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Analysis of free prostate-specific antigen (PSA) after chemical release from the complex with [[alpha].sub.1]-antichymotrypsin (PSA-ACT).

Because of its sensitivity and organ specificity, prostate-specific antigen (PSA) (3) is a very valuable protein marker in human blood for the diagnosis of prostate carcinomas. PSA is also monitored in the follow-up after surgical removal of prostate cancer (CaP) (1-3). However, there are some limitations in the use of PSA in CaP diagnosis. Patients with benign prostatic hyperplasia (BPH) also show PSA concentrations of up to ~15 [micro]g/L in serum. Therefore, PSA concentrations <15 [micro]g/L cannot be used to distinguish between CaP and BPH (3). Furthermore, PSA is present in human blood as a complex mixture of several species. The main immunologically detectable form is a covalent complex of PSA with the serine protease inhibitor (serpin) al-antichymotrypsin (ACT) (4,5). Moreover, the presence of additional PSA-serpin complexes in serum has been reported, albeit in much lower concentrations than the PSA-ACT complex [see, for example, Refs. (4,6)]. A complex of PSA with [[alpha].sub.1]-macroglobulin is also present, but to date, it has not been detectable by clinically used immunological tests and, therefore, does not contribute to the PSA values measured by these tests (4, 7, 8). Free (uncomplexed) PSA (F-PSA) also is present, accounting for 5-30% of the total PSA (T-PSA). This PSA form is enzymatically inactive and cannot form complexes with the protease inhibitors because of internal nicking or the existence of proPSA forms [see, for example, Ref. (9)]. The complex distribution of PSA leads to uncertainties in the immunological measurement of PSA (10). It has been reported that some tests display a bias toward overestimation of the F-PSA form because of the specificity of the antibodies used (7,11,12). Furthermore, it is difficult to standardize the tests because of the various forms present (13). Considering that the ratio of free to total PSA (and also the ratio of F-PSA to the PSA-ACT complex) is being used for better differentiation between CaP and BPH than T-PSA alone (4, 5,14), the exact measurement of these two markers is of importance for the value of PSA as a tumor marker. It would therefore be desirable to be able to measure the ratio of free to complexed PSA using only one immunological test, thus eliminating any possible bias between two immunological assays.

Complexes between proteases and serpins have been described as covalent and stable (15,16), although noncovalently linked complexes that do not dissociate in the presence of sodium dodecyl sulfate (SDS) have also been reported (17). There is a considerable amount of information available on the structures and mechanisms of formations of proteinase-serpin complexes [see, for example, Refs. (15,16,18)], but little information is available on PSA-ACT. In the first report on complex formation of PSA with ACT, it was concluded from electrophoretic mobility that PSA forms a covalent linkage to Leu-358 of ACT, giving an SDS-stable complex and releasing the C-terminal peptide during SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (19). It was later shown that PSA-ACT was somewhat unstable when stored in buffers at pH 7.5 and 35 [degrees]C over several weeks, releasing F-PSA measurable by an immunological assay (20). When a 1000-fold excess of ACT was used and the pH of the buffers was adjusted to 6.8, the cleavage could be largely prevented. Similarly, a complex of PSA and [[alpha].sub.1]-protease inhibitor (API) formed in vitro was shown to dissociate ~30-40% when kept at 37'C for 7 days, yielding enzymatically active PSA and an inactive API that was cleaved between Met-358 and Ser-359 (21).

Our investigations of the structural features of PSA in serum led to the development of a method for the rapid chemical release of PSA from the PSA-ACT complex, using alkaline ethanolamine treatment. After immunoaffinity purification (22), the liberated PSA was analyzed by mass spectrometry (MS) and immunological assays.

Materials and Methods

Plasma from a patient with CaP (T-PSA, 1890 [micro]g/L; F-PSA, 190 [micro]g/L) and reference sera were obtained from Bioclinical Partners Inc. PSA from semen and PSA-ACT complex were products from Scripps Laboratories. The CaP and BPH sera panels were supplied by the sera collection of Roche Diagnostics. The monoclonal antibodies used were from the protein chemistry department of Roche Laboratory Diagnostics, Penzberg. Streptavidin-coated magnetic beads (2.5-[micro]m diameter) were the same as those being used in the Roche Laboratory Diagnostics automatic immunoanalyzer ELECSYS[R].

