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Enzymes and Protein Markers |
a Author for correspondence. Fax 914-524-2195; e-mail jeffrey.allard.b{at}bayer.com.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-1-antichymotrypsin (ACT) is
higher in men with CaP than in men with benign prostate disease. We
developed a novel immunoassay for complexed PSA based on the unique
binding properties of a monoclonal antibody that fails to bind free PSA
in the presence of antibodies specific for free PSA. The assay measured
mixtures of free and complexed PSA accurately, and the measured values
of free + complexed PSA in artificial mixtures and in patient sera were
equivalent to the measured value of total PSA. Both the serum
concentration and the proportion of complexed PSA was substantially
higher in patients with CaP compared with patients with benign prostate
disease. The cPSA assay may have utility in improving specificity in
screening for prostate cancer. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several approaches have been suggested to improve the specificity of
PSA testing. These include the use of serum PSA and prostate gland
volume to develop a ratio termed PSA density (10),
longitudinal measurement of serum PSA values to develop the rate of
increase in PSA per year, or PSA velocity (11), and the
application of age-adjusted reference ranges to compensate for the
known increase in prostate gland volume in men over the age
of 50 years (12). Despite promising evidence in the
clinical literature, none of these approaches has gained widespread
acceptance. Another option to improve PSA specificity stems from the
observations of Stenman et al. (13) and Lilja et al.
(14), who noted that PSA in serum exists in several forms
including free, uncomplexed PSA and PSA complexed to several protease
inhibitors, including -2-macroglobulin, -1-antichymotrypsin,
and -1-antitrypsin. In addition, these groups demonstrated that the
proportion of PSA complexed with ACT increases as a function of the
total PSA concentration and that the majority of immunoreactive PSA in
cancer patients is in complex with ACT (13)(14)(15). Accurate
measurement of complexed PSA has proven difficult, however, because of
technical problems with two-site sandwich assays for PSA-ACT. This may
be because of the high concentrations of free ACT found in serum or
because of complexation of ACT with other proteases, such as cathepsin
G, which may cross-react in assays designed to measure PSA-ACT
(16)(17). Several options have been proposed to
overcome these problems, such as the addition of heparin
(16), the use of Super Block (18), addition of
antibodies to cathepsin G (17)(19), and the use
of antibodies selective for PSA-ACT with reduced binding to free PSA or
ACT alone (20). None of these approaches, however, has been
found to eliminate the problems of over- and underrecovery of PSA-ACT
complexes in serum. Because of these problems, subsequent
investigations focused on the use of a ratio of free and total PSA.
Numerous studies have now demonstrated that the addition of free PSA to
the test panel of total PSA and digital rectal examination yields
improved specificity for discrimination of patients with benign and
malignant prostate disease
(19)(21)(22).
In the studies reported here, we explored a different approach to the measurement of complexed PSA. Previous results from our laboratory have shown that the Bayer Immuno 1TM PSA Assay uses a monoclonal antibody for capture that recognizes both free and complexed PSA, but which, when bound to free PSA, precludes the binding of other antibodies specific for the free form of PSA (23). We exploited the properties of this antibody to develop a novel assay for the measurement of PSA in complex with protease inhibitors. We show here that this assay is accurate, and that both the absolute amount as well as the proportion of complexed PSA is higher in the serum of patients with CaP than in those without cancer.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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automated immunoassays for total and free psa
The Bayer Immuno 1 PSA Assay (Bayer Diagnostics) is a commercially
available sandwich immunoassay that uses a monoclonal antibody for
capture (termed the MM1 monoclonal antibody in the studies reported
here) and affinity purified goat polyclonal antibodies conjugated to
alkaline phosphatase for detection. This assay, which has been
described in detail previously, provides equimolar detection of
free and complexed PSA, based on properties of the monoclonal antibody
used for capture (23)(24). The free PSA assay
used in these studies used antibody PSA19, a free PSA-specific
monoclonal antibody, for the capture phase. All other conditions were
the same as those used in the total PSA assay; this free PSA assay
format has been described in detail previously (24).
patient samples
Serum samples from 86 consenting patients with biopsy-confirmed
CaP and from 103 patients with biopsy-confirmed benign prostate disease
were purchased from Bioclinical Partners. Samples were drawn before the
biopsy procedure and were stored at -80 °C for up to 4
years before use. Information on the stage and grade of
CaP was not available for these samples. All samples were
thawed and tested in each of the three immunoassays (total, free, and
complexed PSA) in one run on the Immuno 1 system and during one
freeze-thaw cycle.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The epitope recognized by free PSA-specific antibodies has been termed
the E epitope. If the capture antibody in the Immuno 1 PSA Assay
inhibits the binding of anti-E-specific antibodies, then the binding of
antibodies to the E epitope may inhibit binding of the MM1 antibody. To
test this hypothesis, we added E epitope-specific monoclonal antibodies
over a range of concentrations to Immuno 1 PSA Assay calibrators
containing only free PSA at concentrations of 25 and 10 µg/L (25 and
10 ng/mL). After a 30-min incubation at room temperature, we measured
the reactivity of free PSA in the Immuno 1 PSA Assay. Results shown in
Fig. 1
A demonstrate that antibodies PSA19, PSA20, and PSA30 did reduce
the reactivity of free PSA at 25 µg/L (25 ng/mL) in the Immuno 1
total PSA Assay. None of these antibodies reached saturation in their
inhibition of free PSA reactivity, even at 200 mg/L, which represents a
2000-fold molar excess of antibody to PSA. This suggested that
antibodies PSA19, PSA20, and PSA30 may be of low affinity relative to
that of MM1. In addition, maximum inhibition of free PSA reactivity was
90% using these antibodies. Additional optimization showed that
antibody PSA20 inhibited 93% of free PSA reactivity at 300 mg/L, but
this concentration is too high for practical application to automated
immunoassay formats. We therefore tested additional anti-E-specific
monoclonal antibodies, and results with the free PSA-specific antibody
ME2 are shown in Fig. 1B
. Preincubation of free PSA at 10 µg/L (10
ng/mL) with the ME2 antibody gave virtually quantitative inhibition of
free PSA reactivity in the Immuno 1 PSA Assay at antibody
concentrations of <10 mg/L. These results formed the basis of an
automated immunoassay specific for complexed PSA, shown
diagrammatically in Fig. 2
. An excess of ME2 antibody is used to reduce reactivity of free
PSA in the Immuno 1 PSA Assay to <3%; therefore, >97% of the
reactivity in the cPSA assay is because of complexed PSA.
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performance of the cPSA immunoassay using supplemented
samples with varying proportions of free and complexed psa
In our initial efforts to optimize an immunoassay for complexed
PSA, anti-E antibodies were preincubated with samples before
measurement on the Immuno 1 instrument. In subsequent studies, we
optimized the addition of anti-E antibody to provide a fully automated
immunoassay format. In this format, the anti-E antibody is added
directly into the compartment containing the alkaline
phosphatase-labeled polyclonal anti-PSA used for the detection phase,
to give a final concentration of anti-E antibody of 38 mg/L. We then
prepared mixtures with varying proportions of free PSA and PSA-ACT at a
total concentration of approximately 4 µg/L (4 ng/mL) PSA and tested
these preparations in the Immuno 1 total, free, and complexed PSA
assays. