Chemistry: analytical and immunological testing – Involving immune complex formed in liquid phase – Separation of immune complex from unbound antigen or antibody
Reexamination Certificate
1998-07-20
2003-11-18
Caputa, Anthony C. (Department: 1642)
Chemistry: analytical and immunological testing
Involving immune complex formed in liquid phase
Separation of immune complex from unbound antigen or antibody
C436S536000, C436S538000, C436S518000, C436S529000, C436S525000, C435S007100, C435S007200, C435S007210
Reexamination Certificate
active
06649420
ABSTRACT:
TECHNICAL FIELD
The present invention relates to novel methods and devices for detecting non-complexed prostate specific antigen (PSA), which can be used either alone or in conjunction with total PSA tests to identify patients having either benign prostatic diseases (BPD), such as benign prostatic hyperplasia, prostatitis, or glandular atrophy or prostatic adenocarcinoma (CAP). In a biological sample, one can find not only non-complexed PSA, but also PSA which has formed a complex with &agr;1-antichymotrypsin (ACT). The present invention removes or precipitates complexed PSA (PSA-ACT) and ACT from a fluid sample, thereby removing any possible interference due to the binding of complexed PSA to assay reagents. The method requires contacting a biological fluid sample possibly containing a mixture of complexed PSA and non-complexed PSA either with an immuno-precipitating reagent or a device having attached an ACT specific binding partner which in both cases specifically binds only to ACT and the ACT portion of complexed PSA, thereby precipitating ACT and the bound complexed PSA and, thus, leaving any non-complexed PSA unbound and in solution. Then, the sample is measured for non-complexed PSA by means of a conventional specific binding reaction. The device can be a filter having a modified filter media that binds ACT and complexed PSA, while permitting the sample to flow or be pulled through the filter media and into assay reagents. Alternatively, the device can be a removable media that is placed into a sample, allowed to remain long enough to bind any ACT and complexed PSA present, and then withdrawn from the sample prior to running a PSA specific binding assay.
BACKGROUND ART
PSA is recognized as a molecular marker for CAP. Blood or serum based immunoassays measuring the total PSA level have been commercially available for a number of years. However, the detection of total PSA does not necessarily mean that a patient has CAP. In order to distinguish CAP, a total PSA test has to satisfy two elements: a high sensitivity—the ability to detect disease when present, and a high specificity—the ability to detect true negatives and avoid false positives. From clinical experience, total PSA tests have become generally accepted as being predictive of CAP if the total PSA level is greater than 10.0 ng/ml. Total PSA values between 0.0 ng/ml and about 3.9 ng/ml have been considered generally predictive of no disease being present, with a value of about 3.5 ng/ml being used for men under 60 years old and about 2.5 ng/ml being used for men under 50 years old. (See Oesterling, J. E., Cooner, W. H., Jacobsen, S. J., Guess H. A., and Lieber, M.M.: “
Influence of Patient Age on the Serum PSA Concentration and Important Clinical Observations
”: Urol. Clin. North Am.; Vol. 20: 671-680, 1993.)
PSA is primarily organ-specific, not cancer specific. Thus, PSA in blood or serum can result not only from CAP, but also from normal or hyperplastic prostate tissues. Historically, a total PSA test cannot reliably distinguish BPD from CAP at less than 10.0 ng/ml. Studies have found that 43% (136/319) of patients with organ-confined CAP have a total PSA value within the normal range of less than 4.0 ng/ml. Moreover, about 25% (148/597) of men with BPD have a total PSA value above 4.0 ng/ml. (See Oesterling, J. E.: “
Prostate Specific Antigen: A Critical Assessment of the Most Useful Tumor Marker for Adenocarcinoma of the Prostate
”, J. Urol., Vol:145: 907-923, 1991.) Standard medical practice is to biopsy patients over 60 years old having total PSA levels of between 4.0 ng/ml and 10.0 ng/ml because about 30% of those patients have CAP. Likewise, patients between 50 years and 60 years old whose total PSA falls between 3.5 ng/ml and 10.0 ng/ml and patients under 50 years old whose total PSA falls between 2.5 ng/ml and 10.0 ng/ml are also biopsied under current medical practice.