All other products used were from Roche Molecular Biochemicals or Merck, if not indicated otherwise.

The following buffers were used for the immunosorption: (a) incubation buffer [phosphate-buffered saline (PBS) containing 10 g/L bovine serum albumin and 1 g/L Tween 20, pH 7.4]; and (b) washing buffer (PBS containing 20 mmol/L octylglucoside, pH 7.4).

CLEAVAGE OF PSA-ACT REFERENCE MATERIAL IN BUFFER

PSA-ACT (4 [micro]L of a 1 g/L solution in PBS) was added to 76 [micro]L of the respective cleavage buffers described below and incubated between 0 and 240 h at 25 [degrees]C. The cleavage buffers consisted of PBS with the nucleophiles added to the indicated concentrations described under Results (0.1-1 mol/L). After addition of the nucleophiles, the buffer was adjusted to pH 9 with 1 mol/L hydrochloric acid. At the end of the incubation, the samples were analyzed by reversed-phase HPLC and the ENZYMUN[R] assays for F-PSA and T-PSA.

REVERSED-PHASE HPLC

A Poros R1/H reversed-phase column (2.1 X 30 mm; Perseptive Biosystems) was used for the analysis of the PSA-ACT cleavage assays. The flow rate was 0.5 mL/min, and absorbance was monitored at 215 nm. Eluent A consisted of 1 g/L trifluoroacetic acid in distilled water, and eluent B consisted of 0.85 g/L trifluoroacetic acid in a mixture of acetonitrile-distilled water (7:3, by volume). The following linear step gradient was used: 0-2 min, 20% eluent B; 2-5 min, 20-55% eluent B; 5-13 min, 55-100% eluent B; 13-15 min, 100% eluent B. A 10-[micro]L sample was injected for each analysis.

CLEAVAGE OF PSA-ACT IN THE CaP AND BPH SERA

A solution of 270 [micro]L of PBS and 30 [micro]L of 2 mol/L ethanolamine (pH 12) was added to 300 [micro]L of serum (final pH of 10.3) and incubated at 25 [degrees]C for 24 h. After that time, the samples were analyzed without further treatment by the ENZYMUN assays for F-PSA and T-PSA.

IMMUNOLOGICAL ASSAYS

The ENZYMUN assays for F-PSA and T-PSA were performed with an ES 600 automatic analyzer from Roche Diagnostics GmbH as described by the supplier.

ISOLATION OF PSA AFTER CLEAVAGE OF THE PSA-ACT COMPLEX

A suspension of streptavidin-coated magnetic beads (1.25 mL; 10.7 g/L) was placed in a 10-mL tube and washed with PBS. After the addition of 2 mL of biotinylated anti-F-PSA IgG (monoclonal antibody from mouse; 25 mg/L) the suspension was incubated for 30 min. The beads were collected, washed three times, and incubated with 3.8 mL of the CaP plasma for 1 h to bind the F-PSA to the beads. After removal of the beads, the supernatant was incubated with 200 [micro]L of 2 mol/L ethanolamine (pH 12) at 25 [degrees]C for 24 h. The pH of the reaction mixture was then adjusted to 7.8 with 0.1 mol/L hydrochloric acid, and the released F-PSA was isolated by immunosorption: A suspension of magnetic beads (1.25 mL; c = 10.7 g/L) was again placed in a 10-mL tube and washed as described. After the addition of another 2 mL of biotinylated anti-FPSA IgG (25 mg/L in incubation buffer) the suspension was incubated for 30 min. The beads were washed three times and incubated for 1 h with the cleavage reaction mixture described above. The suspension was washed as described and treated with 250 [micro]L of 1 mol/L propionic acid for 1 h. After magnetic separation of the beads, the supernatant was removed, lyophilized in a vacuum concentrator, and stored at -20 [degrees]C if further analysis was not performed immediately after lyophilization.

SDS-PAGE AND DIGESTION BY ENDO LYS C

SDS-PAGE was performed under nonreducing conditions, using the MiniPROTEAN II gel electrophoresis system and preformed 4-20% gradient gels from Bio-Rad, essentially using the protocol described by Laemmli (23). The gels were silver stained, and the PSA band was digested with endo Lys C, an endoproteinase from Lysobacter enzymogenes, as described by Shevchenko et al. (24).

