Clinical Chemistry 50: 125-129, 2004.
First published November 18, 2003; 10.1373/clinchem.2003.026146
(Clinical Chemistry. 2004;50:125-129.)
© 2004 American Association for Clinical Chemistry, Inc.
Immunopeptidometric Assay for Enzymatically Active Prostate-Specific Antigen
Ping Wua,1,
Lei Zhu1,
Ulf-H
kan Stenman1 and
Jari Leinonen1
1 Department of Clinical Chemistry, Helsinki University Central Hospital, FIN-00029 Helsinki, Finland.
aAddress correspondence to this author at: Department of Clinical Chemistry, Biomedicum, A417a, Helsinki University Central Hospital, Haartmaninkatu 8, PB 700, FIN-00029 Helsinki, Finland. Fax 358-9-47171731; e-mail ping.wu{at}helsinki.fi.
 |
Abstract
|
|---|
Background: Determinations of certain forms of prostate-specific antigen (PSA) have been shown to increase the specificity for prostate cancer (PCa). One such variant, proteolytically active PSA, is a potentially useful tumor marker, but it is not specifically recognized by antibodies. Using phage display libraries, we previously identified a "family" of peptides that bind specifically to active PSA. We used these to develop an immunopeptidometric assay (IPMA) that specifically detects this form of PSA.
Methods: Microtitration plates coated with a PSA antibody were used to capture PSA, and a PSA-binding glutathione S-transferase (GST) fusion peptide was used as a tracer. Bound tracer was detected with an antibody to GST labeled with a europium chelate. PSA isoenzymes with high and low enzymatic activity were used to study binding specificity.
Results: The IPMA detected enzymatically active PSA but not internally cleaved PSA and pro-PSA, which are enzymatically inactive. The assay detected 1–10% of free PSA in serum from PCa patients.
Conclusions: Peptides identified by phage display can be used to develop assays with unique specificities for enzymatically active PSA. IPMA represents a new assay principle with wide potential utility.
|
Introduction
|
|---|
Immunoassays are extremely specific and facilitate accurate measurement of individual proteins in the presence of more than a 1 million-fold excess of other proteins. This situation is typical of tumor markers and hormones present in serum (1). The use of monoclonal antibodies (MAbs)
1
has facilitated development of assays that differentiate between variants of the same protein, but apparently because antibodies recognize epitopes on the surface of proteins, recognition of differences in the internal structure of proteins is hard to achieve with antibodies.
Prostate-specific antigen (PSA) is a 33-kDa serine protease and a sensitive serum marker for prostate cancer (PCa). Measurement of serum PSA is widely used for early detection and monitoring of patients with PCa. However, the usefulness of PSA for early diagnosis of PCa is limited because increased serum concentrations are also caused by benign diseases such as benign prostatic hyperplasia (BPH) (2)(3). The cancer specificity of PSA can be improved by measurement of its complex with 
1-antichymotrypsin, the proportion of which is higher whereas that of free PSA is lower in cancer than in benign diseases (4)(5). This is thought to result from differences in the types of PSA released into the circulation by benign and malignant prostatic cells. PSA secreted by PCa cells is thought to be more active enzymatically than that leaking from the benign prostate; thus, a larger proportion forms a complex with 1-antichymotrypsin (6). PSA isolated from BPH nodule fluids has been shown to contain a high proportion of cleaved forms (7), and free PSA in serum from patients with BPH has also been shown to be more extensively cleaved than that in PCa serum (8). In addition, part of the free PSA in plasma consists of inactive proforms (9)(10), but the presence of some enzymatically active PSA in the circulation can also be detected by capture with an antibody and detection with a sensitive substrate (11). Because of its association with cancer, this form of PSA is of potential clinical utility, but it has not been possible to determine it directly by immunoassay.
We have previously identified PSA-binding peptides by screening phage display peptide libraries and expressing them as glutathione S-transferase (GST) fusion proteins. The peptides bind specifically to enzymatically active free PSA and enhance its enzymatic activity (12)(13). In this study we used two of these peptides as ligands to develop novel immunopeptidometric assays (IPMAs).
