Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-06-14
2003-10-07
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S518000, C435S023000
Reexamination Certificate
active
06630298
ABSTRACT:
TECHNICAL FIELD
The present invention pertains generally to viral diagnostics. In particular, the invention relates to an antigen/antibody combination assay for accurately diagnosing hepatitis C virus infection.
BACKGROUND OF THE INVENTION
Hepatitis C Virus (HCV) is the principal cause of parenteral non-A, non-B hepatitis (NANBH) which is transmitted largely through blood transfusion and sexual contact. The virus is present in 0.4 to 2.0% of blood donors. Chronic hepatitis develops in about 50% of infections and of these, approximately 20% of infected individuals develop liver cirrhosis which sometimes leads to hepatocellular carcinoma. Accordingly, the study and control of the disease is of medical importance.
HCV was first identified and characterized as a cause of NANBH by Houghten et al. The viral genomic sequence of HCV is known, as are methods for obtaining the sequence. See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. HCV has a 9.5 kb positive-sense, single-stranded RNA genome and is a member of the Flaviridae family of viruses. At least six distinct, but related genotypes of HCV, based on phylogenetic analyses, have been identified (Simmonds et al.,
J. Gen. Virol
. (1993) 74:2391-2399). The virus encodes a single polyprotein having more than 3000 amino acid residues (Choo et al.,
Science
(1989) 244:359-362; Choo et al.,
Proc. Natl. Acad. Sci. USA
(1991) 88:2451-2455; Han et al.,
Proc. Natl. Acad. Sci. USA
(1991) 88:1711-1715). The polyprotein is processed co- and post-translationally into both structural and non-structural (NS) proteins.
In particular, as shown in
FIG. 1
, several proteins are encoded by the HCV genome. The order and nomenclature of the cleavage products of the HCV polyprotein is as follows: NH
2
-C-E1-E2-p7-NS2-NS3-NS4a-NS4b-NS5a-NS5b-COOH. Initial cleavage of the polyprotein is catalyzed by host proteases which liberate three structural proteins, the N-terminal nucleocapsid protein (termed “core”) and two envelope glycoproteins, “E1” (also known as E) and “E2” (also known as E2/NS1), as well as nonstructural (NS) proteins that contain the viral enzymes. The NS regions are termed NS2, NS3, NS4 and NS5. NS2 is an integral membrane protein with proteolytic activity. NS2, either alone or in combination with NS3, cleaves the NS2-NS3 sissle bond which in turn generates the NS3 N-terminus and releases a large polyprotein that includes both serine protease and RNA helicase activities. The NS3 protease serves to process the remaining polyprotein. Completion of polyprotein maturation is initiated by autocatalytic cleavage at the NS3-NS4a junction, catalyzed by the NS3 serine protease. Subsequent NS3-mediated cleavages of the HCV polyprotein appear to involve recognition of polyprotein cleavage junctions by an NS3 molecule of another polypeptide. In these reactions, NS3 liberates an NS3 cofactor (NS4a), two proteins (NS4b and NS5a), and an RNA-dependent RNA polymerase (NS5b).
A number of general and specific polypeptides useful as immunological and diagnostic reagents for HCV, derived from the HCV polyprotein, have been described. See, e.g., Houghton et al., European Publication Nos. 318,216 and 388,232; Choo et al.,
Science
(1989) 244:359-362; Kuo et al.,
Science
(1989) 244:362-364; Houghton et al.,
Hepatology
(1991) 14:381-388; Chien et al.,
Proc. Natl. Acad. Sci. USA
(1992) 89:10011-10015; Chien et al.,
J. Gastroent. Hepatol
. (1993) 8:S33-39; Chien et al., International Publication No. WO 93/00365; Chien, D. Y., International Publication No. WO 94/01778. These publications provide an extensive background on HCV generally, as well as on the manufacture and uses of HCV polypeptide immunological reagents. For brevity, therefore, the disclosure of these publications is incorporated herein by reference.
