CA2405849A1 - Reagents for the simultaneous detection of hcv core antigens and antibodies - Google Patents
Reagents for the simultaneous detection of hcv core antigens and antibodies Download PDFInfo
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- CA2405849A1 CA2405849A1 CA 2405849 CA2405849A CA2405849A1 CA 2405849 A1 CA2405849 A1 CA 2405849A1 CA 2405849 CA2405849 CA 2405849 CA 2405849 A CA2405849 A CA 2405849A CA 2405849 A1 CA2405849 A1 CA 2405849A1
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- hcv
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Abstract
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.
Description
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
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
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
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
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 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.
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 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 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) -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.
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 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Rrg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser (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 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg (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 Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg
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 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.
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 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 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) -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.
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 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
Met Ser Thr Asn Pro Lys Pro Gln Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Pro Ile Pro Lys Ala Arg Arg Pro Glu Gly Arg Thr Trp Ala Gln Pro Gly Tyr Pro Trp Pro Leu Tyr Gly Asn Glu Gly Cys Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg Arg Arg Ser Arg Asn Leu Gly Lys Val Ile Asp Thr Leu Thr Cys Gly Phe Ala Asp Leu Met Gly Tyr Ile Pro Leu Val Gly Ala Pro Leu Gly Gly Ala Ala Rrg Ala Leu Ala His Gly Val Arg Val Leu Glu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Val Pro Ala Ser (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 Arg Arg Pro Gln Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg (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 Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg
Claims (8)
1. An antibody/antigen composition comprising a mixture of HCV protein and modified HCV core protein and anti-core antibodies wherein said anti-core antibodies do not recognize HCV core sequence amino acids 10-43.
2. The antibody composition as claimed in claim 1 wherein said HCV protein is selected from the group consisting of c200-3 and NS5 and said modified HCV core protein is selected from the group consisting of c22KS~ 47,48 and c22KSR47L.
3. The antibody composition as claimed in claim 1 wherein said anti-core antibodies comprise a monoclonal antibody.
4. The antibody composition as claimed in claim 1 wherein said anti-core antibodies comprise a polyclonal antibody.
5. A method of immunoassay for HCV which comprises a) immobilizing onto a solid phase an antibody/antigen composition according to claim 1, b) contacting said antibody/antigen composition with blood or serum from a patient to form an HCV antibody/antigen complex, c) adding anti-human IgG carrying a label, and d) detecting said complex.
6. The immunoassay method as claimed in claim 5 wherein said solid phase is a microwell, mircrotitre plate, membrane or bead.
7. The immunoassay method as claimed in claim 5 wherein said label is an enzyme, a dye, colored particle, radionuclide or fluorescent substance.
8. A kit containing the antibody/antigen composition as claimed in claim 1.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US34794301P | 2001-11-07 | 2001-11-07 | |
| US60/347,943 | 2001-11-07 | ||
| US10/268,562 | 2002-10-10 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2405849A1 true CA2405849A1 (en) | 2003-05-07 |
Family
ID=23365969
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2405849 Abandoned CA2405849A1 (en) | 2001-11-07 | 2002-11-05 | Reagents for the simultaneous detection of hcv core antigens and antibodies |
Country Status (3)
| Country | Link |
|---|---|
| AR (1) | AR037215A1 (en) |
| BR (1) | BR0206887A (en) |
| CA (1) | CA2405849A1 (en) |
-
2002
- 2002-11-05 BR BR0206887A patent/BR0206887A/en not_active Application Discontinuation
- 2002-11-05 CA CA 2405849 patent/CA2405849A1/en not_active Abandoned
- 2002-11-05 AR ARP020104219 patent/AR037215A1/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| AR037215A1 (en) | 2004-10-27 |
| BR0206887A (en) | 2003-09-30 |
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| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |