation or terminal
carboxy-amidation. The peptides of interest are described in
relation to the composite amino acid sequence shown in FIG. 1. The
amino acid sequences are given in the conventional and universally
accepted three-letter code. In addition to the amino acids shown,
other groups are defined as follows: Y is, for example, NH.sub.2,
one or more N-terminal amino acids, or other moieties added to
facilitate coupling. Y may itself be modified by, for example,
acetylation. Z is a bond, (an) amino acid(s), or (a) chemical
group(s) which may be used for linking. X is intended to represent
OH, NH.sub.2, or a linkage involving either of these two
groups.
[0013] Peptide I corresponds to amino acids 1 to 20 and has the
following amino acid sequences:
TABLE-US-00001 (I)
Y-Met-Ser-Thr-Ile-Pro-Lys-Pro-Gln-Arg-Lys-Thr-Lys-
Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Z-X.
[0014] Peptide II corresponds to amino acids 7 to 26 and has the
amino acid sequence:
TABLE-US-00002 (II)
Y-Pro-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-
Pro-Gln-Asp-Val-Lys-Phe-Pro-Gly-Z-X.
[0015] Of particular interest is the oligopeptide IIA:
TABLE-US-00003 (IIA)
Y-Gln-Arg-Lys-Thr-Lys-Arg-Asn-Thr-Asn-Arg-Arg-Z-X.
[0016] Peptide III corresponds to amino acids 13 to 32 and has the
sequence:
TABLE-US-00004 (III)
Y-Arg-Asn-Thr-Asn-Arg-Arg-Pro-Gln-Asp-Val-Lys-Phe-
Pro-Gly-Gly-Gly-Gln-Ile-Val-Gly-Z-X.
[0017] Peptide IV corresponds to amino acid 37 to 56 and has the
sequences:
TABLE-US-00005 (IV)
Y-Leu-Pro-Arg-Arg-Gly-Pro-Arg-Leu-Gly-Val-Arg-Ala-
Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Z-X.
[0018] Peptide V corresponds to amino acids 49 to 68 and has the
sequence:
TABLE-US-00006 (V)
Y-Thr-Arg-Lys-Thr-Ser-Glu-Arg-Ser-Gln-Pro-Arg-Gly-
Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Z-X.
[0019] Peptide VI corresponds to amino acid 61 to 80 and has the
following sequence:
TABLE-US-00007 (VI)
Y-Arg-Arg-Gln-Pro-Ile-Pro-Lys-Val-Arg-Arg-Pro-Glu-
Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Z-X.
[0020] Peptide VII corresponds to amino acids 73 to 92 and has the
sequence:
TABLE-US-00008 (VII)
Y-Gly-Arg-Thr-Trp-Ala-Gln-Pro-Gly-Tyr-Pro-Trp-Pro-
Leu-Tyr-Gly-Asn-Glu-Gly-Cys-Gly-Z-X.
[0021] Peptide VIII corresponds to amino acids 1688 to 1707 and has
the sequence:
TABLE-US-00009 (VIII)
Y-Leu-Ser-Gly-Lys-Pro-Ala-Ile-Ile-Pro-Asp-Arg-Glu-
Val-Leu-Tyr-Arg-Glu-Phe-Asp-Glu-Z-X.
[0022] Peptide IX corresponds to amino acids 1694 to 1713 and has
the sequence:
TABLE-US-00010 (IX)
Y-Ile-Ile-Pro-Asp-Arg-Glu-Val-Leu-Tyr-Arg-Glu-Phe-
Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-Z-X.
[0023] Peptide X corresponds to amino acids 1706 to 1725 and has
the sequence:
TABLE-US-00011 (X)
Y-Asp-Glu-Met-Glu-Glu-Cys-Ser-Gln-His-Leu-Pro-Tyr-
Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Z-X.
[0024] Peptide XI corresponds to amino acids 1712 to 1731 and has
the sequence:
TABLE-US-00012 (XI)
Y-Ser-Gln-His-Leu-Pro-Tyr-Ile-Glu-Gln-Gly-Met-Met-
Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Z-X.
[0025] Peptide XII corresponds to amino acids 1718 to 1737 and has
the sequence:
TABLE-US-00013 (XII)
Y-Ile-Glu-Gln-Gly-Met-Met-Leu-Ala-Glu-Gln-Phe-Lys-
Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Z-X.
