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Characterization and immunological determination of the complex between prostate-specific antigen and ₂-macroglobulin

Wan-Ming Zhang^1,a, Patrik Finne¹, Jari Leinonen¹, Satu Vesalainen², Stig Nordling³, Sakari Rannikko² and Ulf-Hkan Stenman¹

Departments of
¹ Clinical Chemistry,
² Urology, and
³ Pathology, Helsinki University Central Hospital, FIN-00290, Helsinki, Finland.
^a Author for correspondence. Fax 358-0-4714804; e-mail wmzhang{at}helsinki.fi.

Prostate-specific antigen (PSA) rapidly forms a complex with ₂-macroglobulin (A₂M) in vitro; however, PSA complexed with A₂M (PSA-A₂M) is not detected by conventional immunoassays for PSA because it is encapsulated by the A₂M. In this study, we show that denaturation of PSA-A₂M at high pH renders PSA immunoreactive. Part of the complexed PSA is released in free form and part remains bound to denatured A₂M. These forms can be measured by a conventional immunoassay for PSA. This finding enabled us to design a dissociation assay for the detection of PSA-A₂M, which was based on the removal of immunoreactive PSA in serum by immunoadsorption, denaturation of PSA-A₂M at high pH, and measurement of the released PSA immunoreactivity by a conventional PSA immunoassay. This PSA-A₂M assay was calibrated with PSA-A₂M formed in vitro. The detection limit of the assay was 0.14 µg/L. Inter- and intraassay coefficients variation were 4–9% and 8–14%, respectively. When purified PSA was incubated with A₂M, the loss of PSA immunoreactivity was highly correlated with the PSA-A₂M formed, as measured by the dissociation assay for PSA-A₂M (r = 0.99; P <0.0001). The concentration of PSA-A₂M in serum correlated with that of total PSA both in prostate cancer (PCa) and benign prostatic hyperplasia (BPH); however, the ratio of PSA-A₂M in relation to total PSA was significantly higher in BPH than in PCa (P <0.0003). ROC curve analysis suggested that measurement of the ratio of PSA-A₂M to total PSA in serum improves the diagnostic accuracy for PCa compared with assays for total PSA only.

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