Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-04-01
2002-03-26
Caputa, Anthony C. (Department: 1642)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007400, C435S023000, C436S064000, C436S536000, C530S412000
Reexamination Certificate
active
06361955
ABSTRACT:
The present invention concerns an immunological process for the determination of prostate-specific antigen (PSA), in particular for the determination of free PSA, the total PSA level and the concentration of PSA-serpin complexes by incubation of the sample with at least one antibody specifically binding to free PSA but not binding to complex PSA. The process is characterized in that a nucleophile containing amine is added to the sample before the determination of the total PSA concentration. Furthermore, the invention concerns the use of amine-containing nucleophiles for the production of free PSA as well as a process for preparing free PSA from complex PSA by incubation of a sample containing complex PSA with an amine-containing nucleophile.
The prostate-specific antigen is a glycoprotein with a molecular weight of 29 kDa. It is built in the epithelial prostate cells and is a component of the seminal fluid. PSA has the enzymatic activity of a neutral serine protease.
The main function of PSA is the cleavage of the seminogelines I and II as well as of fibronectin which—as essential ejaculation components—block the sperm mobility. By the hydrolysis of these proteins PSA liquefies the semen coagulum and thus enables the sperm mobility.
The enzymatically active PSA is inactivated in the serum by various inhibitors. The so-called serpins (=serine protease inhibitors) belong to the most important inhibitors which inactivate PSA by forming covalent complexes. The main amount (60-95%) of the immunologically detectable PSA is bound in the serum to &agr;1-antichymotrypsin (=ACT), which belongs to the serpins.
Further complexes can be formed with &agr;1-antitrypsin and protein C inhibitor which are, however, in comparison to PSA-ACT, only of secondary importance in the serum.
In addition, PSA also forms a complex with a different type of protease inhibitor, i.e. the &agr;2-macroglobulin (&agr;2-M). Information on the complex concentration in the serum are different in the literature which is particularly due to the immunological inaccessibility of the PSA in this complex. The serum also contains enzymatically inactive free PSA which cannot form complexes. In the following “total PSA” means the sum of free PSA and serpin-complex PSA since the PSA complex with &agr;2-M is not registered in any immunological determination performed until now. (Teware and Bluestein, J. Clin. Ligand Assay 1995, vol. 18, p. 186-196).
&agr;1-antichymotrypsin is a glycoprotein with a molecular weight of approx. 60 kDa. As one of the main inhibitors in the acute phase ACT plays an important role in the control of inflammation. ACT also forms complexes with chymotrypsin, cathepsin G and glandular callicrein hK2. In human serum the molar ACT concentration is 10,000 times higher than that of PSA.
Due to the occurrence of different PSA forms the conditions for determination of the PSA concentration in human sera for the diagnosis of a prostate carcinoma or a benign disease are very complex. This fact evokes a number of diagnostic difficulties and reduces the value of this marker to a certain extent. Thus, it is known that some of the available assays for the determination of PSA give different values of free and complex PSA due to the specificity of the antibodies used thou et al., Clin. Chem. 1993, Vol. 39/12, p. 2483-2491; McCormack et al., Urology 1995, Vol. 45, p. 729-744; Tewari and Williams, Clin. Chem. 1998, Vol. 44, p. 191-192; Blase and Sokoloff, Clin. Chem. 1998, Vol. 44, p.192-193). In addition, an exact and uniform standardization is difficult because of the occurrence of the different forms mentioned (Chen et al., Clin. Chem. 1995, Vol. 41, p. 1273-1282).
The reliability of the PSA serum values as an indicator of a prostate carcinoma (PCa) up to a concentration of approx. 15 ng/ml is particularly problematic since such concentrations of total PSA can also result from the occurrence of a benign prostate hyperplasia (BPH). A prostate carcinoma screening by simple determination of the PSA value in the serum is not possible due to the lacking specificity in this concentration range; further investigations which are partly complex and painful (biopsy) must be performed to find out whether a PCa has occurred or not.
During the last years the determination of the ratio of free, i.e. uncomplexed PSA to complexed PSA or total PSA could improve the specificity to a certain extent (see e.g. WO 92/01936). It is, however, still clearly below a specificity value which would be necessary to avoid a larger number of unnecessary and time-/cost-intensive further examinations. The improvement of the specificity is based on the fact that in sera of patients with BPH the average ratio of free to complex PSA (PSAfree/PSAtotal) is higher than in sera of PCa patients.
For state of the art establishment of the PSAfree/PSAtotal ratio two different measurings must be performed. First of all the concentration of free PSA must be determined. If this test for free PSA is carried out in the sandwich format at least one of the two antibodies must be specific for free PSA and must not bind to complex PSA. Simultaneously, the total PSA concentration must be established in a second separate measurement. In this case the antibodies used in the sandwich test bind to free and complex PSA. Alternatively, the total concentration can also be determined by measuring the PSA-ACT concentration. In the WO 92/01936 such an antibody binding to free and complex PSA as well as an antibody specifically binding to ACT is used for the detection of the PSA-ACT concentration.
The total PSA amount can be achieved by adding the measuring value of the PSA-ACT complex and the measuring value of free PSA.
A big disadvantage of the state of the art process is that two different tests are performed which involve each at least two antibodies with a different specificity. The use of different reagents, e.g. antibodies, in several test procedures for the determination of a diagnostically relevant parameter very probably leads to problems when comparing tests of different manufacturers, particularly when such a complex diagnostic problem like that of PSA is regarded. In addition, the fault probability of the determination of reagents rises with the number of test systems used and reagents required. Furthermore, the provision of a large number of different, generally monoclonal antibodies is very cost- and time-intensive.
The task was therefore to develop an improved and simplified immunological process for the detection of PSA, in particular for the detection of free PSA, the total PSA concentration and the concentration of PSA-serpin complexes to overcome the state of the art disadvantages to a large extent.
The task is fulfilled with an improved immunological process for quantitative determination of PSA by incubation of the sample with at least one antibody binding specifically to free PSA but not to complex PSA, wherein an amine-containing nucleophile is added to the sample before the determination of the total PSA concentration. This procedure is preferably used for the quantitative determination of the concentration of free PSA, total PSA and of the PSA-serpin complexes.
The complex of PSA and serpins, in particular ACT, is stable and withstands drastic temperature and pH conditions (McCormack et al., Urology 1995, Vol. 45, p. 729-744). Surprisingly, it has been shown that the PSA-serpin complex can be cleaved by adding amine-containing nucleophilic reagents.
HPLC analyses have shown that the resulting PSA is stable. Surprisingly, this is also the case in the presence of serum, i.e. no new complexes are built. This can be recognized by the value of free PSA after the incubation under cleavage conditions which corresponds to the total PSA value before the cleavage. This result is surprising because free PSA in the serum actually should have been complexed again due to the very high excess of the protease inhibitors &agr;1-antichymotrypsin and (&agr;2-macroglobulin. This is at least the case when additional PSA is added to a human serum sample (Mai
Hösel Wolfgang
Peter Jochen
Unverzagt Carlo
Amick Marilyn L.
Caputa Anthony C.
Holleran Anne L.
Roche Diagnostics Corporation
Roche Diagnostics GmbH
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