Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-02-02
2003-03-25
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C436S018000, C436S176000
Reexamination Certificate
active
06537745
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related generally to the field of immunoassays and specifically to buffers for stabilizing antigens, in particular hepatitis C virus (HCV) antigens, for use in anti-HCV immunoassays.
BACKGROUND OF THE INVENTION
In general, immunoassays are produced by first determining epitopes that are specifically associated with a virus and then determining which of the epitopes is preferred for the assay being developed. When the particular epitopes are isolated, their sequences are determined, and genetic material for producing the epitopes is produced. Methods of producing proteins by either chemical or biological means are known, as are assays used to detect the presence of antibodies to particular epitopes. Highly selective and sensitive immunoassays generally contain major immunodominant epitopes of the pathogen suspected of infecting a patient.
For the virus HCV, major immunodominant linear epitopes have been identified from the core, NS3 (nonstructural), NS4 and NS5 regions of the virus polyprotein. HCV core protein and putative matrix proteins have been assayed against human serum samples containing antibodies to HCV and several immunodominant regions within the HCV proteins have been defined. Sallberg, et al.,
J. Clin. Microbiol.,
1992, 30, 1989-1994, incorporated by reference herein in its entirety. Protein domains of HCV-1 polyproteins including domains C, E1, E2/NS1, NS2, NS3, NS4, and NS5 have been identified and their approximate boundaries have been provided in WO 93/00365, incorporated by reference herein in its entirety. In addition, individual polypeptides having sequences derived from the structural region of HCV have been designed in order to obtain an immunodominant epitope useful in testing sera of HCV patients. Kotwal, et al.,
Proc. Natl. Acad. Sci. USA,
1992, 89, 4486-4489, incorporated by reference herein in its entirety.
The current assay of choice for HCV antibody detection is the Ortho 3.0 ELISA, a manual assay. Chiron-produced recombinant HCV antigens for use in the ELISA are c200 (ns-3, c100), c22 and NS-5. The c33c and c22 antigens are very immunogenic. Antibodies to c33c and c22 are also found in early seroconversion panels. The prevalence of HCV antibodies varies from 58% to 95% with the highest detection rate obtained for the c33c polypeptide followed by the c22 polypeptide. Chien. et al.,
Proc. Natl. Acad. Sci. USA,
1992, 89, 10011-10015, incorporated by reference herein in its entirety. However, problems of stabilizing HCV antigens in the liquid phase have been encountered. The lack of stability of HCV antigens in the liquid phase is a major disadvantage of the current HCV antibody detection assay. Therefore, developing an antigen buffer for the anti-HCV immunoassay has been attempted utilizing the same antigens as the Ortho 3.0 ELISA wherein the buffer stabilizes the HCV antigens. In addition, adapting the reagents, buffer and protocols to already existing automated machines, such as the ACS:Centaur has been attempted. Accordingly, there is currently a need to improve the stability of HCV antigens in the liquid phase for use in anti-HCV immunoassays. Such improved assay reagents and methods provide for better detection of HCV antibodies in screening of blood supplies and other biological fluids. It is contemplated that the buffers be can used for other antigens which may be unstable in the liquid phase, e.g. human immunodeficiency virus (HIV) antigens
SUMMARY OF THE INVENTION
In one aspect, the present invention is directed to an antigen diluent or buffer capable of stabilizing antigens in the liquid phase, in particular HCV recombinant antigens, comprising a reducing agent.
In another aspect, the present invention is directed to immunoassays using an antigen diluent or buffer containing a reducing agent.
In another aspect, an improved immunoassay kit is provided, the improvement comprising using an antigen diluent or buffer for HCV antigens containing a reducing agent.
DETAILED DESCRIPTION OF THE INVENTION
The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al.,
Molecular Cloning: A Laboratory Manual
(2nd Edition, 1989);
DNA Cloning: A Practical Approach
, Vols. I & II (D. Glover, ed.);
Methods In Enzymology
(S. Colowick and N. Kaplan eds., Academic Press, Inc.);
Handbook of Experimental Immunology,
Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); and
Fundamental Virology,
2nd Edition, Vols. I & II (B. N. Fields and D. M. Knipe, eds.).
Reagent stability over time is a critical issue. The c33c antigen diluted in buffer and tested the same day was functional using Magic Lite Assay protocols described below. However the reagent, when stressed at 37° C., lost more than 50% immunoreactivity to early seroconversion panels. The c33c in the liquid phase may slowly “aggregate” or become insoluble. Known components were tried in order to stabilize c33c immunoreactivity such as sugars, gelatin, glycerol, cross-linking reagents and anti-oxidants. It was discovered that keeping the c33c antigen in the reduced form can maintain immunoreactivity for periods over 24 hours, even up to at least 7 days, at 37° C. on early c33c seroconversion panels (matching Ortho 3.0 ELISA performance). The reducing agent reduces the disulfide bonds among cysteine groups within the c33c molecule, perhaps improving c33c immunoreactivity and solubility. There was no indication of antigen stability at 37° C. C for such lengths of time of conventional lite reagents in the liquid phase prior to the advent of the antigen diluent for c33c. Similar experiments were performed for c200 and a multiple epitope fusion antigen (MEFA-6) as shown below. Thus, the present invention provides antigen diluents or buffers for stabilizing HCV antigens for use in anti-HCV immunoassays. The antigen diluents or buffers of the present invention can be used in immunoassays such as, for example, ELISA and CLIA.
The present invention is directed to antigen diluents or buffers providing for improved stability of HCV antigens in the liquid phase. As used herein, “antigen diluents or buffers” refers to the solution in which the antigen is contained; it may or may not possess buffering capacity. In particular, the invention is directed to antigen diluents or buffers for improved stability for the recombinant HCV antigens in the Ortho 3.0 ELISA, and the like. The present invention was achieved by adding a reducing agent such as, for example, dithiothreitol (DTT) to the antigen diluent or buffer.
In a preferred embodiment of the invention, the HCV antigen diluent or buffer comprises a reducing agent. In another preferred embodiment of the invention, the HCV antigen diluent or buffer comprises sodium phosphate (pH 6.5), ethylenediaminetetraacetic acid (EDTA), DTT, gelatin, ammonium thiocyanate, sodium azide and SDS. However, these individual reagents can be replaced by similar reagents performing essentially the same function. For example, DTT can be replaced with additional reducing agents such as, for example, thioglycerol, mercaptoethenol, and the like. Sodium phosphate can be replaced by sodium borate and other buffers. Gelatin can be replaced with BSA and other blocking agents of non-specific binding. Sodium thiocyanate can be replaced with ammonium thiocyanate and other chaotropic agents. SDS can be replaced by a number of detergents such as, for example, Tween-20, and other detergents. Sodium azide can be replaced by other anti-bacterial agents. In addition, EDTA can be replaced by ethylene glycol-bis(&bgr;-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) and other chelating agents. One skilled in the art is familiar with reagents which can be substituted for those of the present invention.
In a preferred embodiment of the present invention, the HCV antigen dilue
Arcangel Phillip
Chien David Y.
Tirell Stephen
Zeigler Wanda
Blackburn Robert P.
Chiron Corporation
Harbin Alisa A.
Wortman Donna C.
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