MALDI-TOF MS

The samples were analyzed in a Voyager[TM] Biospectrometry[TM] Workstation VESTEC matrix-assisted laser desorption-induced time of flight (MALDI-TOF) mass spectrometer equipped with delayed extraction, operating in the positive mode of detection. The spectrometer contains a nitrogen laser operating at 337 nm. TOF spectra were produced at 25 kV acceleration voltage by averaging 80 single spectra. A matrix consisting of a saturated solution of ferulic acid (4-hydroxy-3-methoxycinnamic acid) in formic acid-water-acetonitrile (1:3:2, by volume) was used for all determinations. PSA from semen was used as a reference solution at a concentration of 2 pmol of protein per milliliter of distilled water. The eluates from the immunosorption procedures were dissolved in 10 [micro]L of distilled water. An aliquot of this protein solution (0.5 [micro]L) was mixed with 1 [micro]L of the matrix solution on the target plate and allowed to dry at room temperature before insertion into the mass spectrometer. All spectra were calibrated externally using bovine serum albumin, [[M+H].sup.+] = 66 431 Da, and horse skeletal apomyoglobin, [[M+H].sup.+] = 16 953 Da, as references.

Results

CHEMICAL CLEAVAGE OF PSA-ACT REFERENCE

MATERIAL

In analogy to the complex formation between human chymotrypsin and ACT (25), the complex of PSA and ACT was described as formed by the esterification of Ser-189 of PSA with Leu-358 of ACT. This led to the cleavage of ACT between Leu-358 and Ser-359, as indicated by SDS-PAGE and N-terminal sequence analysis (19). The molecular masses of the three substances we observed were in agreement with this report: MALDITOF MS revealed molecular masses of 80.8 kDa for PSA-ACT, 28.3 kDa for PSA, and 56.9 kDa for ACT. The molecular mass of the complex was 4.4 kDa less than the sum of the masses of PSA and ACT, and this points to the loss of the terminal peptide of ACT starting from Ser-359, which possesses a molecular mass of 4.4 kDa.

In further agreement with previous reports (19), we found that the PSA-ACT complex is rather stable during storage in buffer. Incubation in PBS buffer at pH 7.3 or 11.3 at 25 [degrees]C for 60 h revealed no cleavage or loss of the complex as indicated by reversed-phase HPLC analysis (data not shown). Similarly, F-PSA did not show any alteration of the HPLC peak after storage under similar conditions. However, a substantial cleavage of the PSA-ACT complex was observed when nucleophilic compounds such as ethanolamine were added to the storage buffer, as is illustrated in Fig. 1.

The PSA-ACT complex disappeared almost completely after storage at 25 [degrees]C in PBS (pH 9.0) containing 1 mol/L ethanolamine, and two peaks representing PSA and ACT increased in size with longer storage times. Cleavage of PSA-ACT was also achieved by other reagents similar to ethanolamine, as shown in Table 1.

Interestingly, nucleophiles such as methylamine or the hydroxyl ion are not able to catalyze the cleavage effectively. Compounds with at least two nucleophilic groups seem to be required. Furthermore, strong nucleophiles such as hydroxylamine or hydrazine appear to degrade the complex altogether, because the PSA-ACT peak disappeared entirely without the concomitant appearance of PSA and ACT peaks. The cleavage rate of the PSA-ACT complex was dependent on the ethanolamine concentration. Reducing the concentration of ethanolamine from 1 mol/L to 0.1 mol/L reduced the product rate to 30% of the initial value (data not shown).

[FIGURE 1 OMITTED]

IMMUNOLOGICAL ANALYSIS OF THE RELEASED PSA

In addition to HPLC analysis, PSA was determined immunologically in the cleavage assays to probe the released PSA for immunologically recognizable epitopes. The measurements of total and free PSA were performed with an ENZYMUN analyzer, and the results are displayed in Fig. 2.

As shown in Fig. 2, the PSA released from the complex was recognized in the ENZYMUN assays for F-PSA as well as for T-PSA. In further accordance with the HPLC analysis, no PSA could be detected after treatment of the complex with hydroxylamine, indicating the degradation of PSA. Ethanolamine treatment seemed to retain the immunological properties of F-PSA, as indicated in Fig. 2. Nearly identical values were found in the total and free PSA ENZYMLJN assays after incubation of F-PSA for 60 h in the presence of 0.1 mol/L ethanolamine at pH 9.