[0026] Peptide XIII corresponds to amino acids 1724 to 1743 and has
the sequence:
TABLE-US-00014 (XIII)
Y-Leu-Ala-Glu-Gln-Phe-Lys-Gln-Lys-Ala-Leu-Gly-Leu-
Leu-Gln-Thr-Ala-Ser-Arg-Gla-Ala-Z-X.
[0027] Peptide XIV corresponds to amino acids 1730 to 1749 and has
the sequence:
TABLE-US-00015 (XIV)
Y-Gln-Lys-Ala-Leu-Gly-Leu-Leu-Gln-Thr-Ala-Ser-Arg-
Gln-Ala-Glu-Val-Ile-Ala-Pro-Ala-Z-X.
[0028] Peptide XV corresponds to amino adds 2263 to 2282 and has
the sequence:
TABLE-US-00016 (XV)
Y-Glu-Asp-Glu-Arg-Glu-Ile-Ser-Val-Pro-Ala-Glu-Ile-
Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Z-X.
[0029] Peptide XVI corresponds to amino acids 2275 to 2294 and has
the sequence:
TABLE-US-00017 (XVI)
Y-Leu-Arg-Lys-Ser-Arg-Arg-Phe-Ala-Gln-Ala-Leu-Pro-
Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Z-X.
[0030] Peptide XVII corresponds to amino acids 2287 to 2306 and has
the sequence:
TABLE-US-00018 (XVII)
Y-Val-Trp-Ala-Arg-Pro-Asp-Tyr-Asn-Pro-Pro-Leu-Val-
Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Z-X.
[0031] Peptide XVIII corresponds to amino acids 2299 to 2318 and
has the sequence:
TABLE-US-00019 (XVIII)
Y-Glu-Thr-Trp-Lys-Lys-Pro-Asp-Tyr-Glu-Pro-Pro-Val-
Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Z-X.
[0032] Peptide XIX corresponds to amino acids 2311 to 2330 and has
the sequence:
TABLE-US-00020 (XIX)
Y-Val-His-Gly-Cys-Pro-Leu-Pro-Pro-Pro-Lys-Ser-Pro-
Pro-Val-Pro-Pro-Pro-Arg-Lys-Lys-Z-X
[0033] Of particular interest is the use of the mercapto-group of
cysteines or thioglycolic acids used for acylating terminal amino
groups for cyclizing the peptides or coupling two peptides
together. The cyclization or coupling may occur via a single bond
or may be accomplished using thiol-specific reagents to form a
molecular bridge.
[0034] The peptides may be coupled to a soluble carrier for the
purpose of either raising antibodies or facilitating the adsorption
of the peptides to a solid phase. The nature of the carrier should
be such that it has a molecular weight greater than 5000 and should
not be recognized. by antibodies in human serum. Generally, the
carrier will be--a protein. Proteins which are frequently used as
carriers are keyhole limpet hemocyanin, bovine gamma globulin,
bovine serum albumin, and poly-L-lysine.
[0035] There are many well described techniques for coupling
peptides to carriers. The linkage may occur at the N-terminus,
C-terminus or at an internal site in the peptide. The peptide may
also be derivatized for coupling. Detailed descriptions of a wide
variety of coupling procedures are given, for example, in Van
Regenmortel, Briand, J. P., Muller, S., and Plaue, S., Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 19,
Synthetic Polypeptides as Antigens, Elsevier Press, Amsterdam,
N.Y., Oxford, 1988.
[0036] The peptides may also be synthesized directly on an
oligo-lysine core in which both the alpha as well as the
epsilon-amino groups of lysines are used as growth points for the
peptides. The number of lysines comprising the core is preferably 3
or 7. Additionally, a cysteine may be included near or at the
C-terminus of the complex to facilitate the formation of homo- or
heterodimers. The use of this technique has been amply illustrated
for hepatitis B antigens (Tam, J. P., and Lu, Y- A., Proc. Natl.
Acad. Sci. USA (1989) 86:9084-9088) as well as for a variety of
other antigens (see Tam, J. P., Multiple Antigen Peptide System: A
Novel Design for Synthetic Peptide Vaccine and Immunoassay, in
Synthetic Peptides, Approaches to Biological Problems, Tam, J. P.,
and Kaiser, E. T., ed. Alan R. Liss Inc., New York, 1989).
[0037] Depending on their intended use, the peptides may be either
labeled or unlabeled. Labels which may be employed may be of any
type, such as enzymatic, chemical, fluorescent, luminescent, or
radioactive. In addition, the peptides may be modified for binding
to surfaces or solid phases, such as, for example, microtiter
plates, nylon membranes, glass or plastic beads, and
chromatographic supports such as cellulose, silica, or agarose. The
methods by which peptides can be attached or bound to solid support
or surface are well known to those versed in the art.
