Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-01-10
2003-10-14
Ungar, Susan (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
Reexamination Certificate
active
06632623
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to an immunoassay for the quantitative determination of the amount of the complex (PSA-A
2
M) between prostate specific antigen (PSA) and &agr;
2
-macroglobulin (A
2
M) in a serum sample. The invention relates further to an improved method for differentiating patients with cancer of the prostate (PCa) from patients with benign prostatic hyperplasia (BPH) or healthy male subjects without PCa.
BACKGROUND OF THE INVENTION
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
In the following description, the abbreviation “PSA” means prostate-specific antigen; A
2
M &agr;
2
-macroglobulin; PSA-A
2
M complex between PSA and A
2
M; ACT &agr;
1
-antichymotrypsin; PSA-ACT complex between PSA and ACT; API &agr;
1
-protease inhibitor; PSA-API complex between PSA and API; MAb monoclonal antibody; BSA bovine serum albumin; TBS 50 mmol/L Tris-HCl buffer, pH 7.4 containing 150 mmol/L sodium chloride and 8 mmol/L sodium azide; IFMA immunofluorometric assay; SDS sodium dodecyl sulfate; SDS-PAGE: SDS polyacrylamide gel electrophoresis; BPH benign prostatic hyperplasia; PCa prostate cancer; CV coefficient variation; ROC receiver operating characteristic; AUC area under the curve; and sandwich assay for PSA-A
2
M a sandwich assay using a monoclonal anti-PSA antibody as catcher and a labelled antibody to A
2
M for detection.
In the definition of the invention and in the results disclosed below, the term “total PSA” shall be understood to mean the total amount of immunodetectable PSA in a sample before any denaturation or other treatment of the sample to render other, under normal conditions non-immunodetectable forms of PSA immunodetectable.
Prostate-specific antigen (PSA) is a 30 kDa single chain glycoprotein mainly produced by prostatic epithelium (1-3). It is a serine protease with chymotrypsin-like enzymatic activity and a member of the glandular kallikrein family (4-6). In vitro PSA forms complexes with protease inhibitors such as &agr;
2
-macroglobulin (A
2
M), pregnancy zone protein, &agr;
1
-antichymotrypsin (ACT), &agr;
1
-protease inhibitor (API) and protein C inhibitor (6-9).
In serum most of the immunoreactive PSA occurs in complex with ACT (PSA-ACT), while the rest is either free or in complex with API (PSA-API) (10-12). Five major antigenic regions have been identified on the PSA molecule and only one of them is covered by ACT in PSA-ACT (13). The PSA-ACT and PSA-API complexes can be readily detected by specific sandwich assays or by conventional PSA immunoassays (9, 10, 14). Specific measurement of complexed and free PSA in serum improves the diagnostic accuracy for prostate cancer (PCa) compared to assay of total PSA (10, 12, 14, 15).
A
2
M is a tetramer assembled from pairwise disulfate-bridged 180 kDa subunits (16, 17). Binding of proteases to A
2
M is initiated by a proteolytic attack of the bait region of A
2
M resulting in a comformational change and entrapping of the enzyme within the A
2
M molecule (18). This sterically hinders access of high molecular weight substance such as high molecular weight inhibitors or antibodies to the enzymes (19). The PSA-A
2
M complex is not detected by conventional PSA immunoassays (8, 11). When PSA-A
2
M is denatured with sodium dodecyl sulfate (SDS), PSA epitopes are exposed rendering it reactive with PSA antibodies (11, 20). PSA-A
2
M has been qualitatively detected in male serum with high levels of PSA by immunoblotting after SDS polyacrylamide gel electrophoresis (SDS-PAGE) (20).