|
Materials and Methods
|
|---|
purification of psa
Seminal plasma was precipitated with ammonium sulfate at 25% and 70% saturation, and the fraction precipitating at 70% saturation was dissolved in 50 mmol/L Tris buffer (pH 8) containing 3 mmol/L sodium azide (partially purified PSA). After dialysis against 50 mmol/L Tris buffer, the partially purified PSA was applied to an immunoaffinity column prepared by coupling MAb 5E4 (14) to CNBr-activated Sepharose (Amersham Pharmacia Biotech). The column was washed with 30 bed volumes of washing buffer (50 mmol/L Tris buffer containing 0.5 mol/L NaCl), and PSA was eluted with 1 g/L trifluoroacetic acid. The PSA isoenzymes were further separated by ion-exchange chromatography on a Resource Q column (Amersham Pharmacia Biotech) (15). Pro-PSA was purified from LNCaP cell culture medium by use of an immunoaffinity column with anti-PSA MAb 4G10 (free PSA-specific antibody) (14). Affinity-purified LNCaP PSA was dialyzed against 50 mmol/L phosphate buffer, pH 5.6 (buffer A), and applied to a Resource S column (Amersham Pharmacia Biotech). Pro-PSA was eluted with a linear gradient of buffer A and buffer B (buffer A containing 0.5 mol/L NaCl). The fraction eluting at 0.2–0.3 mol/L NaCl contained mainly pro-PSA, which could be activated by treatment with bovine trypsin (Sigma) as described previously (16).
measurement of enzymatic activity
Purified PSA (100 ng) was incubated in microtitration wells coated with MAb 5E4 for 1 h at 22 °C with slow shaking. After the wells were washed twice, bound pro-PSA was activated by the addition of bovine trypsin (10 ng) in 200 µL of buffer (5 g/L bovine serum albumin in Tris-buffered saline) and incubated for 20 min at 22 °C. After washing, a fluorescent peptide substrate (11) (Enzyme Systems Products) was added to a final concentration of 400 µmol/L, and the fluorescence (355/460 nm) was monitored for 120 min in a Victor 1420 Multilabel counter (Perkin-Elmer Life Science, Wallac).
serum samples
We studied 15 serum samples from PCa patients with total PSA concentrations of 9–475 µg/L and from 10 apparently healthy women. We also analyzed a female serum sample to which PSA was added. Partially purified seminal PSA was added to serum at a final concentration of 15 mg/L. After incubation for 48 h at 37 °C, the sample was fractionated by gel filtration on a 1.6 x 60 cm Superdex-200 column (Amersham Pharmacia Biotech) equilibrated with 50 mmol/L Tris-HCl buffer (pH 7.7) containing 150 mmol/L NaCl (Tris-buffered saline). The flow rate was 15 mL/h, and 2-mL fractions were collected into tubes containing 200 µL of assay buffer with a 10-fold protein concentration. The column was calibrated with IgG (160 kDa) and bovine serum albumin (67 kDa) as molecular size markers.
psa-binding peptides
PSA-binding peptides were identified by screening phage display peptide libraries (12). Two peptides, B-2 and C-4, were constructed as GST fusion proteins, expressed in Escherichia coli BL 21 cells and purified by glutathione affinity chromatography (Amersham Pharmacia Biotech) as described previously (12).
immunofluorometric assays
Immunofluorometric assays for PSA were performed as described previously (17)(18). In the IPMA, 25 µL of sample or calibrator (0.5–250 µg/L; enzymatically active PSA) and 200 µL of assay buffer [50 mmol/L Tris-HCl (pH 7.7), 150 mmol/L NaCl, 67 µmol/L bovine serum albumin, 1 µmol/L bovine IgG] were added to MAb-5E4-coated wells and incubated for 1 h at 22 °C. For assay of chromatographic fractions, a sample volume of 200 µL was used. After incubation, the wells were washed, and 1 µg of GST-peptide in 200 µL of assay buffer was added. After incubation for 60 min at 22 °C, the wells were washed, and 50 ng of europium-labeled anti-GST antibody (Amersham Pharmacia Biotech) in 200 µL of assay buffer was added and incubated for 60 min. The wells were washed four times, and enhancement solution (Perkin-Elmer Life Science, Wallac) was added. Slow orbital shaking was used during all incubations. After 5 min, time-resolved fluorescence was measured with a Victor 1420 Multilabel counter. The detection limit was defined as the concentration corresponding to the fluorescent signal of assay buffer plus 2 SD calculated from 12 replicates.
Purified PSA isoenzymes (A, B, C, D, and E) at a concentration of 150 µg/L were used to study the reactivity of PSA-binding peptides with different PSA isoenzymes in the IPMA. Pro-PSA purified from LNCaP cell medium was studied before and after activation with trypsin.
|
Results
|
|---|
The detection limit of the IPMA was 0.6 µg/L, and the assay was linear up to 250 µg/L (Fig. 1
). The intra- and interassay imprecision (CV) of the assay were 7–12% (n = 10) and 8–13% (n = 6), respectively, at concentrations of 2–100 µg/L.