[0038] Of particular interest is the use of mixtures of peptides
for the detection of antibodies specific for hepatitis C virus.
Mixtures of peptides which are considered particularly advantageous
are: [0039] A. II, III, V, IX, and XVIII [0040] B. I, II, V, IX,
XI, XVI, and XVIII [0041] C. II, III, IV, V, VIII, XI, XVI, and
XVIII [0042] D. II, IX, and XVIII [0043] E. II, III, IV, and V
[0044] F. VIII, IX, XI, XIII, and XIV [0045] G. XV, XVI, XVII,
XVIII, and XIX
[0046] Antibodies which recognize the peptides can be detected in a
variety of ways. A preferred method of detection is the
enzyme-linked immunosorbant assay (ELISA) in which a peptide or
mixture of peptides is bound to a solid support. In most cases,
this will be a microtiter plate but may in principle be any sort of
insoluble solid phase. A suitable dilution or dilutions of serum or
other body fluid to be tested is brought into contact with the
solid phase to which the peptide is bound. The incubation is
carried out for a time necessary to allow the binding reaction to
occur. Subsequently, unbound components are removed by washing the
solid phase. The detection of immune complexes is achieved using
antibodies which specifically bind to human immunoglobulins, and
which have been labeled with an enzyme, preferably but not limited
to either horseradish peroxidase, alkaline phosphatase, or
beta-galactosidase, which is capable of converting a colorless or
nearly colorless substrate or co-substrate into a highly colored
product or a product capable of forming a colored complex with a
chromogen. Alternatively, the detection system may employ an enzyme
which, in the presence of the proper substrate(s), emits light. The
amount of product formed is detected either visually,
spectrophotometrically, electrochemically, or luminometrically, and
is compared to a similarly treated control. The detection system
may also employ radioactively labeled antibodies, in which case the
amount of immune complex is quantified by scintillation counting or
gamma counting.
[0047] Other detection systems which may be used include those
based on the use of protein A derived from Staphylococcus aureus
Cowan strain I, protein G from group C Staphylococcus sp. (strain
26RP66), or systems which make use of the high affinity
biotin-avidin or streptavidin binding reaction.
[0048] Antibodies raised to carrier-bound peptides can also be used
in conjunction with labeled peptides for the detection of
antibodies present in serum or other body fluids by competition
assay. In this case, antibodies raised to carrier-bound peptides
are attached to a solid support which may be, for example, a
plastic bead or a plastic tube. Labeled peptide is then mixed with
suitable dilutions of the fluid to be tested and this mixture is
subsequently brought into contact with the antibody bound to the
solid support. After a suitable incubation period, the solid
support is washed and the amount of labeled peptide is quantified.
A reduction in the amount of label bound to the solid support is
indicative of the presence of antibodiesin the original sample. By
the same token, the peptide may also be bound to the solid support.
Labeled antibody may then be allowed to compete with antibody
present in the sample under conditions in which the amount of
peptide is limiting. As in the previous example, a reduction in the
measured signal is indicative of the presence of antibodies in the
sample tested.
[0049] Another preferred method of antibody detection is the
homogeneous immunoassay. There are many possible variations in the
design of such assays. By way of example, numerous possible
configurations for homogeneous enzyme immunoassays and methods by
which they may be performed are given in Tijssen, P., Practice and
Theory of Enzyme Immunoassays, Elsevier Press, Amersham, Oxford,
N.Y., 1985. Detection systems which may be employed include those
based on enzyme channeling, bioluminescence, allosteric activation
and allosteric inhibition. Methods employing liposome-entrapped
enzymes or coenzymes may also be used (see Pinnaduwage, P. and
Huang, L, Clin. Chem. (1988) 34/2: 268-272, and Ullman, E. F. et
al., Clin. Chem. (1987) 33/9: 1579-1584 for examples).
[0050] The synthesis of the peptides can be achieved in solution or
on a solid support. Synthesis protocols generally employ the use
t-butyloxycarbonyl- or 9-fluorenylmethoxy-carbonyl-protected
activated amino acids. Tne procedures for carrying out the
syntheses, the types of side-chain protection, and the cleavage
methods are amply described in, for example, Stewart and Young,
Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical
Company, 1984; and Atherton and Sheppard, Solid Phase Peptide
Synthesis, IRL Press, 1989.
