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Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step.

Prostate-specific antigen (PSA)[3] belongs to the glandular kallikrein gene family. It is produced mainly by the prostate gland, and its main biological function has been shown to be the cleavage of the gel-forming proteins semenogelin 1 and 2 in semen (1-3). Multiple cleavages cause dissolution of the gel structure and liquefaction and, thus, release of motile spermatozoa.

PSA has been shown to also possess other biological functions. A possible physiologic substrate for PSA is insulin-like growth factor binding protein-3 (IGFBP-3) (4-7). Cleavage of IGFBP-3 leads to inactivation and loss of its ability to bind to IGF-I, a stimulant of cell growth. Recently, PSA was shown to cleave IGFBP-4 in addition to IGFBP-3 (8). These findings have led to the hypothesis that PSA may promote the growth of prostate metastases by increasing the bioavailability of IGF-I. In addition, reports describing the cleavage of fibronectin and laminin by PSA (9) have led to speculation that PSA can mediate invasion and metastasis of prostate cancer (PCa) cells. In contrast to these reports, PSA was shown to possess antiangiogenic activity, which conceivably contributes to the slow progression of PCa (10). Thus, the actual biological functions and consequences of the enzymatic activity of PSA, other than cleavage of the semenogelins, have not been identified, as reviewed recently (11).

PSA cleaves its substrates at specific sites. The active site of PSA is closely related to that of chymotrypsin (12), and PSA has been shown to possess a chymotrypsin-like substrate specificity. On the other hand, human glandular kallikrein 2 (hK2), which shares 79% amino acid structure homology with PSA, has a trypsin-like substrate specificity (13, 14). hK2 is an in vitro activator and possibly also a physiologic activator of proPSA that cleaves the 7-amino acid prosequence APLILSR of PSA (15).

The predominant form of PSA in serum is the PSA-[[alpha].sub.1]-antichymotrypsin (ACT) complex (16,17). With the aid of various immunoassays that measure free and complexed PSA forms, the diagnostic accuracy for the detection of PCa has improved. The free PSA (PSA-F) forms characterized from blood and tissue include intact PSA forms, namely proPSA forms and inactive mature PSA (18-20), as well as internally cleaved forms (21, 22). Although the diagnostic value of these free forms of PSA is still under study, in PCa, a higher proportion of intact PSA is found in the circulation compared with benign prostatic hyperplasia (BPH), in which more internally cleaved forms of PSA occur (19, 20, 23). It is thought that all PSA in blood measured by current routine immunoassays is inactive. The hypothesis is that all active PSA in blood would be fully complexed because of the large molar excess of serpins and [[alpha].sub.2]-macroglobulin. Complexation of PSA with serpins inactivates PSA, and complexation with [[alpha].sub.2]-macroglobulin renders PSA inaccessible to immunorecognition by the routinely used immunoassays. The remaining free portion is thought to be inactive because it has not complexed.

Studies that aim to determine the exact concentration of active PSA in body fluids have been reported only recently (24, 25). In this study, we developed an enzymatic assay for active PSA that is more sensitive than previously reported assays. In addition, an activation step was included in the assay that enabled us to determine the concentrations of proPSA after it had been converted to active, mature PSA by hK2.

Materials and Methods


The amino acid substrates were custom synthesized by Enzyme Systems Products (Livermore, CA). The substrates had 7-amino-4-methyl coumarin (AMC) attached via an amide bond to the carboxyl group of the C-terminal amino acid, position P1. The sequence of the tetrapeptide substrate, from position P4 to position P1, was Ser-SerTyr-Tyr (SSYY). The sequence was planned and modified from a publication by Coombs et al. (26), who showed that this sequence was most favorably cleaved by PSA. The sequences of two other substrates have been published previously (27). A heptapeptide with a sequence of Lys-Gly-Ile-Ser-Ser-Gln-Tyr (KGISSQY) was most favorably cleaved by PSA, but lacked specificity for PSA because it was even a better substrate for chymotrypsin. A hexapeptide with a sequence of His-Ser-Ser-Lys-Leu-Gln (HSSKLQ) was the most efficient fluorogenic substrate for PSA to cleave without compromising specificity.


