Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Patent
1993-03-19
1996-03-26
Vogel, Nancy T.
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
436514, 436801, G01N 33543, G01N 33558, G01N 33566
Patent
active
055019838
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to an immunoassay of prostate-specific antigen (PSA), in which specific reagent materials (antibodies) are used that allow the measurement of free PSA as well as the PSA proteinase inhibitor complex.
It also relates to the use of free PSA and the PSA proteinase inhibitor complex and their ratio as a useful marker in diagnosis of patients with prostate cancer.
BACKGROUND OF THE INVENTION
The prostate specific antigen (PSA) was first purified from prostatic tissue (Wang et al. Invest Urol 1979), but the same protein was almost simultaneously and independently characterized in the seminal plasma (Hara et al. J Lab Clin Med 1989; Graves et al. N Engl J Med 1985). PSA is now known to be a 33-kDa glycosylated single chain serine protease (Lilja, J Clin Invest 1985; Watt et al. Proc Natl Acad Sci (USA) 1986). The 237 amino-acid polypeptide backbone has extensive similarities with that of the glandular kallikreins (Lundwall et al. FEBS Lett 1987; Schaller et al. Eur J Biochem 1987). Unlike the trypsin-like glandular kallikreins, which display Arg-restricted substrate specifity (MacDonald et al. Biochem J 1988), PSA displays chymotrypsin-like substrate specificity (Akiyama et al. FEBS Lett 1987; Christensson et al. Manuscript 1990; Lilja et al. J Biol Chem 1989). PSA has been predicted to be produced as a presumably inactive zymogen (Lundwall et al. FEBS Lett 1987). Active PSA is secreted into the seminal plasma (Lilja, J Clin Invest 1985) where it is one of the most abundant proteins of the prostate (Lilja et al. The Prostate 1988; Dube/ et al. J Androl 1987). The biological activity of PSA in semen relates to its limited proteolytic fragmentation of the predominant proteins secreted by the seminal vesicles (Lilja, J Clin Invest 1985; Lilja etal. J Clin Invest 1987; McGee etal. Biol Reprod 1988).
Secondary to the release from the prostate epithelium PSA may also be detected in the circulation (Papsidero etal. Cancer Res 1980). Measurements of the serum concentration of PSA have now found widespread use in monitoring of patients with prostate cancer, although increased serum concentrations of PSA have also been reported in benign prostatic hyperplasia and secondary to surgical trauma of the prostate (Duffy, Ann Clin Biochem 1989; Brawer et al. Urology suppl 1989). However, it is presently unknown whether the immunoreactivity in serum represents the PSA-zymogen, the active PSA or PSA inactivated by extracellular proteinase inhibitors and contradictory results have been reported on the molecular mass of this immunoreactivity. Papsidero reported in 1980 the PSA-immunoreactivity to elute as a single 90 to 100 kDa peak (Papsidero etal. Cancer Res 1980), whereas Alfthan and Stenman reported the predominant part of this immunoreactivity to elute as a 30-kDa protein (Alfthan etal. Coin Chem 1988) when subjected to gel filtration chromatography.
In the proceeding invention we showed that PSA has the ability to form complexes with proteinase inhibitors that occur in high concentration in the human extracellular fluids and that PSA occurs in these fluids both in a free and complexed form. In addition, the invention proved to be very useful in diagnosis of prostate cancer patients.
SUMMARY OF THE INVENTION
According to the method of the invention, immunoassays are applied to measure free PSA as well as PSA as a proteinase inhibitor complex. Free PSA and the PSA complex are according to the invention measured by a non-competitive immunoassay employing at least two different monoclonal antibodies. The invention is further characterized in that the PSA proteinase inhibitor complex of interest is formed either with .alpha..sub.2 -antichymotrypsin, .alpha..sub.1 -protease inhibitor (API) or .alpha..sub.2 -macroglobulin. Moreover, the invention is characterized by that free PSA, the PSA-proteinase inhibitor complex and their ratio are applied in the diagnosis of patients with prostate cancer.
DESCRIPTION OF THE DRAWINGS
FIG. 1 presents a polyclonal antibody and three monoclonal antibodies used
REFERENCES:
patent: 4446122 (1984-05-01), Chu et al.
Stenman et al., Scand. J. Clin. Lab. Invest. 51, Suppl. 205, 86-94 (1991).
Dialog Information Service, File 351, WPIL, Accession No. 007662133 (WAKP) Wako Pure Chem. Ind. KK, "Determining Antigen Specific to Human Prostate Gland . . ." Japanese 63/215967 and 880908 8842 (Basic).
New York Academy of Sciences, N.Y. Annals., vol. 417 (1983), Chu et al., "Circulating Antibody to Prostate Antigen in Patients with Prostatic Cancer", (pp. 383-389).
Patent Abstracts of Japan, vol. 11, No. 233, P600, Abstract of Japanese Patent 62-46263, publ. 1987-02-28; Chugai Pharmaceut. Col. Ltd.
Watt et al. (1986). Proc. Nat. Acad. Sci. USA 83:3166.
Lilja, H. (1985). J. Clin. Invest. 76:1899.
Lilja Hans
Stenman Ulf-Hakan
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