Received on July 21, 2004
Accepted on September 9, 2004

Proteomics and Protein Markers

Analysis of Subforms of Free Prostate-Specific Antigen in Serum by Two-Dimensional Gel Electrophoresis: Potential to Improve Diagnosis of Prostate Cancer

¹ Department of Urology, University Hospital Charité, Humboldt University, Berlin, Germany
² Roche Diagnostics, Penzberg, Germany
³ Institut of Laboratory Medicine and Pathobiochemistry, University Hospital Charité, Humboldt University, Berlin, Germany, and Current address: Institute of Medical and Chemical Laboratory Diagnostics, State Hospital, Klagenfurt, Austria

^* To whom correspondence should be addressed. E-mail: klaus.jung{at}charite.de.

Background: The aim of this study was to develop a method to separate and quantify subforms of free prostate-specific antigen (fPSA) in serum by two-dimensional electrophoresis and to assess the diagnostic accuracy of these subforms for prostate cancer (PCa) diagnosis in comparison with total PSA (tPSA) and the ratio of fPSA to tPSA (%fPSA).

Methods: Sera from 50 patients with and without PCa, respectively, were studied. PSA was isolated by immunoadsorption on streptavidin-coated magnetic beads with biotinylated anti-PSA antibodies and separated by two-dimensional electrophoresis. After semidry blotting, the intensities of the fPSA spots were quantified by chemiluminescence using an imager analyzer.

Results: The method detected subforms to a concentration of 0.1 µg/L fPSA with an imprecision (CV) <16%. We detected 15 immunoreactive fPSA spots of different intensities. Spots F2 and F3 were present in all samples. F2 was lower in samples from non-PCa patients (median, 23%) than in samples from PCa patients (49%), whereas F3 behaved inversely (non-PCa, 73%; PCa, 45%). Ratios of F2 to F3 and F2/F3 to %fPSA, respectively, showed improved diagnostic accuracy compared with tPSA and %fPSA. Better differentiation by F2/F3 or by F2/F3 to %fPSA was particularly evident in patients with %fPSA values >15%. There were no associations between the PCa grading scale and fPSA subforms.

Conclusions: fPSA subforms separated by two-dimensional electrophoresis may improve both sensitivity and specificity in prostate cancer diagnostics compared with tPSA and %fPSA. The development of a practicable assay based on the immunologic properties of these different fPSA subforms seems to be promising.