Experimental
I. Peptide Synthesis
[0051] All of the peptides described were synthesized on Pepsyn K
polyamide-Kieselguhr resin (Milligen, Novato, Calif.) which had
been functionalized with ethylenediamine and onto which the
acid-labile linker 4-(alpha-Fmoc-amino-2',4'-dimethoxybenzyl)
phenoxyacetic acid had been coupled (Rink, Tetrahedron Lett. (1987)
28:3787). t-Butyl-based side-chain protection and Fmoc
alpha-amino-protection was used. The guanidino-group of arginine
was protected by the 2,2,5,7,8-pentamethylchroman-6-sulfonyl
moiety. The imidazole group of histidine was protected by either
t-Boc or trityl and the sulfhydryl group of cysteine was protected
by a trityl group. Couplings were carried out using performed
O-pentafluorophenyl esters except in the case of arginine where
diisopropylcarbodiimide-mediated hydroxybenzotriazole ester
formation was employed. Except for peptide I, all peptides were
N-acetylated using acetic anhydride. All syntheses were carried out
on a Milligen 9050 PepSynthesizer (Novato, Calif.) using continuous
flow to procedures. Following cleavage with trirluoroacetic acid in
the presence of scavengers and extraction with diethylether, all
peptides were analyzed by C.sub.18-reverse phase
chromatography.
II Detection of Antibodies to Hepatitis C Virus
A. Use of Peptides Bound to a Nylon Membrane.
[0052] Peptides were dissolved in a suitable buffer to make a
concentrated stock solution which was then further diluted in
phosphate-buffered saline (PBS) or sodium carbonate buffer, pH 9.6
to make working solutions. The peptides were applied as lines on a
nylon membrane (Pall, Portsmouth, United Kingdom), after which the
membrane was treated with casein to block unoccupied binding sites.
The membrane was subsequently cut into strips perpendicular to the
direction of the peptide lines. Each strip was then incubated with
a serum sample diluted 1 to 100, obtained from an HCV-infected
individual. Antibody binding was detected by incubating the strips
with goat anti-human immunoglobulin antibodies conjugated to the
enzyme alkaline phosphatase. After removing unbound conjugate by
washing, a substrate solution containing
5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium was
added.
[0053] Positive reactions are visible as colored lines
corresponding to the positions of the peptides which are
specifically recognized. The reaction patterns of thirty-six
different sera are tabulated in Table 1. The results shown in Table
1 are further summarized in Table 2.
B. Use of Peptides in an Enzyme-Linked Immunosorbent Assay
(ELISA).
[0054] Peptide stock solutions were diluted in sodium carbonate
buffer, pH 9.6 and used to coat microtiter plates at a peptide
concentration of is 2 micrograms per milliliter A mixture
consisting of peptides II, V, IX, and XVIII was also used to coat
plates. Following coating, the plates were blocked with casein.
Fifteen HCV-antibody-positive sera and control sera from seven
uninfected blood donors were diluted 1 to 20 and incubated in wells
of the peptide-coated plates. Antibody binding was detected by
incubating .the plates with goat anti-human immunoglobulin
antibodies conjugated to the enzyme horseradish peroxidase.
Following removal of unbound conjugate by washing, a solution
containing H.sub.2O.sub.2 and 3,3'5,5'-tetramethylbenzidine was
added. Reactions were stopped after a suitable interval by addition
of sulfuric acid. Positive reactions gave rise to a yellow color
which was quantified using a conventional microtiter plate reader.
The results of these determinations are tabulated in Table 3. To
correct for any aspecific binding which could be attributable to
the physical or chemical properties of the peptides themselves, a
cut-off value was determined for each peptide individually. This
cut-off absorbance value was calculated as the average optical
density of the negative samples plus 0.200. Samples giving
absorbance values higher than the cut-off values are considered
positive. The results for the fifteen positive serum samples are
further summarized in Table 4.
[0055] While it is evident that some of the peptides are recognized
by a large percentage of sera from HCV-infected individuals, it is
also clear that no single peptide is recognized by all sera. In
contrast, the peptide mixture was recognized by all fifteen sera
and, for six of the fifteen sera, the optical densities obtained
were equal to or higher than those obtained for any of the
peptides, individually. These results serve to illustrate the
advantages of using mixtures of peptides for the detection of
anti-HCV antibodies.
C. Binding of Antibodies in Sera From HCV-Infected Patients to
Various Individual Peptides and Peptide Mixtures in an ELISA.
