Note: Descriptions are shown in the official language in which they were submitted.
CA 02405849 2002-11-05
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Reagents for the Simultaneous Detection of HCV Core Antigens and Antibodies
Background of the Invention
Hepatitis C Virus (HCV) is the leading cause of transfusion-associated and
community-acquired hepatitis. HCV antibodies and antigens are used in
immunoassays to test blood that might be used in transfusion in order to
detect the
presence of HCV. This type of blood screening has significantly reduced the
spread
of this virus due to blood transfusion.
During the .last 10 years there has been considerable progress made in
to improving the sensitivity of blood screening assays but there is a window
period of
60-80 days from the time of infection in which there are no detectable
antibodies.
During this window period the persons are highly infective. To close this
window,
nucleic acids based tests are being developed to detect HCV infection. These
nucleic
acids based tests are very laborious and have not yet been adapted for routine
blood
screening assays. Recently methods have been developed for detecting HCV core
antigen. HCV core antigen can be detected in most HCV antibody negative HCV
RNA positive individuals. From the published research it appears that HCV core
antigen detection can be used as an earlier indication of HCV infection. (S.R.
Lee et
al., Vox Sanguinis, Efficacy Of A Hepatitis C Virus Core Antigen Enzyme-Linked
2o Immunosorbent Assay For The Identification Of The 'Window-Phase' Blood
Donation. 2001; 80: 19-23.) Though core antigen detection can detect HCV
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infection during the window period, the detection of core antigen becomes
difficult
once the anti-core antibodies appear. The core antigen gets complexed with the
anti-
core antibodies and it requires strong dissociating reagents to separate the
core antigen
from the antibodies.
Therefore, there remains a need for a more effective assay that detects HCV
infection. We describe herein a combination assay that detects anti-HCV
antibodies
and HCV core antigen in a single assay, therefore enabling for the detection
of HCV
infection much more effectively than the current HCV core antigen or anti-HCV
antibody assays.
1o Summar3r of the Invention
The present invention is directed to core protein sequences and anti-core
antibodies that can be used to detect core antigen and anti core antibodies in
serum
collected from HCV infected individuals.
Detailed Description
15 Anti-core antibodies are major contributors to the sensitivity of the anti-
HCV
antibodies assays. These antibodies are generally among the earlier antibodies
to
develop. HCV anti-core antibodies are directed against multiple epitopes. Most
of
the HCV anti-core activity can be accounted for by the amino terminal 1/3'~ of
the
HCV core protein. HCV infected individuals have antibodies to multiple
epitopes in
2o HCV core protein sequence. It is possible to detect HCV anti-core
antibodies by
using part of the HCV core sequence. Analysis of serum from HCV infected
individuals using overlapping pentadecapeptides showed that the antibodies to
HCV
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core are distributed over many of the peptides. Four peptides towards the
amino
terminus end have greater reactivity than the peptides towards the carboxy
terminus
of the HCV c22-3 protein. This region of HCV core protein when further
analyzed
by a peptide walk analysis using overlapping octapeptides showed that 6-8
amino
acids stretch of sequences between amino acids 10-43 represent multiple
epitopes in
HCV core sequence. Though the antibodies to HCV core were spread all across
the
core protein sequence, it should be possible to delete or alter amino acid
sequences
outside the 10-43 sequence with little impact on the ability of these modified
antigens to detect anti-core antibodies. Based on the serological information
a
to sensitive assay for the simultaneous detection of core antigen and anticore
antibodies can be designed as follows.
A mixture of HCV core protein as recombinant protein or synthetic peptides
along with anti-core monoclonal or polyclonal antibodies is coated onto
microwells.
The anti-core antibodies selected for this assay are selected from a group of
antibodies that do not recognize core sequence 10-43. This solid phase is then
used
to capture anti HCV core antibodies and core antigen from patient serum or
plasma
specimens using standard ELISA format. The captured proteins, human anti-HCV
core antibodies and core protein, are detected using immunochemical methods.
The
anti-HCV core human antibodies are detected using peroxidase labeled anti-
human a
IgG. The core antigen is detected using peroxidase labeled anti-core
monoclonal
antibodies. The anti-core antibodies used for detection are selected from a
group of
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antibodies that do not recognize as 10-43 and are different from the
antibodies used
to capture the core antigen onto the solid phase. The core antigen used to
detect
anti-HCV core antibodies includes most of the sequence 10-43 with or without
additional HCV core sequences. The core sequences outside of as 10-43 are
s modified by deleting or altering amino acid sequence in the region
recognized by
the anti-core antibodies used to capture or detect core antigen.
Therefore, we have developed an ELISA based assay for the detection of
HCV infection. This new assay can detect HCV infected blood earlier than the
currently used anti-HCV antibodies detection assay. The method described
herein
to can detect HCV infection earlier because in addition to the detection of
anti-HCV
antibodies, this assay method also detects HCV core antigen. A specific
detergent
from the polyoxyethylene class is used in the HCV assay. The detergent used
should be able to release the HCV core antigen from the virus by possibly
disrupting
the envelope protein and/or the lipid layer but this detergent should not
affect the
is ability of the HCV recombinant antigens to capture the anti-HCV antibodies.