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Dose–response curve for the IPMA of PSA.
PSA isoenzyme B was used as a calibrator. The background of  2500 cps has been subtracted.
|
|
The reactivities of the PSA isoenzymes (A–E), which display variable enzyme activities, were analyzed by the IPMA. GST-peptides B-2 (CVFAHNYDYLVC) and C-4 (CVAYCIEHHCWTC) bound equally to the intact isoenzymes A and B (defined as 100%), whereas the response was 25%, 15%, and 7% with PSA isoenzymes C, D, and E, respectively (Fig. 2
). Pro-PSA purified from LNCaP cell culture medium showed low enzymatic activity, 7% of that for PSA-B. After activation by trypsin, the activity increased to 93% (Fig. 3
). Approximately 6–9% of pro-PSA was recognized by the IPMA, and after activation with trypsin recognition increased to 71–88% compared with that of PSA-B (Fig. 4
).
View larger version (28K):
[in this window]
[in a new window]
|
Figure 2. Reactivity of various PSA isoenzymes in the IPMA.
Equal amounts (150 µg/L) of each purified PSA isoenzyme (A, B, C, D, and E) were analyzed by IPMA. The response of isoenzyme B was defined as 100%.
|
|
View larger version (14K):
[in this window]
[in a new window]
|
Figure 3. Enzymatic activity of purified LNCaP PSA before and after trypsin activation.
Enzymatic activity of PSA purified from LNCaP cell culture medium was measured with a fluorescent peptide substrate before and after activation with trypsin.
|
|
View larger version (38K):
[in this window]
[in a new window]
|
Figure 4. Reactivity of LNCaP PSA in the IPMA.
Equal amounts of PSA purified from LNCaP cell culture medium were analyzed by IPMA before and after activation with trypsin. The response of PSA isoenzyme B was defined as 100%.
|
|
When serum was analyzed by IPMA, active PSA was detected in samples containing >10 µg/L free PSA. The concentrations measured by IPMA corresponded to 1–10% of the free PSA (Fig. 5
). No PSA was detected by the IPMA in 10 female sera.
View larger version (10K):
[in this window]
[in a new window]
|
Figure 5. Concentrations of free and active PSA in sera from PCa patients.
Free PSA was assayed by immunofluorometric assay, and active PSA was assayed by the IPMA.
|
|
To characterize the fraction of PSA reacting in the IPMA, an excess of purified PSA (15 g/L) was added to serum. During incubation with serum for 48 h, most of the added PSA was rendered undetectable by reaction with 2-macroglobulin, whereas 27% of the added PSA was detectable by immunoassay. Approximately 19% of the detectable PSA was complexed with 1-antichymotrypsin, and 81% was free. Measurement of active PSA by IPMA in fractions separated by gel filtration showed that 10% of free PSA was in the active form. No active PSA was detected in the fractions containing complexed PSA (Fig. 6
).
View larger version (16K):
[in this window]
[in a new window]
|
Figure 6. Fractionation of serum supplemented with purified PSA by gel filtration and assay of total, free, and active PSA in the fractions.
The fractions were assayed for total and free PSA by immunofluorometric assay and for active PSA by the IPMA. Arrows indicate the elution volumes of molecular size markers (150 and 67 kDa).
|
|
|
Discussion
|
|---|
This study shows that artificial peptides identified by phage display libraries can be used together with antibodies as ligands for development of sensitive assays, which we call IPMAs. These assays can be used to measure the enzymatically active subfraction of PSA, which to date has not been detected by established antibody-based assays. We selected peptides reacting with enzymatically active PSA from a phage display library (12) and prepared GST-peptide fusion proteins, which were used as labels in combination with a monoclonal capture antibody. Both active and inactive PSA were bound by the antibody, but only the active PSA fraction was detected by the peptide. Both peptides used, B-2 and C-4, reacted to the same extent with the various PSA isoenzymes isolated from seminal plasma, and isoenzymes with variable enzymatic activities were detected to an extent corresponding to their enzymatic activities. We also studied whether pro-PSA isolated from LNCaP culture medium reacted in the assay. Pro-PSA does not occur in seminal plasma, but it can be isolated from spent medium of LNCaP cells. Approximately 7% of this pro-PSA was recognized, which corresponded to its enzymatic activity. After activation of pro-PSA by trypsin, the enzymatically active PSA was recognized by the assay.