[0056] Five peptides were used individually and in seven different
combinations to coat microtiter plates. The plates were
subsequently incubated with dilutions of fifteen HCV
antibody-positive sera in order to evaluate the relative merits of
using mixtures as compared to individual peptides for antibody
detection. The mixtures used and the results obtained are shown in
FIG. 2.
[0057] In general, the mixtures functioned better than individual
peptides. This was particularly evident for mixture 12 (peptides I,
III, V, IX, and XVIII) which was recognized by all twelve of the
sera tested. These results underscore the advantages of using
mixtures of peptides in diagnostic tests for the detection of
antibodies to HCV.
D. Use of a Mixture of Peptides in an ELISA Assay for the Detection
of Anti-HCV Antibodies.
[0058] A mixture of peptides II, III, V, IX, and XVIII was prepared
and used to coat microtiter plates according to the same procedure
used to test the individual peptides. A total of forty-nine sera
were tested from patients with clinically diagnosed but
undifferentiated chronic non A non B hepatitis as well as
forty-nine sera from healthy blood donors. Detection of antibody
binding was accomplished using goat anti-human immunoglobulin
antibodies conjugated to horseradish peroxidase. The resulting
optical density values are given in Table 5. These results indicate
that the mixture of peptides is not recognized by antibodies in
sera from healthy donors (0/49 reactives) out is recognized by a
large proportion (41/49, or 84%) of the sera from patients with
chronic NANBH. These results demonstrate that the peptides
described can be used effectively as mixtures for the diagnosis of
HCV infection.
E. Detection of Anti-HCV Antibodies in Sera From patients With
Acute NANB Infection Using Individual Peptides Bound to Nylon
Membranes and a Mixture of Peptides in an ELISA Assay, and
Comparison With a Commercially Available Kit.
[0059] Peptides were applied to nylon membranes or mixed and used
to coat microtiter plates as previously described. The peptide
mixture consisted of peptides II, III, V, IX, and XVIII. Sera
obtained from twenty-nine patients with acute non-A, non-B
hepatitis were then tested for the presence of antibodies to
hepatitis C virus. These same sera were also evaluated using a
commercially available kit (Ortho, Emeryville, Calif., USA).
[0060] The results of this comparative study are given in Table 6.
In order to be able to compare the peptide-based ELISA with the
commercially available kit, the results for both tests are also
expressed as signal to noise ratios (S/N) which were calculated by
dividing the measured optical density obtained for each sample by
the cut-off value. A signal-to-noise ratio greater or equal to 1.0
is taken to represent a positive reaction. For the commercially
available kit, the cut-off value was calculated according to the
manufacturer's instructions. The cut-off value for the
peptide-based ELISA was calculated as the average optical density
of five negative samples plus 0.200.
[0061] The scale used to evaluate antibody recognition of
nylon-bound peptides was the same as that given in Table 1. Of the
twenty-nine samples tested, twenty-five (86%) were positive in the
peptide-based ELISA and recognized one or more nylon-bound
peptides. In contrast, only fourteen of the twenty-nine sera scored
positive in the commercially available ELISA. These results serve
to illustrate the advantages of using peptide mixtures for the
detection of anti-HIV antibodies as well as the need to include in
the mixtures peptides which contain amino acid sequences derived
from different regions of the HCV polyprotein.