[FIGURE 2 OMITTED]

CLEAVAGE OF PSA-ACT IN CaP PLASMA

To determine whether the PSA-ACT complex in human blood can be cleaved similar to the complex formed in vitro, plasma from a CaP patient (T-PSA, 1890 [micro]g/L; F-PSA, 190 [micro]g/L) was treated with 0.1 mol/L ethanolamine for 60 h at different pH values, and the release of F-PSA was monitored using the ENZYMUN assays. The results of the incubations are shown in Fig. 3.

It can be seen that only a minor release of F-PSA is observed after incubation with 0.1 mol/L ethanolamine at pH 8.4. Raising the pH to 9.3 increases the value of F-PSA considerably within 24 h of incubation. After incubation for 48 h at pH 10.3 in the presence of ethanolamine, the F-PSA value is close to the T-PSA value of 1890 [micro]g/L. The reaction proceeded at an appreciable velocity, as indicated by the 0 h values at pH 9.3 and 10.3, which already were well above the starting value of 190 [micro]g/L. Release occurred between the addition of the ethanolamine and the withdrawal and freezing of the sample aliquot for the immunological analysis.

[FIGURE 3 OMITTED]

CLEAVAGE OF PSA-ACT IN CaP AND BPH SERA WITH LOWER PSA CONTENT

We applied the ethanolamine cleavage of PSA-ACT to the measurement of T-PSA, using only the assay for F-PSA. Thus, a panel of BPH and CaP sera was incubated for 24 h in the presence of 0.1 mol/L ethanolamine as described in Materials and Methods, and the F-PSA content was analyzed by the ENZYMUN assay. In addition, the free and total PSA values of the untreated sera were determined in parallel. The results of these experiments are shown in Table 2. The T-PSA values measured as F-PSA after treatment correlated very well with the values determined in the ENZYMUN assay for T-PSA (r = 0.97 for both panels).

MALDI-TOF MS ANALYSIS OF PSA RELEASED FROM PSA-ACT COMPLEX OF CaP PLASMA

The PSA released from the PSA-ACT complex of the CaP plasma used above was isolated by immunosorption as described previously (22). F-PSA was first removed from the plasma by immunosorption, confirming that no F-PSA remained. Subsequently, the plasma was treated with ethanolamine as described above, and the released PSA was isolated by immunosorption using a biotinylated antibody with high affinity to F-PSA only. The PSA was isolated by SDS-PAGE, analyzed by MALDI-TOF MS, and compared to the PSA reference material from semen. The results are displayed in Fig. 4.

The two samples showed almost identical molecular masses and similar peak patterns. An additional shoulder at ~28.2 kDa was present in the spectrum of the PSA obtained from the complex. This was later shown to be caused by contamination with apolipoprotein A, which bound nonspecifically to the magnetic beads and moved similarly to PSA in SDS-PAGE (compare also Fig. 5).

After SDS-PAGE separation, the F-PSA from the two sources was compared in detail by protease digests with endo Lys C. Fig. 5 shows that the peptide patterns of the two PSA samples were very similar and matched the theoretical values predicted from the PSA sequence (78% coverage of the sequence).

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The additional peptides observed in the PSA sample matched the peptides predicted for the endo Lys C digest of human apolipoprotein A, thus revealing that this protein of 28.2 kDa was a contaminant of the PSA sample originating from the CaP plasma (see Fig. 5). Altogether, the data confirm that the PSA released from the PSA-ACT complex is very similar, if not identical, to the reference material obtained from semen.

