Development of Methods for Characterization of Prostate Specific Antigen in Urine

Prostate cancer is the most detected cancer in males and one of the leading causes of cancer mortality in the western society. Prostate Specific Antigen (PSA) is since the 1980´s widely used as a serum marker but can not distinguish between prostate cancer and benign prostate hyperplasia. PSA is a glycoprotein with a molecular weight of 30 kDa and consists of an active 237 amino acid residues polypeptide with five disulphide bonds and approximately 7-12 % carbohydrates. The aim of the project was to develop an immunochromatographic test for measuring the total concentration of PSA in urine and to verify, by using size exclusion chromatography, if PSA in urine was free or complexed with other proteins. However, the first issue was to deal with the variable urine composition and the occurrence of precipitate in urine which can bind proteins. Six different immunochromatographic test systems were developed and the concentration of PSA was measured in urine specimens from normal individuals and from patients with prostate cancer by using two selected systems. The most sensitive immunochromatographic system showed a detection limit of 1.2 ng/L which is 130 times more sensitive than presently available commercially enzyme immunoassays. The precipitates in urine were dissolved by pH-adjustment and addition of chelator and detergent. Only 0.5% PSA was detected in the supernatant of centrifuged urine in comparison to when the precipitates where dissolved and these findings confirm that PSA in urine is in precipitate. The median concentration in normal male urine was 106µg/L. The highest value of 991µg/L was obtained in urine from a patient with prostate cancer but several of the urine specimens from patients showed non-detectable values. Four normal and patient urine specimens were separated by size exclusion chromatography and the fractions were measured by two immunochromatographic test systems. For all specimens a single peak was eluted in the same position which was consistent with a 30kDa molecular weight protein and no larger complex of PSA could be found. However, it was found that measurable PSA during storage in the fraction tubes could disappear rapidly