TABLE-US-00021 TABLE 1 Recognition of peptides bound to nylon
membranes by sera from persons infected by HCV. PEPTIDE Serum nr. I
II III IV V VI VII VIII IX 1 3 1 1 2 3 1 0.5 2 1 0.5 2 0.5 4 6 2 1
0.5 7 0.5 1 2 1 0.5 3 2 8 0.5 1 3 1 1 1 1 10 1 0.5 3 1 13 0.5 0.5 2
0.5 15 0.5 2 1 16 2 1 0.5 0.5 1 0.5 2 0.5 18 1 1 3 0.5 2 0.5 23 0.5
1 1 0.5 24 1 0.5 2 1 0.5 0.5 0.5 2 25 1 0.5 2 0.5 26 1 27 0.5 0.5 1
3 2 29 0.5 3 2 1 1 0.5 30 0.5 0.5 1 1 0.5 31 1 0.5 32 1 2 33 0.5 34
1 1 1 3 1 35 1 1 2 1 1 1 0.5 36 1 2 1 1 37 1 1 44 1 2 1 0.5 46 0.5
2 0.5 0.5 0.5 2 47 0.5 0.5 0.5 1 48 1 2 2 0.5 2 49 1 1 0.5 0.5 0.5
50 1 2 1 2 0.5 51 2 0.5 0.5 0.5 52 2 0.5 0.5 54 2 0.5 0.5 1 0.5 56
ND ND ND ND ND ND ND 2 Se- rum nr. X XI XII XIII XIV XV XVI XVII
XVIII XIX 1 0.5 2 2 1 1 2 0.5 1 2 2 1 3 1 0.5 4 1 6 2 7 2 1 1 2 0.5
1 1 8 2 1 1 1 1 0.5 10 1 0.5 2 2 2 2 13 1 1 0.5 0.5 15 0.5 1 0.5 16
1 2 2 1 2 18 1 0.5 23 1 0.5 0.5 0.5 24 1 25 0.5 2 1 1 2 26 0.5 27 1
2 1 0.5 29 2 1 1 1 2 2 1 1 1 30 0.5 0.5 0.5 31 1 0.5 1 1 32 1 0.5
0.5 33 1 1 0.5 34 2 0.5 35 1 1 36 0.5 1 1 1 0.5 37 44 46 47 48 49
0.5 0.5 0.5 1 50 1 1 1 1 1 0.5 0.5 51 1 1 0.5 52 0.5 54 1 1 1 1 1 1
56 0.5 1 2 1 Blank: no reaction; 0.5: weakly positive; 1: clearly
positive; 2: strong reaction; 3: intense reaction; ND: not
determined
TABLE-US-00022 TABLE 2 Summary of antibody binding to nylon-bound
HCV peptides by sera from infected patients. Peptide No. reactive
sera % reactive sera I 13/35 37 II 22/35 63 III 27/35 77 IV 24/35
69 V 14/35 40 VI 11/35 31 VII 11/35 31 VIII 19/36 53 IX 9/36 25 X
17/36 47 XI 15/36 42 XII 1/36 3 XIII 13/36 36 XIV 7/36 19 XV 9/36
25 XVI 20/36 56 XVII 14/36 39 XVIII 14/36 39 XIX 8/36 22
TABLE-US-00023 TABLE 3 Comparison of Individual Peptides in an
ELISA Assay for the Detection of Antibodies to HCV. peptide sample
ident I II III IV V VI VII VIII IX 1 0.786 1.119 1.284 0.265 0.042
0.04 0.05 0.571 0.659 2 0.044 0.039 0.11 0.041 0.037 0.038 0.039
0.479 0.78 3 0.815 0.944 0.825 0.399 0.654 0.487 0.32 0.705 0.965 7
1.122 1.23 0.588 0.682 0.659 0.182 0.107 0.907 1.42 8 1.155 1.159
1.2 0.508 1.272 0.433 0.623 0.61 0.863 10 1.089 1.236 1.083 0.044
0.508 0.042 0.073 1.49 1.529 11 0.048 0.051 0.476 0.052 0.119 0.039
0.1 0.634 0.711 15 0.224 0.602 0.813 0.093 0.068 0.077 0.147 0.807
1.225 23 0.62 0.8 0.924 0.568 0.759 0.442 0.683 0.089 0.121 24
1.042 1.132 1.026 0.518 0.916 0.302 0.253 1.013 1.364 49 0.624 0.73
0.884 0.171 0.372 0.055 0.04 0.084 0.064 13 0.76 0.857 0.815 0.087
0.422 0.098 0.045 0.473 0.489 31 0.84 1.114 0.445 0.672 0.046 0.041
0.042 0.184 0.15 47 1.303 1.53 1.236 0.751 0.83 0.629 0.073 0.545
0.739 56 1.169 1.301 1.364 1.269 1.374 0.85 1.066 1.45 1.523 bd A28
0.054 0.043 0.139 0.045 0.135 0.042 0.041 0.086 0.115 bd A169 0.041
0.042 0.134 0.044 0.038 0.04 0.041 0.061 0.07 bd A170 0.04 0.044