All dilutions were made in buffer containing 50 mmol/L Tris-HCI (pH 7.5) and 1.5 mol/L NaCl (kinetic buffer). The effects of various NaCl concentrations were determined, and 1.5 mol/L was selected as giving the highest PSA activity. We added 2 g/L bovine serum albumin (BSA) to the kinetic buffer to prevent attachment of the proteins to plastic surfaces.


The production and purification of proPSA and hK2 in a baculovirus expression system have been described previously (15). Purified seminal plasma PSA was from the University Hospital Malmb (Malmb, Sweden). A fraction of seminal PSA that contained only an intact form of PSA, called pool B PSA (28), was a kind gift from Prof. U.H. Stenman (Helsinki University Central Hospital, Helsinki, Finland).


An anti-PSA antibody bound to microtiter plate wells was used to capture the PSA present in the sample. Antibody H117 binds to epitope region 6 (29), which corresponds to the N-terminal region of PSA. Contrary to most anti-PSA antibodies, H117 has been shown to enhance the enzymatic activity of PSA (30). In the first step, biotinylated antibody (300 ng/well in 100 [micro]L of kinetic buffer) was attached to streptavidin wells in a 1-h incubation, after which plates were washed four times. Kinetic buffer (100 [micro]L/well) and samples (100 [micro]L/well) were added and incubated overnight at 4 [degrees]C. Plates were washed four times, after which the fluorogenic substrate was added. Substrates were diluted to a final concentration of 400 [micro]mol/L, and 200 [micro]L was added to each well. The hydrolysis of the substrate by PSA was measured with a Victor2 Multilabel reader (Perkin-Elmer Life Sciences), using an excitation wavelength of 355 rim and an emission wavelength of 460 nm. Wells were measured every 5 min for 1 h. Between measurements, the plate was incubated with slow shaking at 37'C. A calibration curve based on the fluorescence of known amounts of AMC (Sigma) was used in the determination of AMC released (pmol) per min. The fluorescence of AMC was determined to be 410 counts/pmol, and this value was used in the calculation of product formation.

The PSA concentration was plotted against the measured activity (pmol of AMC released per min), and the analytical detection limit of the assay was determined from the calibration curve. Analytical sensitivity was calculated as 2 SD of the standard diluent divided by the slope of the calibration curve. The limit of quantification was determined by plotting the measured amount of active PSA against the CV for two replicates of the sample.


In the hK2 activation studies, hK2 was added after the sample incubation step. Plates were incubated at 37 [degrees]C for 2 h, after which they were washed, the substrate was added, and the measurement was performed, as described above. The different incubation times and the amount of hK2 added were optimized.


ProPSA and seminal plasma PSA were used as calibrators, and the concentrations were determined with previously characterized assays. The assay for PSA-F used an antibody combination of 5A10 and H117, and the assay for total PSA (PSA-T; PSA-F plus PSA-ACT complex) used antibodies H117 and H50 (17). The assay for intact PSA used antibody combination 5A10 and 5C3, of which 5C3 was specific for PSA that is not internally cleaved at [Lys.sup.145]-[Lys.sup.146] (20).


Monoclonal antibody (Mab) 5A10 is shown to bind to epitope region 1 (29), which is accessible only when PSA is free and not complexed with serpins. Thus, Mab 5A10 is a PSA-F-specific antibody. Mab 5A10 has previously been shown to block the activity of PSA toward synthetic substrates (25, 27, 30). Mab 5A10 was added to the wells (500 ng/well) after 10 cycles of measurement, after which wells were measured for another 10 cycles.


We assayed 15 male EDTA-plasma samples with increased PSA-T concentrations but without clinical background information. Ten of these samples had highly increased PSA-T concentrations (median, 139 [micro]g/L; group A), and in 5 samples, PSA-T concentrations were <20 /[micro]/L (median, 9.7 [micro]g/L; group B). In addition, three EDTA-plasma samples from patients with BPH who had undergone transurethral resection of the prostate (TUR-P) were analyzed. These samples were taken immediately after the procedure, which causes a rapid increase in blood PSA. As a control, we also analyzed three EDTA-plasma samples from healthy females (Table 1).