Sensitive, specific methods for screening and identifying carriers of HCV and HCV-contaminated blood or blood products would provide an important advance in medicine. Post-transfusion hepatitis (PTH) occurs in approximately 10% of transfused patients, and HCV has accounted for up to 90% of these cases. Patient care as well as the prevention and transmission of HCV by blood and blood products or by close personal contact require reliable diagnostic and prognostic tools. Accordingly, several assays have been developed for the serodiagnosis of HCV infection. See, e.g., Choo et al.,
Science
(1989) 244:359-362; Kuo et al.,
Science
(1989) 244:362-364; Choo et al.,
Br. Med. Bull.
(1990) 46:423-441; Ebeling et al.,
Lancet
(1990) 335:982-983; van der Poel et al.,
Lancet
(1990) 335:558-560; van der Poel et al.,
Lancet
(1991) 337:317-319; Chien, D. Y., International Publication No. WO 94/01778; Valenzuela et al., International Publication No. WO 97/44469; and Kashiwakuma et al., U.S. Pat. No. 5,871,904.
A significant problem encountered with some serum-based assays is that there is a significant gap between infection and detection of the virus, often exceeding 80 days. This assay gap may create great risk for blood transfusion recipients. To overcome this problem, nucleic acid-based tests (NAT) that detect viral RNA directly, and HCV core antigen tests that assay viral antigen instead of antibody response, have been developed. See, e.g., Kashiwakuma et al., U.S. Pat. No. 5,871,904; Beld et al.,
Transfusion
(2000) 40:575-579.
However, there remains a need for sensitive, accurate diagnostic and prognostic tools in order to provide adequate patient care as well as to prevent transmission of HCV by blood and blood products or by close personal contact.
SUMMARY OF THE INVENTION
The present invention is based in part, on the finding that HCV seroconversion antibodies are typically anti-core and anti-NS3 (helicase). Accordingly, the invention provides an HCV core antigen and NS3 antibody combination assay that can detect both HCV antigens and antibodies present in a sample using a single solid matrix.
Accordingly, in one embodiment, the subject invention is directed to an immunoassay solid support comprising at least one HCV anti-core antibody and at least one isolated HCV NS3/4a epitope bound thereto. The antibody and NS3/4a epitope can be any of the herein described molecules. Additionally, the solid support may include any of the multiple epitope fusion antigens described herein, such as the multiple epitope fusion antigen comprising the amino acid sequence depicted in
FIGS. 7A-7F
.
In certain embodiments, the solid support comprises at least two HCV anti-core antibodies bound thereto. Moreover, the anti-core antibody may be a monoclonal antibody. Additionally, the NS3/4a epitope may be a conformational epitope, such as a conformational NS3/4a epitope comprising the amino acid sequence depicted in
FIGS. 4A-4D
.
In another embodiment, the invention is directed to an immunoassay solid support comprising at least two HCV anti-core monoclonal antibodies and at least one HCV NS3/4a conformational epitope comprising the amino acid sequence depicted in
FIGS. 4A-4D
, bound thereto.
In still a further embodiment, the invention is directed to a method of detecting HCV infection in a biological sample. The method comprises: (a) providing an immunoassay solid support as described above; (b) combining a biological sample with the solid support under conditions which allow HCV antigens and antibodies, when present in the biological sample, to bind to the at least one anti-core antibody and the NS3/4a epitope, respectively; (c) adding to the solid support from step (b) under complex forming conditions (i) a first detectably labeled antibody, wherein the first detectably labeled antibody is a detectably labeled HCV anti-core antibody, wherein the labeled anti-core antibody is directed against a different HCV core epitope than the at least one anti-core antibody bound to the solid support; (ii) an antigen that reacts with an HCV antibody from the biological sample reactive with the NS3/4a epitope; and (iii) a second detectably labeled antibody, wherein the second detectably labeled antibody is reactive with the antigen of (i
Arcangel Phillip
Chien David Y.
Coit Doris
George-Nascimento Carlos
Medina-Selby Angelica
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Robins Roberta L.
Wortman Donna C.
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