0.117 0.04 0.036 0.04 0.04 0.081 0.05 bd A171 0.041 0.046 0.148
0.043 0.037 0.045 0.045 0.077 0.065 bd A166 0.047 0.046 0.124 0.044
0.038 0.042 0.041 0.056 0.066 bd A165 0.041 0.046 0.123 0.043 0.035
0.051 0.042 0.051 0.091 AVG 0.044 0.045 0.131 0.043 0.053 0.043
0.042 0.069 0.076 STD 0.005 0.002 0.011 0.002 0.037 0.004 0.002
0.013 0.021 cut off 0.109 0.101 0.214 0.099 0.214 0.105 0.098 0.158
0.189 sample ident X XI XII XIII XIV XV XVI XVII XVIII XIX 1 0.048
0.04 0.043 0.068 0.044 0.041 1.063 0.956 1.383 1.346 2 0.169 0.563
0.039 0.042 0.515 0.039 0.64 0.319 0.154 0.49 3 0.468 0.668 0.041
0.093 0.341 0.043 0.292 0.038 0.046 0.038 7 0.663 0.646 0.041 0.235
0.068 0.575 0.042 0.041 0.872 1.271 8 0.752 1.175 0.046 0.42 0.102
0.068 0.552 0.671 0.417 0.058 10 0.689 0.834 0.041 0.044 0.314
0.793 0.886 0.037 1.335 1.356 11 0.199 0.967 0.125 0.454 0.088
0.111 0.274 0.093 0.838 0.065 15 0.315 0.688 0.046 0.154 0.202
0.065 0.372 0.097 0.155 0.077 23 0.422 0.896 0.041 0.049 0.101
0.068 0.311 0.038 0.052 0.05 24 0.236 0.397 0.054 0.123 0.076 0.051
0.418 0.053 0.1 0.085 49 0.209 0.731 0.044 0.113 0.039 0.044 0.299
0.038 0.192 0.041 13 0.529 0.735 0.043 0.044 0.186 0.043 0.086
0.037 0.066 0.04 31 0.255 0.69 0.041 0.04 0.061 0.136 0.292 0.038
0.224 0.501 47 0.044 0.041 0.041 0.041 0.498 0.04 0.268 0.042 1.288
1.206 56 0.079 1.069 0.058 0.568 0.038 0.039 0.218 0.036 0.087
0.039 bd A28 0.044 0.042 0.044 0.052 0.043 0.043 0.307 0.042 0.045
0.061 bd A169 0.043 0.042 0.041 0.04 0.041 0.041 0.255 0.038 0.056
0.042 bd A170 0.04 0.039 0.04 0.038 0.038 0.144 0.292 0.036 0.058
0.039 bd A171 0.043 0.041 0.043 0.039 0.04 0.045 0.286 0.037 0.05
0.04 bd A166 0.041 0.041 0.042 0.04 0.041 0.041 0.207 0.039 0.046
0.041 bd A165 0.041 0.04 0.042 0.039 0.043 0.039 0.253 0.034 0.06
0.098 AVG 0.042 0.041 0.042 0.041 0.041 0.059 0.267 0.038 0.053
0.054 STD 0.001 0.001 0.001 0.005 0.002 0.038 0.033 0.002 0.006
0.021 cut off 0.095 0.094 0.095 0.106 0.097 0.223 0.416 0.084 0.121
0.167
TABLE-US-00024 TABLE 4 Summary of antibody-binding to individual
peptides in an ELISA assay. Peptide No. reactive sera % reactive
sera I 13 87 II 13 87 III 14 93 IV 10 67 V 10 67 VI 7 47 VII 8 53
VIII 13 87 IX 12 80 X 13 87 XI 13 87 XII 1 7 XIII 7 47 XIV 8 53 XV
2 13 XVI 5 33 XVII 4 27 XVIII 10 67 XIX 6 40
TABLE-US-00025 TABLE 5 Use of a peptide mixture for the detection
of antibodies to HCV in sera from chronic NANBH patients and
comparison to sera from healthy blood donors. Chronic NANB Sera
Control Sera Serum nr. Optical Density Serum nr. Optical Density
101 0.041 1 0.049 102 1.387 2 0.047 103 1.578 3 0.049 104 1.804 4
0.046 105 1.393 5 0.049 107 1.604 6 0.045 108 1.148 7 0.043 109
1.714 8 0.053 110 1.692 9 0.049 112 0.919 10 0.047 113 1.454 11
0.060 114 0.936 12 0.044 115 0.041 13 0.049 116 1.636 14 0.051 118