Discussion

To date, the biochemical characterization of PSA complexed to ACT in human blood has been difficult because it has only been possible to analyze the entire PSA-ACT complex or to separate the two components by treatments that cause the loss of the structural integrity of PSA. Therefore, to date nearly all structural information on PSA has been obtained by investigating PSA from semen (26), from cell cultures (27), or from recombinant sources [for review, see Ref. (28)]. The chemical method described here allows the cleavage of the PSA-ACT complex in human blood and the isolation of the liberated PSA, making it amenable for structural investigations. The immunological measurements performed on liberated PSA to date illustrate that at least these epitopes of PSA, which are recognized by the antibodies in the two immunoassays used, retain their intact structures. The HPLC analysis on reversed-phase also did not reveal any difference in the elution time or pattern of the released PSA compared with the PSA of semen. Finally, the MALDI-TOF MS measurements of the released intact PSA and the peptides after proteolytic digestion also did not show any difference for both sources, again confirming that no major structural alterations of the PSA took place during the chemical cleavage of the complex. Moreover, it can be assumed that the structure of the PSA released from the complex is very similar to that of the F-PSA present in semen, a fact that is not too surprising considering that it has been shown that only the enzymatically active PSA isoenzymes react with ACT (29). Thus, close similarity was found for the peptides analyzed by MALDI-TOF MS (see Fig. 5) and also for the glycosylated peptide (amino acids 11-45), although this could not be observed in the peptide pattern because of the low response in the MALDI-TOF MS at low concentrations. However, the glycopeptide obtained from reference PSA (semen) was detected by MALDI-TOF MS, and it showed the sialylated complex biantennary N-glycan structure described previously (26). Because the intact PSA molecules of the two sources showed an almost identical molecular mass and a similar peak pattern in HPLC and MALDI-TOF MS, it can be assumed that only minor differences are present between these two molecules. Regarding the structural integrity, it will be interesting to learn whether the released PSA still displays protease activity, and if it does, to what extent. Furthermore, it will be worthwhile to probe the released PSA with more antibodies, specifically those recognizing conformational epitopes to probe their integrity.

The data obtained for the cleavage of the PSA-ACT complex in human plasma or sera (see Fig. 3 and Table 2) suggest that the complex was cleaved to near completion. On average, the values measured after ethanolamine treatment were -85% of the total PSA values detected in the untreated sera. There could be several effects contributing to this slightly lower value: (a) A small amount of PSA-ACT complex could still be present because of incomplete cleavage. (b) Other PSA-serpin complexes, which are also present in human sera (4, 6) but represent <5% of T-PSA (30), might not be cleaved by the treatment. (c) The released PSA might form complexes with [[alpha].sub.2] macroglobulin or serpins. However, new complex formation does not seem to take place to a substantial extent because otherwise the amount of F-PSA present at the end of the incubations would be much smaller. This inability to form new complexes could be attributable to the loss of proteolytic activity of the released PSA or the "cleavage conditions" that are always present in the assays. This is in contrast to the reported cleavage of the PSA-API complex, where the released PSA seemed to form complexes again when the cleavage was performed in serum (21). Which of these possible reasons hold true as explanations for the lower value detected need to be analyzed in further investigations.

It can also be concluded from the data that the complex of PSA and [[alpha].sub.2] macroglobulin present in serum does not seem to release PSA under the cleavage conditions because otherwise the PSA values after ethanolamine treatment should be higher than the T-PSA values of the untreated sera.

The data also reveal that the presence of serum was favorable for the cleavage of PSA-ACT, because it was almost complete after 24 h, whereas only ~40% of PSA-ACT was cleaved when PBS buffer with the same pH and ethanolamine concentration was used. A similar favorable effect of serum was also reported for the cleavage of PSA-API (21). Incidentally, the addition of bovine serum albumin (10 g/L) to the assays also seemed to accelerate the cleavage of PSA-ACT in buffer (data not shown).

The novel procedure described allows the measurement of free and "total" PSA with one type of assay, thus eliminating any bias that might exist when two types of assays are used (7, 11, 12, 31 ). Whether the ratio of F-PSA to T-PSA determined by this method might somewhat improve the differentiation between BPH and CaP compared with the determination of the ratio using two different types of assays is uncertain and needs to be analyzed in larger panels of BPH and CaP sera. However, the main value of this new method must be regarded more in the fact that it makes PSA complexed to ACT in serum amenable for studies in the form of F-PSA, which allows further structural analysis (e.g., of glycosylation), which to date has been very difficult or not possible.

Received November 9, 1999; accepted January 24, 2000.

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(2.) Chu TM. Prostate-specific antigen and early detection of prostate cancer. Tumor Biol 1997;18:123-34.

(3.) Sokoll U, Chan DW. Total, free and complexed PSA: analysis and clinical utility. J Clin Ligand Assay 1998;21:171-9.

(4.) Stenman UH, Leinonen J, Alfthan H, Ranniko S, Tuhkanen K, Alfthan 0. A complex between prostate-specific antigen and [[alpha].sub.1] antichymotrypsin is the major form of prostatic-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51: 222-6.