The best substrate of the three tested was KGISSQY. The lack of specificity reported by Denmeade et al. (27) was not a concern because we used a PSA-specific antibody that captures only PSA. Before adding the substrate, we washed the wells to remove all unbound substances that might be able to cleave the substrate. Ten nanograms of PSA/well (100 [micro]g/L) cleaved 9.1 pmol of KGISSQY substrate per min. Peptide HSSKLQ was hydrolyzed much more slowly by PSA. Only 0.06 pmol of peptide HSSKLQ substrate was hydrolyzed per min by the same amount of PSA. In addition, peptide SSYY was not hydrolyzed as efficiently as peptide KGISSQY (3.7 pmol of substrate cleaved per min by 10 ng of PSA); thus, KGISSQY was selected and used in further studies.

The calibration curve and the CV for three replicates of each calibrator are shown in Fig. 1, using the substrate KGISSQY-AMC. The lowest PSA calibrator had a concentration of 0.68 [micro]g/L, and this concentration could still clearly be detected. The lower limit of detection was 0.21 [micro]g/L, calculated as 2 SD of the calibration diluent divided by the slope of the calibration curve. The limit of quantification was 0.5 [micro]g/L, determined from a precision plot as the lowest analyte concentration measured with CV [less than or equal to]20%.

Mab H117, unlike most anti-PSA antibodies, has previously been shown to increase the enzymatic activity of PSA with synthetic substrates to 166% compared with reactions without Mab H117 (30). We also found that with Mab H117, ~185% of PSA activity was measured compared with assays without any capture antibody.

Various salt concentrations in the kinetic buffer were tested (0.1-4 mol/L). As can be seen from Fig. 2, PSA activity increased with increasing NaCl concentrations until the NaCl concentration reached 2 mol/L. Therefore, we selected 1.5 mol/L NaCl as the optimal concentration and used that concentration in subsequent experiments. Similar results have been reported previously (24).

We used 2 g/L BSA to avoid nonspecific attachment of proteins to plastic surfaces. When no BSA was used, the activity was ~40% lower than with 2 g/L BSA. When we incubated BSA with PSA, no cleavage products could be detected by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (data not shown); thus, BSA acted only as a carrier protein.


Recently it was reported that glycerol increases PSA activity and that optimal glycerol concentrations were as high as 200-300 mL/L (24). We tested glycerol concentrations in the range of 0-200 mL/L and detected a 57% decrease in PSA activity when 200 mL/L glycerol was added to kinetic buffer containing the substrate. A slight decrease (4%) in PSA activity was detected even with 10 mL/L glycerol. Thus, in contrast to the previous report, we found that glycerol decreased the enzymatic activity of PSA toward a synthetic substrate.

ACTIVITY OF PSA PURIFIED FROM SEMINAL PLASMA The activity of seminal plasma PSA and a fraction of seminal plasma PSA, called pool B PSA (28), that contained only intact PSA were compared. Dilution series of the PSA preparations were made. Seminal plasma PSA had ~30% lower activity than pool B PSA. This was expected because seminal plasma PSA has been shown to contain internally cleaved, and thus inactive, forms.



As has been reported previously, proPSA does not possess enzymatic activity (15, 31). We found that when hK2 was added to a well containing recombinant proPSA, a substantial increase of activity was detected. Thus, proPSA attached to H117-coated wells could be activated to active, mature PSA by hK2. Different amounts of hK2 were tested in the activation step. As can be seen from Fig. 3A, 10 ng/well hK2 was enough to activate proPSA at concentrations of 5.5-135 [micro]g/L, and no significant increase in PSA activity was detected with increasing amounts of hK2. In a well containing only hK2, no increase in signal was detected, indicating that hK2 could not cleave the substrate. The optimum hK2 incubation time was 2 h (Fig. 3B). Prolonged incubation led to a slight decrease in activity; thus, 2 h was selected to be the optimum time for the hK2 activation step. The activity of activated proPSA was, surprisingly, ~70% lower than the activity of pool B PSA (data not shown).



The three TUR-P samples with PSA-T concentrations of 27.5-92.4 [micro]g/L were analyzed for free, intact, and active PSA. PSA-T concentrations had been determined earlier with the commercial Prostatus Free/Total PSA Kit (Perkin-Elmer Life Sciences). Most of the PSA in TUR-P samples was in the free form. The intact-PSA assay that measures PSA that has not been internally cleaved at [Lys.sup.145]-[Lys.sup.146] (20) was used to measure the amount of intact PSA in these samples. The intact-to-free PSA ratio was low (mean, 33%), indicating that most of the PSA-F was internally cleaved at [Lys.sup.145]-[Lys.sup.146]. Surprisingly, we found small but detectable amounts of active PSA. The amount of active PSA in PSA-F was ~3%. When hK2 activation was performed for these samples, no significant increase in activity could be detected, indicating that the PSA-F in these samples was not proPSA (Table 1). Recombinant proPSA was used as a control to confirm that the hK2 activation was successful.

