Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-03-28
2004-04-20
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S518000
Reexamination Certificate
active
06723502
ABSTRACT:
BACKGROUND OF THE INVENTION
An estimated 170 million people worldwide have been infected by hepatitis C virus (HCV). In the next few years, the number of U.S. deaths for HCV-caused liver disease and cancer may overtake deaths caused by Acquired Immune Deficiency Syndrome (AIDS).
The transmission of HCV seems to require blood-to-blood contact. Carrying a single strand of ribonucleic acid (RNA), HCV contains just one gene, coding for a polyprotein that is subsequently spliced into at least 10 functional proteins. Clearly, the ability to test the blood supply for HCV is of great importance. Furthermore, the earlier the stage of HCV one can detect, the better.
Therefore, we have developed an ELISA based assay for the detection of HCV infection. This new assay can detect HCV infection earlier than the currently used assays for the screening of blood for HCV infection. Current assays are based on the detection of anti-HCV antibodies in infected blood by capturing these antibodies by HCV protein sequences represented by recombinant proteins or peptides.
For example, the Ortho HCV 3.0 ELISA uses multiple antigens to detect anti-HCV antibodies very early in seroconversion. However, there is a significant window period in viremic individuals before they develop anti-HCV antibodies. This window period can be as long as 60 days. During this period the individual, though highly infective, will go undetected by current HCV screening assays.
SUMMARY OF THE INVENTION
One object of the invention is to provide a diagnostic test that can detect HCV infection during the window period described above by capturing both HCV core antigen and antibodies. We describe herein a method for detecting HCV infection earlier than the currently used antibodies assays. This has been accomplished by providing a test that detects HCV core antigen in addition to the anti-HCV antibodies.
Another object of the invention is to provide a method for determining the presence of HCV in a sample, comprising, contacting the sample with HCV antigens and anti-HCV core antibodies attached to a solid phase, adding a polyoxyethylene ether to said sample, detecting captured antigens and antibodies by adding labeled anti-human IgG and labeled anti-HCV core antibodies and detecting the signal as an indication of the presence of HCV infection.
Another object of the invention is to provide certain detergents that are needed to release the HCV core antigen from the virus by disrupting the envelope protein and/or the lipid layer.
DETAILED DESCRIPTION OF THE INVENTION
It is preferrable in a combination assay for the detergents to release the core antigen from the virus and yet not have a negative impact on the ability of the HCV recombinant proteins to capture anti-HCV antibodies. In a preferred embodiment of the invention, detergents from the polyoxyethylene ethers class are used for this purpose. Commonly available detergents in this class are: BRIJ 30, BRIJ 35, 56, 58, 92, 96, 98, 700 and MYRJ 52, 59, 53, 45. These detergents help in releasing the HCV core antigen from the virus but the presence of these detergents do not negatively impact the anti-HCV assay Certain detergents can effect the antibody detection by either affecting the recombinant antigens coated on the solid phase or inactivating the anti-HCV antibodies in the sample. Some of the detergents like N-Lauryl Sarcosine used in the detection of HCV core assay destroy the ability of HCV recombinant proteins c22-3, c200 and NS5 to detect anti HCV antibodies in Ortho HCV 3.0 ELISA.
As used herein a “sample” refers to any substance which may contain the analyte of interest. A sample can be biological fluid, such as whole blood or whole blood components including red blood cells, white blood cells, platelets, serum and plasma, ascites, urine, cerebrospinal fluid, and other constituents of the body which may contain the analyte of interest.
As used herein “antigen” rerers to any antigenic substance including recombinant proteins and peptides or a mixture thereof.
REFERENCES:
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patent: 2002/0192639 (2002-12-01), Chien et al.
patent: 1 020 727 (2000-07-01), None
Aoyagi et al., Development of a Simple and HIghly Sensitive Immunoassay for Hepatitis C Core Antigen. Journal of Clinical Microbiology 37(6):1802-1808, 1999.*
The Merck Index, Eleventh Edition, Budavari et al., Eds. 1989, Monograph 6681.*
Lee, S.R. et al., “Efficacy of a hepatitis C virus core antigen enzyme-linked immunosorbent assay for the identification of ‘window-phase’ blood donations”, Vox Sanguinis, vol. 80, pp. 19-23, Jan. 2001, XP02210662.
Courouce AM et al., “Efficacy of HCV core antigen detection during the preseroconversion period”, Transfusion, vol. 40, pp. 1198-1202, Oct. 2000, XP002210663.
European Search Report, dated Sep. 16, 2002, for European Appln. No. EP 02252195.
Bahl Chander
Madjor Denise
Niven Patrick
Samson Antonio
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