Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1996-11-12
2003-01-28
Stucker, Jeffrey (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007920, C435S015000, C435S975000, C436S518000, C436S528000, C436S531000, C436S538000
Reexamination Certificate
active
06511812
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates generally to a method for improving the specificity of immunoassays, and more particularly, relates to a method for adding denatured and/or purified bacterial enzyme to a specimen diluent to increase the specificity of immunoassays which utilize recombinant antigens expressed as fusion proteins with a bacterial enzyme.
Historically, immunoassays have been known to be prone to false positive reactions. Viral lysates or recombinant protein preparations, which comprise the typical antigen solutions which are utilize on the solid support, also can contain other proteins. These other proteins can bind to the solid support, introducing the possibility of reactivity with antibodies in the patient's sample. Alternatively, a component in the patient's sample can bind to the solid support and interfere with the assay.
For example, McFarlane et al.,
Lancet
(1990) 335:754-57, reported a high prevalence of antibody to hepatitis C virus (HCV) in patients with autoimmune chronic active hepatitis (AI-CAH). They suggested that the serum of AI-CAH patients may contain a component that gives false-positive results in the assay. McFarlane et al. surmised that the assay might have been non-specifically detecting IgG since they saw a correlation between IgG levels and OD values, or that factors contained in the patient sera were responsible for the results, such as an antibody against some other pathogen which cross-reacts with the antigen used in the assay, or a component which adheres to the solid phase and binds IgG. In addition, some false positive results are due to cross-reactivity between the patient sera and the fusion protein used to express the recombinant antigen utilized in the assay.
Since reactivity such as that described herein can be expected, assays can be designed to circumvent some of the problems generally associated with utilizing the recombinant protein. See, for example, P.C.T. Publication No. WO 92/13275, which corresponds to U.S. Ser. No. 08/059,868 (now U.S. Pat. No. 5,330,893, issued Jul. 19, 1994) which is incorporated herein by reference. In that publication, it is taught that the addition of superoxide dismutase to the specimen diluent is useful in increasing the specificity in assays which utilize recombinant proteins produced as fusion proteins with superoxide dismutase (SOD). One skilled in the art would be led, based on this teaching, to add such a bacterial enzyme to the specimen diluent in order to increase specificity in assays which utilize recombinant proteins.
Surprisingly, however, applicants have discovered that the mere addition of such a bacterial enzyme, even if expressed in the same bacterial host system, may not lead to increased specificity in all types of fusion proteins. Thus, it would be advantageous to provide a method and reagents which could increase the specificity in immunoassays when fusion proteins are utilized as the capture and/or indicator reagent in an assay.
SUMMARY OF THE INVENTION
The present invention provides an improved method for increasing specificity in immunoassays which comprises the steps of (a) mixing the specimen with a diluent comprising a denatured recombinant bacterial enzyme expressed as a fusion protein with said bacterial enzyme, and (b) contacting said diluted specimen with at least one recombinant antigen expressed as a fusion protein with said denatured bacterial enzyme. The recombinant denatured bacterial enzyme comprises a recombinant protein, and can be either CKS or SOD. Further, the concentration of said recombinant denatured bacterial enzyme is from about 0.001 g/L diluent to about 1.0 g/L diluent. More preferably, the concentration of said recombinant denatured bacterial enzyme is from about 0.01 g/L diluent to about 0.1 g/L diluent. Most preferred, the recombinant bacterial enzyme is at a concentration of about 100 &mgr;g/m. The method can detect numerous analytes, including antibody to hepatitis C virus (HCV) and antibody to human immunodeficiency virus (HW). Denaturation can be accomplished by a variety of methods, including heat and urea Further, purified, recombinant bacterial enzyme may be utilized
The present invention also provides a diluent useful for detecting antibodies in a test sample when performing an assay which uses at least one recombinant antigen expressed as a fusion protein with a recombinant bacterial enzyme, which diluent comprises the recombinant bacterial enzyme which has been denature. The concentration of said denatured recombinant bacterial enzyme is about 100 &mgr;g/ml. Purified, recombinant bacterial enzyme may be utilized.
The present invention also provides a test kit for performing an immunoassay which comprises a container containing a denatured recombinant bacterial enzyme selected from the group consisting of CKS and SOD.
DETAILED OF THE INVENTION
The present invention provides an improvement to methods for detecting antibodies in a specimen from an individual wherein a recombinant antigen employed in the method is expressed as a fusion protein by a vector and wherein the recombinant antigen is encoded with a gene of a bacterial enzyme such as CKS or SOD. The improvement comprises the step of mixing the specimen with a diluent comprising denatured recombinant bacterial enzyme (such as, SOD or CKS), wherein said enzyme also may be purified by methods known in the art as discussed herein, including anion-exchange resins and cation-exchange resins.
The denaturation of proteins by heat or chemical means is known in assay diagnostics when preparing serum samples for certain types of assay procedures. Denaturation of proteins occurs when the tertiary bonds of proteins are broken, which results in partial or complete unfolding of the protein. Usually, however, care is taken not to denature the protein solutions used for assays, since such denaturation can change the properties of such proteins, adversely affecting the results of the assay. See, for example, N. W. Tietz, ed.,
Fundamentals of Clinical Chemistry,
2nd Edition, W. B. Saunders Company, Philadelphia (1976), page 270.
Although it is known to add the recombinant bacterial enzyme utilized in the fusion protein to the assay diluent in order to improve assay specificity, it heretofore has not been known that the mere addition of such bacterial enzyme to a diluent may not result in the desired effect of increasing assay specificity by reducing the amount of false positive reactions. A false positive result is defined herein as one in which a sample is repeatably reactive in an ELISA, EIA or PHA but is not confirmed by alternative methods for the presence of analyte antibodies. Alternative testing methods include synthetic peptide EIAs, antibody blocking procedures and recombinant immunoblot assays.
Applicants have observed that in certain instances, addition of recombinant or purified recombinant bacterial enzymes does not result in increased specificity in assays which employ recombinant proteins. Applicants have surprisingly discovered that denaturaion of the recombinant bacterial enzyme results in increased specificity in assays. Also, applicants have discovered that at times it may be advantageous to utilize a purified, denatued, recombinant bacterial enzyme in a specimen diluent to increase the specificity of the assay.
It has been discovered that it is especially advantageous to add denatured, recombinant bacterial enzyme to a diluent (hereinafter defined) to improve the detection of analyte by increasing assay specificity. Immunoassays can employ recombinant antigens which are expressed as a fusion protein by a vector in which the antigen is encoded with the superoxide dismutase (SOD) gene or the CKS (CTP:CMP-3-deoxy--manno-octulosonate cytidylyl transferase or CMP-KDO synthetase) gene (see, U.S. Pat. No. 5,124,255, issued Jun. 23, 1992, which is incorporated herein by reference and U.S. Ser. No. 07/903,043 (now U.S. Pat. No. 5,322,769, issued Jun. 21, 1994), which is incorporated herein by reference). Therefore, those assays which utilize recombinant ant
Inoue Yuzo
Jomura Satoshi
Niedbalski Joseph S.
Schoen Robert C.
Sweeney Susan E.
Abbott Laboratories
Casuto Dianne
Stucker Jeffrey
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