We also studied 15 EDTA-plasma samples from patients with increased PSA-T concentrations. We divided these samples into two groups. In group A, PSA-T concentrations were highly increased, ranging from 91 to 423 [micro]g/L. Group B consisted of five samples with PSA-T <20 [micro]g/L. In group A, surprisingly all the samples except one had small amounts of active PSA, which was ~3% of the amount of PSA-F. However, after the hK2 activation step, we found considerably higher amounts of active PSA. The amount of activated PSA ranged from 4.4 to 34 [micro]g/L, which corresponds to 26-79% of the amount of PSA-F in the samples. Thus, a considerable portion of PSA-F in these samples was proPSA, which could be measured after the activation step. In group B samples, active PSA was not detected. However, after the activation step, ~43% of PSA-F was active and thus represented the proPSA fraction (Table 1). All three tested female samples with undetectable PSA concentrations were assayed for active PSA as a control. We detected no PSA activity in these samples (Table 1).

Most of the PSA-F in group A samples was not cleaved at [Lys.sup.145]-[Lys.sup.146], which is the most common inactivating cleavage site on PSA. The median intact PSA concentration was 39 /,g/L, which produced a high (96%) intact-to-free PSA ratio. In group B, the amount of intact PSA in PSA-F was lower (median, 47%). According to the results from the hK2 activation step, only part of the intact PSA in both groups was in the proform. Thus, there is an inactive form of PSA that is intact at [Lys.sup.145]-[Lys.sup.146] but is not proPSA.

We calculated the correlation between different measured markers: total, free, and intact PSA and proPSA. Groups A and B were combined, and a total of 14 plasma samples were analyzed (one sample was excluded because of a missing intact PSA value). The correlation (r) compared with PSA-T was 0.713 for PSA-F, 0.696 for intact PSA, and 0.393 for proPSA.


Binding of Mab 5A10 to PSA inhibits the enzymatic activity of PSA. To confirm that the activity detected from patient samples is truly PSA specific, we studied whether this activity was blocked with 5A10. Shown in Fig. 4 are the enzymatic activities detected for the 0 (background) and 20 [micro]g/L calibrators and for three samples after hK2 activation. The time point at which Mab 5A10 was added is shown with an arrow. The addition of 5A10 produced a clear decrease in activity of all samples. Thus, the activity detected in patient samples was confirmed to be PSA specific, first with Mab H117 and then with 5A10. The small amount of activity detected in samples before hK2 activation was also similarly blocked. Because Mab 5A10 is an antibody specific for PSA-F forms, it seems that the active PSA portion in blood is the free form and that the activity detected is not coming from complexed PSA forms.


The concentrations and biological functions of active PSA and its different forms in body fluids have not been clarified. Sensitive and specific methods are needed to study this proteome. We have developed a sensitive enzymatic assay for specific and quantitative measurement of active PSA. Our assay also enables quantification of proPSA by applying a hK2 activation step that converts proPSA to active, mature PSA that can be measured.

PSA has very restricted substrate specificity and specific enzymatic properties. The reason for the preferential hydrolysis of hydrophobic residues at the carboxy terminus lies in the structure of the substrate specificity pocket, which contains a serine (32,33). PSA has been shown to possess highest activity toward substrates that have tyrosine at the P1 position (26, 27). Coombs et al. (26) determined that the amino acid sequence that would be most favorably cleaved by PSA would contain amino acids SS(Y/F)YS(G/S), where the peptide bond cleavage occurs between the tyrosine (Y) in the P1 position and serine (S) in the P1' position. These results have been confirmed in other studies. Yang et al. (34) determined that a synthetic hexapeptide with the sequence QFYSSNK, where phenylalanine (F) was in position P2, tyrosine in P1, and serine in P1' would be the optimal PSA substrate. On the other hand, Denmeade and coworkers (25,27) used the amino acid sequence HSSKLQ in their studies; this peptide does not contain a tyrosine but has glutamine (Q) in position P1. This sequence was selected because of its high specificity for PSA. They reported that higher cleavage rates were obtained with sequence KGISSQY, but that this was also a better substrate for serine proteases other than PSA.


