tPSA Assays Research Articles

Development of an immunoassay specific for the PSA-ACT complex without the problem of high background.

We have developed an assay specific for the PSA-ACT (PSA-alpha 1-antichymotrypsin) complex that effectively diminishes the problem of high assay background commonly reported by other investigators. The assay follows a two-site ELISA format. Polyclonal anti-PSA antibodies were coated on the microplate to capture the PSA complex from the serum, whereas the biotinylated anti-ACT polyclonal antibodies and HRP-conjugated streptavidin were used for detection. The high background ordinarily associated with this assay was greatly reduced when milk casein was added in addition to albumin for blocking and when the Super Block was also included in the diluents for sample dilution and dilution of enzyme conjugated detecting antibodies. The assay has a sensitivity of 0.05 ng/mL. The within-run precision ranges from 4.2-7.2% and the between-run precision falls between 5.8-8.5%. Cross reactions with ACT and free PSA (fPSA) are 0.0001% and 0.02%, respectively. The highest concentration of PSA-ACT complex in the maternal sera was < 0.4 ng/mL by this assay, much less than reported in the literature. Using this improved assay, the sum of fPSA and PSA-ACT concentrations were less than that of their corresponding total PSA (tPSA) most of the time. We believe that this improved assay should be used to replace the current tPSA assay for screening, monitoring, and managing patients with prostate cancer.

Comparison of prostate-specific antigen (PSA) measured by four combinations of free PSA and total PSA assays

We compared prostate-specific antigen (PSA) assay systems [i.e., free PSA (f-PSA) and the corresponding total PSA (t-PSA) assay] from four different manufacturers as well as the f-PSA/t-PSA ratios with regard to their ability to discriminate between benign prostate hyperplasia (BPH) and prostate cancer (PCA). ROC analysis showed similar areas under the curves (AUCs) with different assay systems. For the entire patient population the AUCs of the f-PSA/t-PSA ratio were not or slightly increased compared with the sole measurement of t-PSA (t-PSA, 0.792-0.820; f-PSA/t-PSA ratio, 0.685-0.859). In contrast, for only those patients who showed t-PSA concentrations within the diagnostic gray area of 4-25 micrograms/L t-PSA, the AUCs were greater for the f-PSA/ t-PSA ratio than for measurement of t-PSA alone (t-PSA, 0.608-0.647; f-PSA/t-PSA ratio, 0.690-0.806). These results were confirmed by the predictive values of the negative results (NPVs) of the t-PSA assays and the f-PSA/t-PSA ratios (assay thresholds corresponding to a 95% detection limit). Compared with the sole t-PSA measurement there was no mentionable increase in the NPVs due to the f-PSA/t-PSA ratio for the entire patient population, but an increase up to 49% when limited to t-PSA concentrations within 4-25 micrograms/L. We therefore conclude that the f-PSA/t-PSA ratio may be helpful for differential diagnosis of BPH and PCA within the diagnostic gray area of 4-25 micrograms/L t-PSA.

Serum free prostate-specific antigen in the diagnosis of prostate cancer.

To determine the value of the ratio of free prostate-specific antigen (fPSA) to total PSA (tPSA) in the diagnosis of benign prostatic hyperplasia (BPH) and prostate cancer in a cohort of patients undergoing prostatic transrectal ultrasonography (TRUS). The study comprised 153 patients (99 with BPH and 54 with prostate cancer) undergoing diagnostic TRUS of the prostate. Patients with a tPSA of > 30 ng/mL were excluded from analysis. Free PSA was assayed using an immunoassay specific for unbound PSA (CanAg Diagnostics, Sweden). Total PSA was measured using the HybriTech Tandem-R PSA immunoradiometric assay in routine clinical use and this estimate was validated using the CanAg tPSA assay. The measurements of tPSA from both assay systems correlated closely. The f/tPSA ratios in patients with prostate cancer were significantly lower than in those with BPH (median values 0.152 and 0.2, respectively, P<0.01). In patients with prostate cancer, the median f/t PSA levels apparently declined with increasing tPSA levels but in those with BPH, the levels of tPSA were not significantly associated with the f/tPSA ratio; the ratios did not vary significantly with age in either group. A f/tPSA ratio at a threshold of 0.16 had positive and negative predictive values of 44% and 74%, respectively; the corresponding values for a tPSA of > 4 ng/mL were 30% and 52%. The f/tPSA ratio differs significantly between patients with BPH and cancer but because there is a considerable overlap of f/tPSA ratios between the groups, f/tPSA values alone were not sufficiently specific to be used as a single diagnostic test.

Evaluation of a two-site immunoradiometric assay for measuring noncomplexed (free) prostate-specific antigen

Serum prostate-specific antigen (PSA) in men is present as two different molecular forms separable by gel-filtration chromatography (GFC). We have evaluated a two-site IRMA that measures only the noncomplexed (free) form of PSA (F-PSA). Verification that the F-PSA assay measures solely F-PSA was obtained by assaying GFC-fractionated serum samples with both the F-PSA IRMA and a commercial PSA assay that measures total PSA (T-PSA: F-PSA plus alpha 1-antichymotrypsin-complexed PSA). The F-PSA assay detected only the 30-kDa peak corresponding to the free form of PSA, whereas the T-PSA assay detected two peaks: complexed PSA at approximately 90 kDa and F-PSA at approximately 30 kDa. The F-PSA assay had an analytical detection limit of 0.03 microgram/L and a measuring range up to 50 micrograms/L. The intraassay CV was 1.7-10% in the concentration range of 0.2-30 micrograms/L. The interassay CV was 3.4-12.5% in the same concentration range. Dilution and recovery studies showed no significant deviation from linearity across the assay range. The assay was insensitive to interference from hemoglobin, bilirubin, and total lipids up to concentrations of 5, 0.2, and 10 g/L, respectively. No significant loss of immunological activity (analyte stability) was seen day-to-day ( < or = 5) or after repeated freeze/thaw ( < or = 5) cycles. We conclude that the F-PSA IRMA is an accurate, precise, and reliable tool for measuring F-PSA in human serum.