The design of two-site immunoassays for the determinalion of circulating PSA-A
2
M complexes have recently been reported (27 and 31). These assys combine antibodies recognizing epitopes on PSA with antibodies recognizing epitopes on A
2
M (the native and/or the transformed molecule). Such assays are feasible since some epitopes of PSA, while in complex with A
2
M, are still to some extent accessible to certain anti-PSA antibodies. It is also possible to enhance this recognition by partly denaturing the PSA-A
2
M complex e.g. by the use of SDS treatment. However, the analytical sensitivities using these assays are still clearly inferior compared to state-of art assays of circulating free, ACT-complexed or total PSA assays, which fact is explained by the poor accessability of anti PSA antibodies to PSA in complex with A
2
M. The large excess of free A
2
M or A
2
M complexed to other serpines may also introduce non-specific interferences with the potential appearance of false signal elevations unrelated to the presence of PSA-A
2
M.
The International Patent Publication WO 92/01936 (30) discloses the use of the ratio between free PSA and total PSA, between a PSA-proteinase inhibitor complex and total PSA, or between free PSA and PSA-proteinase inhibitor complex, as a marker to distinguish between PCa and BPH. As one of the PSA-proteinase inhibitor complexes is mentioned PSA-A
2
M. However, no method for its detection was described.
OBJECT AND SUMMARY OF THE INVENTION
The object of the present invention is to provide a novel sensitive method for the quantitative determination of the PSA-A
2
M complex in an individual's serum sample.
Another object of the invention is to provide an improved method for differentiating PCa patients from BPH patients or healthy male subjects without PCa, based on the use of a new marker comprising the information on the amount of the PSA-A
2
M complex in an individual's serum sample.
Thus, according to one aspect, this invention concerns an immunoassay for quantitative determination of the amount of the complex (PSA-A
2
M) between prostate specific antigen (PSA) and &agr;
2
-macroglobulin (A
2
M) in a sample. Said method is characterized by the steps of
removing the immunoreactive PSA from said sample,
treating the PSA-A
2
M complex in the remaining supernatant so as to make the PSA thereof immunoreactive,
determining the immunoreactive PSA derived from the PSA-A
2
M complex by exposing it to an antibody which binds said immunoreactive PSA, and
detecting said PSA.
Furthermore, this invention concerns a method for differentiating patients with cancer of the prostate (PCa) from patients with benign prostatic hyperplasia (BPH) or healthy male subjects without PCa, wherein the individual's body fluid concentration of prostate specific antigen (PSA) has been determined as free PSA and as total PSA. Said method is characterized in that PSA complexed with A
2
M (PSA-A
2
M) in the individual's serum sample has been determined, and that the ratio between PSA-A
2
M and other forms of PSA is calculated, or that the diagnostic value is calculated by logistic regression, neural networks, fuzzy logic, or similar mathematical and statistical methods, using PSA-A
2
M and other forms of PSA, such as total PSA, free PSA and PSA-ACT as input variables.
REFERENCES:
patent: 197 17 436 (1998-10-01), None
patent: 0 196 846 (1985-03-01), None
patent: WO 92/01936 (1992-02-01), None
patent: WO 98/32021 (1998-07-01), None
Tewari, P. et al. (1995). “Immunological characterization and quantitation of prostate specific antigen-&agr;2-macroglobulin complex in prostate cancer patient sera”,Clin. Chem.41(6):226.
Zhou, A.M. et al. (1993). “Multiple Forms of Prostate-Specific Antigen in Serum: Differences in Immunorecognition by Monoclonal and Polyclonal Assays,”Clin. Chem.39(12):2483-91.
Zhang, W-M. et al. (1998). “Characterization and immunological determination of the complex between prostate-specific antigen and &agr;2-macroglobulin,”Clin. Chem.44(12), 2471-79.
Chekhonin, V.P. et al. (1997). “Identification physico-chemical properties and immunochemical characteristics of prostate specific antigen and its complexes with protease inhibitors,”Vopr. Med. Khim.43(3):165-71 (May-Jun.) (article in Russian, abstract in English).
Chen, Z. et al. (1996). “Addition of Purified Prostate Specific Antigen to Serum from Female Subjects: Studies on the Relative Inh
Davis Minh-Tam
Rothwell Figg Ernst & Manbeck
Ungar Susan
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