We used substrate KGISSQY in our experiments. Other proteases that could cleave KGISSQY were not a concern in our assay construct because we used the PSA-specific Mab H117, which captures PSA and binds it to the well, whereas other substances are washed away. To confirm that our assay is truly PSA specific and that the activity detected is not coming from substances that might bind nonspecifically to plastic surfaces, the activity was blocked using another anti-PSA antibody, 5A10. With this antibody, the activity detected in samples and controls clearly diminished (Fig. 4). Thus, the only possible enzyme that can cleave the fluorogenic substrate in our assay format is PSA.

hK2 has been shown to posses a trypsin-like substrate specificity (13, 14), and cleavages of arginine residues or other basic amino acids at the carboxy terminus are preferred because of the aspartate residue at the bottom of the catalytic pocket (35). It has been shown that cleavage of the proPSA-7 and -5 proforms in vitro converts the inactive proPSA to mature, active PSA (15). In another study, in addition to proPSA-7 and -5, proPSA-4 was converted to mature PSA after <2 h of incubation (36). However, activation of proPSA-2 was not successful, indicating that a short propeptide of two amino acids is no longer recognized by hK2.

In this study, we exploited the capability of hK2 to activate proPSA and combined it with our enzymatic assay to measure the amount of proPSA in the samples. On the basis of previous results for proPSA activation using hK2, we concluded that our enzymatic assay measures proPSA with prosequences of four amino acids or longer. ProPSA constituted a considerable portion of PSA-F (47%) in samples with highly increased PSA-T (group A in Table 1) and in samples with PSA-T <20 [micro]g/L (43%; group B in Table 1). However, in samples obtained from patients after TUR-P, the amount of active PSA was not increased after hK2 activation. This indicates the difference between the sample populations. The PSA in blood samples with high PSA-T concentrations most likely comes from PCa tissue, but the PSA in blood samples obtained from TUR-P patients is released from BPH tissue. Recently, serum samples from PCa patients were analyzed by mass spectrometry, and samples were shown to contain proPSA forms, which represented a considerable portion of total PSA-F (19). This was confirmed in our study: we showed that in samples with highly increased PSA-T, most of the PSA-F was in pro-forms. On the other hand, it has been shown that BPH samples contain more internally cleaved forms than intact forms (20,23). Thus, intact proPSA is not a considerable portion of PSA-F in BPH, but internally cleaved forms are. Cleavage at [Lys.sup.145]-[Lys.sup.146] and at another known internal cleavage site, [Lys.sup.182]-[Ser.sup.183], inactivates PSA (22, 37), and these forms are responsible for the free, inactive PSA forms found in increased concentrations in BPH.

Our activation step was not totally successful because we could activate only ~70% of proPSA attached to the well. This could mean that the actual concentrations of proPSA might be higher in patient samples than those that we measured. A recent report described the inability of hK2 to cleave a synthetic peptide with the prosequence APLILSR (38). This result might suggest than hK2 is not the optimal activator of proPSA and that additional proteases are needed to fully activate PSA in vivo. The performance of the activation step requires further studies.

Peter et al. (19) reported that in addition to the full-length 7-amino acid prosequence, various other proPSA forms occur in the blood of PCa patients. They found proPSA with prosequences of 7, 5, 4, 2, and 1 amino acids. In addition, a mature PSA form with a regular amino terminus was found. These results could explain the difference we detected in the amounts of proPSA and intact PSA in patient samples. Intact PSA accounted for 96% of the PSA-F in group A patients. However, on average, only 51% of this intact PSA was in proforms containing the 4- to 7-amino acid propeptide. Thus, there is an inactive form of PSA that is intact at [Lys.sup.145]-[Lys.sup.146] but is not proPSA. The uncleavable proPSA-2 proform and inactive mature PSA forms could be the intact PSA portion that can not be activated with hK2.