Some MAbs recognizing certain PSA variants have been described recently; one MAb does not recognize PSA cleaved at Lys145-Lys146 but binds to intact and other cleaved forms of PSA (19). Another antibody reacts specifically with PSA cleaved after Lys182 (20). However, MAbs with a specificity similar to that of the peptides for enzymatically active PSA have not been obtained. Antibodies bind to the surface of PSA (21) and probably do not recognize structural differences within the grooves and pits of the molecule. Because of their small size, peptides may recognize differences in the inner structure of PSA, such as the active groove. Because the peptides increase the enzymatic activity of PSA (12), it is likely that they bind to the vicinity of the active groove.
In patients with PCa, PSA is thought to be released into the circulation in a more active form than the forms that leak from benign prostatic tissue, which could explain the high proportion of PSA complexed with inhibitors in cancer patients (4)(22). The IPMA detected 1–10% of free PSA in serum from PCa patients with clearly increased PSA. This result is in line with the finding that 3% of free PSA in the circulation is enzymatically active (11). To characterize the fraction of PSA in serum recognized by the IPMA we added a large excess of PSA isolated from seminal fluid to a serum sample and fractionated it by gel filtration. This showed that 10% of the free PSA was recognized by the IPMA, indicating that it was enzymatically active in spite of the large excess of protease inhibitors in plasma.
The sensitivity of the IPMA is higher than expected on the basis of the moderate affinity of the peptides (Ka =10-7 mol/L). This may be explained by the fact that some of the GST fusion proteins occur as dimers and trimers, which facilitate multivalent binding and enhanced avidity (12). Even so, the IPMA is not sensitive enough for measurement of active PSA in sera with the moderately increased concentrations that are typically present at early stages of PCa and BPH. We are working on improving the sensitivity to facilitate testing of samples in the clinically important range of 2–10 µg/L, in which differentiation between cancer and BPH is a problem.
In conclusion, our studies show that PSA-binding peptides selected by phage display can be used as a new type of ligand in sandwich assays together with an antibody as the capturing ligand on the solid phase. This assay detects enzymatically active PSA and is therefore a potential tool for a specific assay of cancer-associated forms of PSA. A large number of peptides reacting with specific proteins have already been identified by phage display. This technique has mainly been used to develop reagents for tumor targeting (23), but our results demonstrate their potential utility for development of novel types of ligand-binding assays.
|
Acknowledgments
|
|---|
This study was supported by The Finnish National Technology Agency (TEKES), The Finnish Cancer Foundation, The Research Foundation (EVO) of Helsinki University Central Hospital, and the University of Helsinki.
|
Footnotes
|
|---|
Previously published online at DOI: 10.1373/clinchem.2003.026146
1 Nonstandard abbreviations: MAb, monoclonal antibody; PSA, prostate-specific antigen; PCa, prostate cancer; BPH, benign prostatic hyperplasia; GST, glutathione S-transferase; and IPMA, immunopeptidometric assay. 
|
References
|
|---|
- Stenman U-H. Immunoassay standardisation. Price C Newman D eds. Principles and practice of immunoassay, 2nd ed 1997:245-268 Macmillan London. .