(5.) Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson 0, Pettersson K, Lovgren T. Prostate-specific antigen in human serum occurs predominantly in complex with [[alpha].sub.1] antichymotrypsin. Clin Chem 1991;37:1618-27.

(6.) Becker C, Lilja H. Individual prostate-specific antigen (PSA) forms as prostate tumor markers. Clin Chim Acta 1997;257:117-32.

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(10.) Wu JT. Assay for prostate-specific antigen (PSA): problems and possible solutions. J Clin Lab Anal 1994;8:51-62.

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(21.) Zhang WM, Leinonen J, Kalkkinen N, Stenman UH. Prostate-specific antigen forms a complex with and cleaves [[alpha].sub.1]-protease inhibitor in vitro. Prostate 1997;33:87-96.

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JOCHEN PETER, [1] * CARLO UNVERZAGT, [1] [dagger] and WOLFGANG HOESEL [2] [double dagger]

[1] Institut fur Organische Chexnie and Biochemie, Technische Universitat Munchen, Lichtenbergstrasse 4, 85748 Garching, Germany.

[2] Roche Diagnostics GmbH, Nonnenwaldstrasse 2, 82372 Penzberg, Germany.

[3] Nonstandard abbreviations: PSA, prostate-specific antigen; CaP, prostate cancer; BPH, benign prostatic hyperplasia; ACT, [[alpha].sub.1]- antichymotrypsin; F-PSA and T-PSA, free and total PSA; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; API, [[alpha].sub.1]-protease inhibitor; PBS, phosphate- buffered saline; and MALDI-TOF MS, matrix-assisted laser desorption-induced time of flight mass spectrometry.

* Present address: National Institute of Environmental Health Sciences (NIH/NIEHS), Bldg. 101, Room F011, Research Triangle Park, NC 27709. ([dagger]) Present address: Lehrstuhl fur Bioorganische Chexnie, Universitat Bayreuth, Gebaude NW 1, 95440 Bayreuth, Germany. ([double dagger]) Author for correspondence. Fax 49-8856-603341; e-mail wolfgang.hoesel@roche.com.
Table 1. Cleavage of the PSA-ACT complex in the presence
of different amine-containing nucleophiles.

 Cleavage after 60 h (b)

Amine-containing nucleophile (a) %
Ethanolamine 28
Diethanolamine 17
Tris[hydroxymethyl]aminomethane 14
Ethylenediamine 8
Hydroxylamine Complex destroyed
Hydrazine Complex destroyed

(a) The concentration of the nucleophiles was 0.1 mol/L, respectively.

(b) Incubation for 60 h at 25 [degrees]C in PBS, pH 9, after
addition of the nucleophiles.

Table 2. Determination of F-PSA values in sera panels of
BPH and CaP after cleavage of the PSA-ACT complex by
treatment with ethanolamine.

 F-PSA, [micro] g/L after
 24-h alkaline
 F-PSA, (a) T-PSA, (a) ethanolamine
Serum mg/L mg/L treatment (b)
BPH sera

1 0.48 1.61 1.28
2 0.81 3.12 2.44
3 1.01 3.71 2.92
4 2.85 7.05 5.84
5 0.45 3.05 2.38
6 0.67 2.32 2.26
7 1.42 5.28 4.84
8 1.62 5.78 5.18
9 0.22 2.03 1.78
10 1.72 8.44 6.64
11 4.01 10.1 7.8
12 1.12 4.33 3.54

CaP sera

1 2.93 45.7 48
2 4.54 37.9 32.18
3 2.33 17.6 12.4
4 0.86 5.28 4.3
5 0.53 3.99 3.2
6 0.84 16.1 13.5
7 1.85 15.4 12.54
8 0.64 2.58 2.4
9 0.81 4.45 3.8
10 0.71 4.05 3.7
11 1.43 7.26 6.54
12 1.96 16.5 13.92
13 0.73 7.17 5.82

(a) Values of the untreated sera.

(b) Treatment with 0.1 mol/L ethanolamine in PBS, pH 10.3, at 25
[degrees]C for 24 h as described in Materials and Methods. At the
end of the incubations, the cleavage assays were used as samples
in the ENZYMUN F-PSA assay without additional treatment.
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Title Annotation:Enzymes and Protein Markers
Author:Peter, Jochen; Unverzagt, Carlo; Hoesel, Wolfgang
Publication:Clinical Chemistry
Date:Apr 1, 2000
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Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step.
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