Liquid purification or separation – Processes – Liquid/liquid solvent or colloidal extraction or diffusing...
Reexamination Certificate
2000-07-24
2002-04-30
Therkorn, Ernest G. (Department: 1723)
Liquid purification or separation
Processes
Liquid/liquid solvent or colloidal extraction or diffusing...
C210S656000, C210S198200, C435S007230, C435S007400
Reexamination Certificate
active
06379550
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to prostate specific antigen (PSA) and its protein complexes as may be found in blood serum, ascites, tissues, tumors and seminal fluid.
Prostate cancer is one of the most frequently diagnosed cancers among U.S. men, and is the second most common male cancer and is a leading cause of male cancer related mortality. In the United States over 35,000 men die annually from prostate cancer. Since its discovery, prostate specific antigen (PSA) has become the most valuable tool for the diagnosis and management of prostate cancer.
PSA, a single chain glycoprotein of approximately 30 kDa, is a member of the human kallikrein gene family, which consists of hKLK
1
, hKLK
2
and hKLK
3
(PSA). All three genes are clustered within 60-kb genome region on chromosome 19 Q13.3-q 13.4. The PSA gene has five exons and encodes a 237 amino acid mature protein which is secreted. PSA protein is glycosylated at a single site (asparagine 45). Each PSA molecule has six immunoreactive binding sites.
PSA is primarily produced by prostate epithelial cells and is secreted into seminal fluid to a high concentration. PSA at low concentrations has been found in endometrium, normal brain tissue, breast tumors, breast milk, adrenal neoplasm and renal cell carcinoma. PSA circulates in the serum as uncomplexed or free form and complexed or bound form. In the serum most of the PSA is complexed with &agr;
1
-antichymotrypsin (ACT) (1,2); and &agr;
2
-macroglobulin (A
2
M) (3). A small portion of PSA is bound to &agr;
1
-protease inhibitor (API). A complex between PSA and protein C-inhibitor (PCI) is present only in seminal plasma.
Despite the wide spread acceptance and use of serum PSA as a marker, for the early detection of prostate cancer, the specificity of this PSA test is relatively low. The yield of cancer in a screening population is only 22-30%; which means that 70-80% of all test results, indicating that a biopsy should be performed, are false positives. Progressively rising PSA levels above the “normal range” of 0-4 ng PSA/ml are one of the earliest signs of prostate cancer. As a regular screening for prostate cancer becomes the standard of care, improved specificity in detection of prostate cancer is needed to avoid costly, unnecessary biopsies. PSA is produced by malignant as well as by non-malignant prostate epithelial cells. Therefore, there is a substantial overlap in total PSA levels between men with prostate cancer, benign prostatic hyperplasia (BPH) and chronic prostatitis. Recently, the measurement of the ratio between total PSA and free PSA has been introduced as a useful clinical tool for the early detection of localized prostate cancer in order to increase reliability of the test.
Different commercial PSA immunoassays usually give different results in the same patients. This underscores the need to standardize PSA assays. It is particularly important that PSA immunoassays be standardized in the range of 0-10 ng PSA/ml, because this is the most critical range for a prostate cancer screening program.
The process of standardization of the PSA-immunoassays requires several steps: 1) a standard method for PSA isolation in its native forms must be defined, 2) a method to preserve native PSA for a reasonable period and 3) a serum based PSA standard is needed. This is essential because most of the clinical decisions for patient care are based upon measurement of serum PSA levels whereas most, if not all, PSA standards currently used are from a seminal plasma source. Additionally, recent data indicate that analytical characteristics of seminal fluid PSA differs from that of serum PSA. Current PSA immunoassays are designed to measure total PSA in the serum. To evaluate differences in such assays, one should use “serum standards” containing different proportions of free and complexed PSA and not free PSA obtained from seminal plasma. It has been proposed that only the complexed forms of PSA be used as the internal antigen calibrator for PSA immunoassays. However, at present, no such standards are available.
Current PSA quantitation methodology estimates either free PSA, total PSA or PSA that complexes with &agr;
1
antichymotrypsin (PSA-ACT). It, however, does not include other known complexes such as &agr;
2
macroglobulin complex with PSA (PSA-A
2
M); &agr;
1
-protease inhibitor complex with PSA (PSA-API) and a complex between PSA and protein C inhibitor present only in seminal fluid (PSA-PCI). This is the reason why total PSA determination is often higher than sum total of free PSA and PSA-ACT complex. In order to correctly define the role of different molecular forms of PSA in immunoassays it is essential that an entire panel of PSA molecular forms (free and complexed forms) found in the patient samples, e.g., serum, be represented in standardization of PSA assays and in preparation of a PSA calibrator.
All currently available methods for the quantitation of PSA involve use of monoclonal and polyclonal antibodies to measure free PSA and PSA-ACT complex, usually in the serum. Regardless of the assay procedure used, the value of total PSA always exceeds the sum of free PSA and PSA-ACT complex by up to 30%. Possible reasons for this discrepancy may be: 1) presence of other known PSA-complexes (PSA-A
2
M, PSA-API), 2) other unknown PSA-complexes, 3) differences in specificity of anti-PSA antibodies-as they are prepared against free PSA from seminal plasma and may have different affinity for free PSA than PSA-complexes, 4) differences in the degradation rate of different PSA molecular forms, 5) use of seminal plasma based PSA as an internal antigen calibrator, 6) use of solid phase for immobilizing the capture antibody which may affect the kinetics of antigen-antibody interaction and 7) any combination of these.
For the most part, the source for the isolation of prostate specific antigen (PSA) for clinical and laboratory use has been seminal plasma and rarely from cultured prostate tumor cells. As of today, all internal standards used for monitoring PSA in patient serum are prepared from seminal plasma. However, the biochemical nature of PSA from seminal plasma and from patient blood may not be identical. Thus far no one has purified PSA or any of its molecular forms directly from the patient serum. Most identification of PSA in patient serum is based upon immunological detection and quantitation using monoclonal and polyclonal antibodies. The validity and accuracy of such measurements largely depends upon the quality and nature of each antibody used. There is no single antibody known today that can capture free PSA and all of its molecular forms.
Thiophilic adsorption chromatography was first introduced by Porath and coworkers in 1985 employing a thiophilic gel containing a sulfone group and at least one thioether function. The synthesis of the gel consisted of coupling 2-mercaptoethanol to agarose that has been activated by divinylsulfone. The adsorption of proteins to the stationary phase of their particular gel and many others is promoted by high concentration of lytropic-salts such as sodium, potassium and ammonium sulfates, whereas desorption is achieved by decreasing salt concentration. Thiophilic adsorption chromatography has been largely used for the purification of immunoglobulins of different classes: IgA, IgG and IgM from different species and for purification of polyclonal antibodies from serum and monoclonal antibodies from tissue cellular supernatants and from ascitic fluid. Ion exchange effects are excluded in view of high salt concentration in the adsorption step. There has, however, been no recorded indication or suggestion that thiophilic gels might have applicability to isolation or purification of PSA.
Thiophilic gels have pendant surface ligands attached to a hydrophilic solid support, e.g. agarose or polyacryamide. The surface structures are ligands containing hydrophilic electron donor and acceptor groups. Commercially there are three different thiophilic gels that contain one, two and three sulfur containing groups respectively.
T
Chadha Kailash C.
Kawinski Elzbieta
Sulkowski Eugene
Dunn Michael L.
Health Research , Inc.
Therkorn Ernest G.
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