- Stamey TA, Yang N, Hay AR, McNeal JE, Freiha FS, Redwine E. Prostate-specific antigen as a serum marker for adenocarcinoma of the prostate. N Engl J Med 1987;317:909-916.[Abstract]
- Hudson MA, Bahnson RR, Catalona WJ. Clinical use of prostate specific antigen in patients with prostate cancer. J Urol 1989;142:1011-1017.[ISI][Medline]
[Order article via Infotrieve]
- Stenman UH, Leinonen J, Alfthan H, Rannikko S, Tuhkanen K, Alfthan O. A complex between prostate-specific antigen and 1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostatic cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.[Abstract/Free Full Text]
- Lilja H, Christensson A, Dahlen U, Matikainen MT, Nilsson O, Pettersson K, et al. Prostate-specific antigen in serum occurs predominantly in complex with 1-antichymotrypsin. Clin Chem 1991;37:1618-1625.[Abstract/Free Full Text]
- Stenman UH, Leinonen J, Zhang WM, Finne P. Prostate-specific antigen. Semin Cancer Biol 1999;9:83-93.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
- Chen Z, Chen H, Stamey TA. Prostate specific antigen in benign prostatic hyperplasia: purification and characterization. J Urol 1997;157:2166-2170.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
- Charrier JP, Tournel C, Michel S, Comby S, Jolivet-Reynaud C, Passagot J, et al. Differential diagnosis of prostate cancer and benign prostate hyperplasia using two-dimensional electrophoresis. Electrophoresis 2001;22:1861-1866.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
- Christensson A, Laurell CB, Lilja H. Enzymatic activity of prostate-specific antigen and its reactions with extracellular serine proteinase inhibitors. Eur J Biochem 1990;194:755-763.[ISI][Medline]
[Order article via Infotrieve]
- Mikolajczyk SD, Marker KM, Millar LS, Kumar A, Saedi MS, Payne JK, et al. A truncated precursor form of prostate-specific antigen is a more specific serum marker of prostate cancer. Cancer Res 2001;61:6958-6963.[Abstract/Free Full Text]
- Niemela P, Lovgren J, Karp M, Lilja H, Pettersson K. Sensitive and specific enzymatic assay for the determination of precursor forms of prostate-specific antigen after an activation step. Clin Chem 2002;48:1257-1264.[Abstract/Free Full Text]
- Wu P, Leinonen J, Koivunen E, Lankinen H, Stenman UH. Identification of novel prostate-specific antigen-binding peptides modulating its enzyme activity. Eur J Biochem 2000;267:6212-6220.[ISI][Medline]
[Order article via Infotrieve]
- Wu P, Stenman U-H, Pakkala M, Närvänen A, Leinonen J. Separation of enzymatically active and inactive prostate-specific antigen (PSA) by peptide affinity chromatography. Prostate 2003;in press..
- Leinonen J, Wu P, Stenman UH. Epitope mapping of antibodies against prostate-specific antigen with use of peptide libraries. Clin Chem 2002;48:2208-2216.[Abstract/Free Full Text]
- Zhang WM, Leinonen J, Kalkkinen N, Dowell B, Stenman UH. Purification and characterization of different molecular forms of prostate-specific antigen in human seminal fluid. Clin Chem 1995;41:1567-1573.[Abstract/Free Full Text]
- Takayama TK, Fujikawa K, Davie EW. Characterization of the precursor of prostate-specific antigen. Activation by trypsin and by human glandular kallikrein. J Biol Chem 1997;272:21582-21588.[Abstract/Free Full Text]
- Leinonen J, Lovgren T, Vornanen T, Stenman UH. Double-label time-resolved immunofluorometric assay of prostate-specific antigen and of its complex with 1-antichymotrypsin. Clin Chem 1993;39:2098-2103.[Abstract]
- Leinonen J, Zhang WM, Stenman UH. Complex formation between PSA isoenzymes and protease inhibitors. J Urol 1996;155:1099-1103.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
- Nurmikko P, Pettersson K, Piironen T, Hugosson J, Lilja H. Discrimination of prostate cancer from benign disease by plasma measurement of intact, free prostate-specific antigen lacking an internal cleavage site at Lys145-Lys146. Clin Chem 2001;47:1415-1423.[Abstract/Free Full Text]
- Linton HJ, Marks LS, Millar LS, Knott CL, Rittenhouse HG, Mikolajczyk SD. Benign prostate-specific antigen (BPSA) in serum is increased in benign prostate disease. Clin Chem 2003;49:253-259.[Abstract/Free Full Text]
- Stenman UH, Paus E, Allard WJ, Andersson I, Andres C, Barnett TR, et al. Summary report of the TD-3 workshop: characterization of 83 antibodies against prostate-specific antigen. Tumour Biol 1999;20(Suppl 1):1-12.
- Stenman UH. Prostate-specific antigen, clinical use and staging: an overview. Br J Urol 1997;79(Suppl 1):53-60.
- Ruoslahti E, Rajotte D. An address system in the vasculature of normal tissues and tumors. Annu Rev Immunol 2000;18:813-827.[CrossRef][ISI][Medline]
[Order article via Infotrieve]
The following articles in journals at HighWire Press have cited this article:
|
|
|
|
 
C. Hekim, J. Leinonen, A. Narvanen, H. Koistinen, L. Zhu, E. Koivunen, V. Vaisanen, and U.-H. Stenman
Novel Peptide Inhibitors of Human Kallikrein 2
J. Biol. Chem.,
May 5, 2006;
281(18):
12555 - 12560.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U.-H. Stenman
Improving Immunoassay Performance by Antibody Engineering
Clin. Chem.,
May 1, 2005;
51(5):
801 - 802.
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