One method for detecting CAP is disclosed in U.S. Pat. No. 5,501,983 to Hans Lilja et alia. In general, the Lilja patent discloses using immunoassays to measure “free PSA” and a complexed form of PSA. “free PSA” is a 33 kDa single chain glycoenzyme that is produced by the epithelial cells lining the acini and prostatic ducts of the prostate gland. Complexed PSA refers primarily to a 90 kDa complex of PSA bound to &agr;1-antichymotrypsin (ACT) protein. Free PSA and complexed PSA, and their proportions are applied in the diagnosis of patients with CAP. Throughout, the specification discloses using a combination of a free PSA to total PSA (F/T) proportion and a complexed PSA to total PSA (C/T) proportion for use in diagnosing CAP. No prostate needle biopsy were performed on the patients, and the patients covered a full range of total PSA values. The text provides no guidance as to specifically how one uses these proportions.
A significant advance in diagnosing BPD in a male human patient without requiring a biopsy is disclosed by Luderer, A. A., et alia in “
Measurement of the Proportion of Free to Total Prostate-Specific Antigen Improves Diagnostic Performance of Prostate-Specific Antigen in The Diagnostic Gray Zone of Total Prostate-Specific Antigen
”, Urol., Vol. 46: 187-194, 1995. This reflex method eliminates the need for about one-third of those patients to undergo such a biopsy. For those patients in the gray diagnostic zone, the method comprises four steps. First, one measures the total PSA level in the blood or serum of the patient. Second, one measures the free PSA level in the blood or serum of a patient, but only if he has a total PSA level of between about 2.5 ng/ml and about 10.0 ng/ml. If the patient has a total PSA level below 2.5 ng/ml, then he is diagnosed to have BPD. If the patient has a total PSA level above 10.0 ng/ml, then he is presumed to have CAP and must be biopsied. Third, one calculates the proportion of free PSA to total PSA. Fourth and finally, one diagnoses the patient as having BPD if the calculated proportion of free PSA to total PSA is equal to or greater than about 25%.
Another method for detecting complexed PSA has been proposed by Yeung, K. K., et alia, in “
Novel Immunoassay for the Measurement of Complexed Prostate-Specific Antigen in Serum
”, Clin. Chem., Vol. 44:1216-1223, 1998. In this assay an antibody is used to bind to a particular epitope, rendering a potential assay interferent unable to be detected by immunoassay antibodies. In particular, antibodies specific for the region on PSA where ACT binds to PSA are used to change the conformation of non-complexed PSA, thereby rendering the non-complexed PSA unable to be detected by immunoassay antibodies that can bind only to complexed PSA. Only antibodies which cause the desired conformational change will work.
The removal of an unwanted component from a biological sample is disclosed in U.S. Pat. No. 5,403,745 to James F. Ollington et alia. When assaying for a cholesterol analyte in a targeted lipoprotein class, there can be present in a sample at least one cholesterol-containing interfering substance in another lipoprotein class. This interfering substance is removed by binding the interfering substance with immobilized antibodies.
DISCLOSURE OF THE INVENTION
The present invention relates to a method for detecting non-complexed PSA in a biological sample using an assay that incorporates a specific binding reaction. For the purposes of this invention, a distinction is made between “free PSA” and “non-complexed PSA”. “Free PSA” is defined as the form of PSA that is measured by antibodies disclosed by Lilja, namely, those which will normally recognize an epitope on PSA when PSA is not complexed with ACT, but will not recognize that same epitope if the PSA is complexed with ACT. “Non-complexed PSA” is the PSA moiety which remains after all ACT moiety in a mixture of free PSA and complexed PSA has been specifically bound by an antibody and removed or precipitated. The distinction between the two is important. In some cases, the Lilja method can detect as free PSA what is really complexed PSA. This erroneous detection occurs because in some samples the
Caputa Anthony C.
Holleran Anne L.
Voyce Brian D.
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