1.242 15 0.056 119 1.568 16 0.050 120 1.290 17 0.049 121 1.541 18
0.055 122 1.422 19 0.054 123 1.493 20 0.058 124 1.666 21 0.050 125
1.644 22 0.044 126 1.409 23 0.043 127 1.625 24 0.045 128 1.061 25
0.046 129 1.553 26 0.049 130 1.709 27 0.050 131 0.041 28 0.047 132
0.044 29 0.050 133 1.648 30 0.053 134 0.043 31 0.051 135 1.268 32
0.053 136 1.480 33 0.055 138 0.628 34 0.064 139 0.042 35 0.063 140
0.040 36 0.057 141 0.039 38 0.048 142 1.659 39 0.045 143 1.457 40
0.046 144 0.722 41 0.046 145 1.256 42 0.051 146 0.373 43 0.057 147
1.732 44 0.050 148 1.089 45 0.050 149 1.606 46 0.045 150 1.725 47
0.041 151 1.449 48 0.064 154 1.639 49 0.040 155 1.775 50 0.036
TABLE-US-00026 TABLE 6 Comparison of anti-HCV antibody detection by
nylon-bound peptides, a peptide-based ELISA, and a commercially
available kit. Se- rum Nylon-bound peptides nr. I III IV V VI VIII
XI XIV XV XVI XVIII 191 0 0 0 0 0 0 0 0 0 0 0 192 0 0 0 0 0 0 0 0 0
0 0 193 0 0 0 0 0 0 0 0 0 0 0 194 0 0 0 0 0 0 0 0 0 0 0 195 1 2 2 3
0 0 0.5 0.5 1 3 1 196 1 2 1 2 0.5 0.5 0.5 0.5 0.5 2 0 197 1 2 1 2 0
0.5 0.5 0.5 1 2 0 198 1 2 2 2 0 0 0 0 1 2 0 211 0.5 1 0.5 0.5 0 2 2
0 2 0 1 213 0 0 0 1 0 0 0 0 0 0 0 214 0 0 0 1 0 0 0 0 0 0 0 215 0 0
0 1 0 0 0 0 0 0 0 216 0 0 0 0.5 0 0 0 0 0 0 0 217 0 0 0 1 0 0 0 0 0
0 0 219 0.5 1 1 2 1 0.5 1 0 0.5 0.5 1 220 0.5 1 1 2 1 0.5 1 0 0.5
0.5 1 221 0 0 0 0.5 0 0 0 0 0 0 0 222 1 1 1 1 0 0 2 0.5 0.5 0 0 223
1 1 1 1 0 0 3 0.5 0.5 0 0 224 1 1 2 1 0 0.5 3 0.5 0.5 0 0 225 0 0 0
0 0 0.5 0.5 0.5 0 0 2 226 0.5 0 0 0 0 2 3 2 0.5 0.5 3 227 0 0 0 0 0
2 2 0.5 0.5 0.5 2 228 0.5 0 0.5 0.5 0 2 2 2 0 0 2 234 0.5 0.5 0 0.5
0 0 3 1 3 1 3 235 0 0 0 0.5 0 0 0 0 0 0 0 236 0 0 0 0.5 0 0 0 0 0 0
0 237 0 0 0 1 0 0 0 0 0 0 0 238 0 0 0 1 0 1 1 0 0 0 0 Optical
density Optical density Serum nr. Peptide ELISA S/N Commercial
ELISA S/N 191 0.045 0.18 0.295 0.47 192 0.042 0.17 0.289 0.46 193
0.039 0.16 0.197 0.32 194 0.044 0.18 0.183 0.29 195 1.692 6.77
3.000* 4.82* 196 1.569 6.28 0.386 0.62 197 1.523 6.09 0.447 0.72
198 1.578 6.31 0.354 0.57 211 1.606 6.42 3.000* 4.82* 213 0.369
1.48 0.127 0.20 214 0.444 1.78 0.101 0.16 215 0.637 2.55 0.101 0.16
216 0.812 3.25 0.092 0.15 217 1.320 5.28 0.875 1.40 219 1.547 6.19
3.000* 4.82* 220 1.536 6.14 3.000* 4.82* 221 1.428 5.71 0.327 0.52
222 1.362 5.45 3.000* 4.82* 223 1.316 5.26 3.000* 4.82* 224 1.304
5.22 3.000* 4.82* 225 1.178 4.71 2.398 3.85 226 1.256 5.14 3.000*
4.82* 227 1.335 5.34 3.000* 4.82* 228 1.400 5.60 3.000* 4.82* 234
1.481 5.92 3.000* 4.82* 235 0.351 1.40 0.257 0.41 236 0.475 1.90
0.245 0.39 237 1.134 4.54 0.351 0.56 238 1.096 4.38 1.074 1.72
Cut-off: 0.250 Cut-off: 0.623 0: no reaction; 0.5: weakly positive;
1: clearly positive; 2: strong reaction; 3: intense reaction; *O.D.
exceeded 3.000 and was out of range. The values given are therefore
minimum values.
Sequence CWU 1
1
* * * * *