Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-10-13
2004-07-20
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S007500, C435S007600, C435S007900, C435S007920, C435S007930, C435S007940, C435S975000, C436S514000, C436S518000, C436S524000, C436S810000, C436S825000
Reexamination Certificate
active
06764825
ABSTRACT:
Throughout this application various publications are referenced. The disclosures of these publications are hereby incorporated by reference into this application in order to more fully describe the state of the art to which the invention pertains.
The present invention relates to a device and diagnostic methods for detecting an antigen, such as prostate specific antigen (PSA), in biological samples. More particularly the present invention provides a PSA immunoassay system in which a range of PSA concentrations in a sample is determined by adjusting the sensitivity of detection of various zones containing different PSA monoclonal antibodies in order to detect and monitor prostate cancer.
BACKGROUND OF THE INVENTION
The prostate specific antigen (PSA) is a glycoprotein of 28,700 Daltons, consisting of 237 amino acid residues mainly secreted by the prostate gland secretory luminar cells lining the secretory duct. (McCormack R T et al.,
Urolozy
45,729-744, 1995).
Small quantities of PSA are normally found in the circulatory system. The amount of serum PSA can increase as carcinomas of the prostate develop and mature. Elevated serum PSA levels have been used to aid in the diagnosis and monitoring of prostate cancer, for example, for the early detection of prostate adenocarcinoma. (Rittenhouse, H. G. et al.,
Critical Reviews in Clinical Laboratory Sciences
, 35(4), 275-368, 1998).
As is conventionally understood or practiced in the field of urology, men having serum PSA concentrations less than 2 ng/ml generally are not diagnosed with, or considered to have, prostate cancer. However, when serum PSA concentration levels increase, the likelihood of being diagnosed with prostate cancer increases. For example, typically, one in nine men that have serum PSA concentration between 2 to 4 ng/ml will be diagnosed with prostate adenocarcinoma. When the concentration of serum PSA is between 4 and 10 ng/ml, one in four men will be diagnosed with prostate cancer, and when the levels increase above 10 ng/ml, the ratio is one out of two men. (Catalona, W. J. et al.,
Journal of the American Medical Association
, 274(15), 1214-1220, 1995 Catalona, W. J. et al.,
J. Urol
., 151, 1283-1290, 1994; Brawer, M. K. et al.,
J. Urol
., 147, 841-845, 1992
; Oesterling, J. F., J. Urol
., 145, 907-923, 1991).
Existing immunoassay systems used to detect or monitor prostate cancer incorporate one or more monoclonal antibodies (mAbs) capable of binding to any of the six different known major PSA epitopes (see Stenman U. H. et al.
Tumor Biology
, 20, suppl. 1, 1-12, 1999).
Generally, immunoassays can be categorized into quantitative or qualitative groups, as discussed below. The quantitative type of immunoassay is typically more expensive and relatively difficult to conduct. The qualitative immunoassays are relatively less expensive and easier to perform, but do not necessarily provide the amount or accuracy of information obtained with the quantitative immunoassays.
With respect to the quantitative type of immunoassays, conventionally known as “sandwich assays” and as conventionally practiced, one antibody is coupled to a solid support, and a second antibody is coupled to a detectable label. A test antigen having separate binding sites (epitopes) for the first and second antibodies is exposed to the antibody coupled to the solid support such that the antigen binds to that antibody. Subsequently, the labeled second antibody is added to the support to permit the binding of the labeled second antibody to the test antigen. Thus, the amount of the antigen present in a sample is a function of the amount of detected label bound to the second antibody bound to the antigen. Examples of such detectable labels include chromophores, radioisotopes, or enzymes which can be converted into a product that can be measured photometrically. When the amount of detected label is compared to the amount of antibody binding in a standard sample containing known amounts of antigen, quantitative results can be obtained. However, as indicated above, this procedure is typically complicated, time consuming, and expensive to perform compared to immuno-chromotography techniques described below, because this assay requires personnel training, complicated instruments, and test samples or standards, to be used during each measurement or assay.
To attempt to reduce the difficulty and expense of the quantitative immunoassay described above, immuno-chromatography methods have been developed. These tests provide qualitative information (e.g. a positive or negative result). The immuno-chromatography method typically utilizes a solid support such as a membrane strip having a region (a “reaction zone”) coated with a first antibody (a “capture antibody”) that is capable of binding to an antigen. The concentration of the capture antibody is empirically determined prior to the manufacture of the device. The concentration of the capture antibody is typically selected based on antibody/antigen binding data corresponding to the detection of an antigen above a single selected concentration threshold. The concentration threshold (i.e., the concentration of antigen that is believed to correlate with a disease condition) is chosen based on clinical or research data used in the diagnosis and/or monitoring of diseases having disease specific antigens. As discussed herein, clinicians typically begin to carefully monitor male patients for prostate cancer when their serum PSA levels are greater than 2 ng/ml. Thus, existing immunochromatography assays utilize a concentration of the capture antibody in the reaction zone that permits detection of PSA above a 2 ng/ml concentration threshold. A second antibody (a “detection antibody”), capable of binding the antigen at a different site, or epitope, from the first antibody, is usually coupled with color particles, such as colloidal gold or blue latex, and is applied in a solution having other factors, such as detergents, to facilitate solubilization of the labeled antibody onto a different region (a “reagent zone”) of the solid support, e.g., near one end of the membrane strip. The sample is then loaded on the membrane near the end that contains the detection antibody. The sample subsequently diffuses through the region with the detection antibody where the antigen binds to the detection antibody, and diff-uses continuously toward the region of the capture antibody. Because the detection antibody is applied to the reagent zone with solution components that increase solubilization of the antibody, the detection antibody is capable of diffusing with the antigen as it diffuses towards the other end of the membrane strip. When the antigen bound by the detection antibody interacts with the capture antibody, it is trapped in the reaction zone. If the test antigen present in the solution does not recognize the capture antibody, or it is present at a concentration lower than the concentration threshold determined by the capture antibody, the test antigen coupled with the detection antibody with the color particles will not bind to the membrane strip region containing the capture antibody, and thus, no staining will be present in the reaction zone, indicating that the antigen is present at a concentration less than the concentration threshold of the capture antibody (i.e. 2 ng/ml). Similarly, if no antigen is present in the sample, no binding will occur with either of the antibodies, and thus, no staining will occur. Therefore, a positive result is indicated by the presence of color in the reaction zone. The intensity of color correlates with the amount of bound antigen in the reaction zone. Thus, it is possible that the user will be able to make a more quantitative interpretation based on the degree of staining intensity.
However, as indicated above, these known immuno-chromatography-based PSA antibody assays only provide information of PSA concentration above a single value, or concentration threshold, for example 2 ng/ml, based on the clinical values discussed above, and do not provide multiple values or concentration thresh
Mandel & Adriano
Nguyen Bao-Thuy L.
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