Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1995-05-12
2001-11-06
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S518000, C436S531000, C436S820000, C530S350000
Reexamination Certificate
active
06312889
ABSTRACT:
DESCRIPTION
1. Technical Field
The present invention is in the field of immunoassays for HCV (previously called Non-A, Non-B hepatitis virus). More particularly, it concerns combinations of HCV antigens that permit broad range immunoassays for anti-HCV antibodies.
2. Background
The disease known previously as Non-A, Non-B hepatitis (NANBH) was considered to be a transmissible disease or family of diseases that were believed to be viral-induced, and that were distinguishable from other forms of viral-associated liver diseases, including that caused by the known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (HBV), and delta hepatitis virus (HDV), as well as the hepatitis induced by cytomegalovirus (CMV) or Epstein-Barr virus (EBV). NANBH was first identified in transfused individuals. Transmission from man to chimpanzee and serial passage in chimpanzees provided evidence that NANBH was due to a transmissible infectious agent or agents. Epidemiologic evidence suggested that there may be three types of NANBH: a water-borne epidemic type; a blood-borne or parenterally transmitted type; and a sporadically occurring (community acquired) type. However, until recently; no transmissible agent responsible for NANBH had been identified, and clinical diagnosis and identification of NANBH had been accomplished primarily by exclusion of other viral markers. Among the methods used to detect putative NANBH antigens and antibodies were agar-gel diffusion, counterimmunoelectrophoresis, immunofluorescence microscopy, immune electron microscopy, radioimmunoassay, and enzyme-linked immunosorbent assay. However, none of these assays proved to be sufficiently sensitive, specific, and reproducible to be used as a diagnostic test for NANBH.
In 1987, scientists at Chiron Corporation (the owner of the present application) identified the first nucleic acid definitively linked to blood-borne NANBH. See, e.g., EPO Pub. No. 318,216; Houghton et al., Science 244:359 (1989). These publications describe the cloning of an isolate from a new viral class, hepatitis C virus (HCV), the prototype isolate described therein being named “HCV1.” HCV is a Flavi-like virus, with an RNA genome.
U.S. patent application Ser. No. 456,637 (Houghton et al.), incorporated herein by reference, describes the preparation of various recombinant HCV polypeptides by expressing HCV cDNA and the screening of those polypeptides for immunological reactivity with sera from HCV patients. That limited screening showed that at least five of the polypeptides tested were very immunogenic; specifically, those identified as 5-1-1, C100, C33c, CA279a, and CA290a. Of these five polypeptides, 5-1-1 is located in the putative NS4 domain; C100 spans the putative NS3 and NS4 domains; C33c is located within the putative NS3 domain and CA279a and CA290a are located within the putative C domain. The screening also showed that no single polypeptide tested was immunologically reactive with all sera. Thus, improved tests, which react with all or more samples from HCV positive individuals, are desirable.
DISCLOSURE OF THE INVENTION
Applicants have carried out additional serological studies on HCV antigens that confirm that no single HCV polypeptide identified to date is immunologically reactive with all sera. This lack of a single polypeptide that is universally reactive with all sera from individuals with HCV may be due, inter alia, to strain-to-strain variation in HCV epitopes, variability in the humoral response from individual-to-individual and/or variation in serology with the state of the disease.
These additional studies have also enabled applicants to identify combinations of HCV antigens that provide more efficient detection of HCV antibodies than any single HCV polypeptide.
Accordingly, one aspect of this invention is a combination of antigens comprising:
(a) a first HCV antigen from the C domain; and
(b) at least one additional HCV antigen selected from the group consisting of
(i) an HCV antigen from the NS3 domain;
(ii) an HCV antigen from the NS4 domain;
(iii) an HCV antigen from the S domain; and
(iv) an HCV antigen from the NS5 domain.
In one embodiment, the combination of HCV antigens is in the form of a fusion protein comprised of the antigens. In an alternative embodiment, the combination of antigens is in the form of the individual antigens bound to a common solid matrix. In still another embodiment, the combination of antigens is in the form of a mixture of the individual antigens.
Another aspect of the invention is a method for detecting antibodies to HCV in a mammalian body component suspected of containing said antibodies comprising contacting said body component with the above-described combination of HCV antigens under conditions that permit antibody-antigen reaction and detecting the presence of immune complexes of said antibodies and said antigens.
Another aspect of the invention is a method for detecting antibodies to HCV in a mammalian body component suspected of containing said antibodies comprising contacting said body component with a panel of HCV antigens, simultaneously or sequentially, comprising:
(a) a first HCV antigen from the C domain; and
(b) at least one additional HCV antigen selected from the group consisting of
(i) an HCV antigen from the NS3 domain;
(ii) an HCV antigen from the NS4 domain;
(iii) an HCV antigen from the S domain; and
(iv) an HCV antigen from the NS5 domain under conditions that permit antibody-antigen reaction and detecting the presence of immune complexes of said antibodies and said antigens.
Another aspect of the invention is a kit for carrying out an assay for detecting antibodies to HCV in a mammalian body component suspected of containing said antibodies comprising in packaged combination
(a) said combination of HCV antigens;
(b) standard control reagents; and
(c) instructions for carrying out the assay.
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Bradley et al., Posttransfusion Non-A, Non-B Hepatitis in Chimpanzees Physicochemical Evidence That the Tubule-Forming Agent Is a Small, Enveloped Virus. Gastroenterology 88:773-779, 1985.*
Choo et al., “Isolation of a cDNA clone derived from a blood borne non-A, non-B viral hepatitis genome”Science(1989) 244:359-362.
Geysen et al., “Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid”Proc. Natl. Acad. Sci. USA(1984) 81:3998-4002.
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Choo Qui-Lim
Houghton Michael
Kuo George
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Monroy Gladys H.
Wortman Donna C.
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