With our optimized assay having a limit of quantification of 0.5 [micro]g/L, we could detect active PSA from blood samples with highly increased PSA-T concentrations. The concentrations of active PSA were very low, from undetectable to 7% of PSA-F (0-2.7 [micro]g/L). However, this result was surprising because it is thought that all active PSA would form complexes with serpins in blood or with [[alpha].sub.2] macroglobulin, in which case it would not be accessible to capture antibody. This activity was blocked with anti-PSA-F Mab 5A10, meaning that PSA-F was responsible for the active fraction of PSA. To exclude the possibility that the activity would come from the dissociation of PSAACT complex and the release of active PSA, we studied the stability of PSA-ACT complex in our assay construct. During the measurement time, we did not detect any activity for purified PSA-ACT complex that was attached to Mab H117 (data not shown). In addition, previous reports have shown that PSA-ACT complex is stable when stored 37 [degrees]C, even for days (39). Thus, the active PSA in blood might be coming from dissociation of complexes other than PSA-ACT, or it might represent an unknown, uncharacterized fraction of PSA. This fraction might be inactive in vivo because of unknown mechanisms but active in our assay construct.

Denmeade et al. (25) reported that enzymatically active PSA is secreted from PCa cells, but that no active PSA can be found in blood. The detection limit of their assay using substrate HSSKLQ-AMC was not low enough to detect low amounts of active PSA in blood samples. They reported that the lower limit of detection in their enzymatic assay was 50 ng of total PSA per assay using substrate HSSKLQ-AMC (27). This is ~1000-fold higher than the lower limit of detection for our assay.

In conclusion, we have developed a sensitive and specific method for measuring active PSA and for measuring proPSA after activating it to mature, active PSA. This activation step enables quantification of proPSA forms with propeptides of four to seven amino acids and thus makes this assay, to our knowledge, the first to measure these proPSA forms. We intend to use our assay to study the roles and concentrations of active PSA and proPSA in different body fluids and tissue extracts. In addition, the diagnostic value of proPSA in blood remains to be clarified.

This work was supported by grants from the Academy of Finland (Projects 45252 and 54229) and in part by grants from the Swedish Research Council (Medicine; Project 7903); Swedish Cancer Society (Project 3555); the Medical Faculty, Lund University; the Research Fund and Cancer Research Fund at University Hospital, Malmo; and Fundacion Federico S.A.

Received February 1, 2002; accepted May 1, 2002.


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[3] Nonstandard abbreviations: PSA, prostate-specific antigen; IGFBP, insulin-like growth factor binding protein; PCa, prostate cancer; hK2, human glandular kal&krein 2; ACT, a,-anti chymotrypsin; PSA-F, free PSA; BPH, benign prostatic hyperplasia; AMC, 7-amino-4 methyl coumarin; BSA, bovine serum albumin; PSA-T, total PSA; Mab, monoclonal antibody; and TUR-P, transurethal resection of the prostate.


[1] Department of Biotechnology, University of Turku, Tykist6katu 6A 6th Floor, FIN-20520 Turku, Finland.

[2] Department of Clinical Chemistry, Lund University, University Hospital Malm6, 20502 Malm6, Sweden.

* Author for correspondence. Fax 358-2-3338050; e-mail
Table 1. Concentrations of total, free, intact, and active
PSA and proPSA, and ratio of proPSA to PSA-F in four
different patient groups. (a)

 Median (range)

 PSA concentration, [micro]g/L

 Total Free Intact

Group A 139 (91.1-423) 40.5 (11.4-64.2) 38.8 (9.7-63.0)
Group B 9.7 (5.3-19.9) 2.1 (0.9-4.3) 1.3 (0.3-2.0)
TUR-P samples 51.9 (27.5-92.4) 46.3 (28.4-94.3) 18.2 (7.7-30.7)
Female samples ND (b) ND ND

 PSA concentration, [micro]g/L

 Active proPSA proPSA/PSA-F, %

Group A 1.1 (ND (b) to 2.7)20 (4.4-34.4) 47 (25.6-78.6)
Group B ND 1.0 (ND to 1.3) 42.6 (ND to 51.8)
TUR-P samples 1.4 (0.8-1.7) 2.3 (0.9-2.9) 3.2 (3.1-5.0)
Female samples ND ND

(a) Group A, patients with highly increased PSA-T (n = 10);
group B, patients with PSA-T <20 [micro]g/L (n = 5); TUR-P
samples, patients who had undergone TUR-P (n = 3);
female samples, healthy females (n = 3).

(b) ND, not detectable.
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Title Annotation:Cancer Diagnostics: Discovery and Clinical Applications
Author:Niemela, Pauliina; Lovgren, Janita; Karp, Matti; Lilja, Hans; Pettersson, Kim
Publication:Clinical Chemistry
Date:Aug 1, 2002
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