Some
of the common detergents such as N-lauryl sarcosine used in the detection of
HCV
core antigen destroy the ability of HCV recombinant proteins such as c22,
c200, and
NSS to detect anti-HCV antibodies in a standard ELISA.
The effectiveness and advantages of the invention are further illustrated by
2o the following examples. The examples are meant to illustrate, but not to
limit, the
scope and spirit of the invention.
CA 02405849 2002-11-05
Example 1
In the present assay, HCV antigens c200-3, NSS and a modified core antigen
such as c22KS0 47,48 or c22KSR47L along with anti-core monoclonal antibodies
(anti core marine monoclonals Pep 10, 12) were coated onto microwells in a
buffer
s solution. After overnight incubation the buffer containing the coating
proteins are
removed and the microwells washed with phosphate buffered saline (PBS)
containing a detergent, TWEEN 20. The antigen/antibody coated microwells were
then treated with bovine serum albumin (BSA)/ sucrose solution to block the
remaining protein binding sites that may be available on the microwells. After
2-24
io hours, the BSA/sucrose solution is removed and the microwells air dried and
stored
under a descicant.
Example 2
Two aliquotes of the specimen to be tested are diluted into 100 uL of PBS
solution containing BSA, superoxide dismutase, yeast extract and 1% BRIJ 58 or
is BRIJ 35 or a mixture of both. The two diluted specimens were then pipetted
into two
HCV antigen/antibody coated wells described above. The microwells were
incubated for 90 minutes at 37 degrees Celsius. The microwells were then
washed
five times with PBS containing 0.5% TWEEN 20 detergent. To one microwell
200uL of a marine anti-human IgG labeled with HRP was added and in the other
2 o well the same volume of anti-HCV core monoclonal antibodies (Anti Pep4)
labeled
with HRP was added. To each well a solution of ORTHO phenylenediamine and
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hydrogen peroxide was added. After incubation in the dark for 30 minutes, the
reaction is stopped by adding 50 microliters of 4N sulfuric acid. An orange
color in
either well indicates that the specimen being tested is infected with HCV. The
microwell tested with anti-human IgG tests for anti-HCV antibodies and the one
tested with and-core conjugate tests for HCV core antigen.
Example 3
HCV core sequences are synthesized based on the HCV core sequence
represented below. These sequences are prepared using recombinant techniques
or
standard peptide synthesis methods. SEQ ID NO.: 1
i o MSTNPKPQKKNKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPR
LGVRATRKTSERSQPRGRRQPIPR;A.RRPEGRTWAQPGYPWPLYG
NEGCGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGF
ADLMGYIPLVGAPLGGAARALAHGVRVLEDGVNYATGNLPGCS
FSIFLLALLSCLTVPAS (AA 1-190 HCV Core Sequence)
is SEQ ID NO.: 2
MSTNPKPQKI~1KRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPR
(AA1-43)
SEQ ID NO.: 3
KNKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPR (AA 10-4.3)
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SEQ ID NO.: 1
MSTNPKPQKKNI~RNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRL
GVRATRKTSERSQPRGRRQPIPh,'ARRPEGRT'WAQPGYPWPLYGNEG
CGWAGWLLSPRGSRPSWGPTDPRRRSRNLGKVIDTLTCGFADLMGY
IPLYGAPLGGAARALAHGYRYLEDGVNYATGNLPGCSFSIFLLALLSC
LTVPAS.
Modifications can be made in the italicized and underlined amino acid sequence
of
HCV core protein. These modifications can be alterations of amino acids or
deletion
i o of sets of amino acids to remove sites of reagent anti-core antibodies.
The antigen
used for the assay is paired with the monoclonal antibodies that have their
binding
sites outside the core region 10-43. The modified core antigens along with the
appropriate anti-core antibodies are used for the simultaneous detection of
HCV core
antigen and anti-HCV antibodies in specimens infected with HCV.
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Ortho-Clinical Diagnostics, Inc.
(ii) TITLE OF INVENTION: REAGENTS FOR THE SIMULTANEOUS DETECTION
OF HCV CORE ANTIGENS AND ANTIBODIES
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: OGILVY RENAULT
(B) STREET: Suite 1600, McGill College Avenue 1981
(C) CITY: Montreal
(D) STATE: Quebec
(E) COUNTRY: Canada
(F) ZIP: H3A 2Y3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: CA 2,405,849
(B) FILING DATE: 05-NOV-2002
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/347,943
(B) FILING DATE: 07-NOV-2001
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ogilvy Renault,
(C) REFERENCE/DOCKET NUMBER: 1011-3953CA
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (514) 845-7126
(B) TELEFAX: (514) 288-$389
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 190 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C virus
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro
50 55 60
Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly
65 70 75 80
Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys
115 120 125
Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu
130 135 140
Gly Gly Ala Ala Rrg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp
145 150 155 160
Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile
165 170 175
Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser
180 185 190
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 43 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C virus
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn
1 5 10 15
Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
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20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg
35 40
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: unknown
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hepatitis C virus
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro
1 5 10 15
Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly
20 25 30
Pro Arg
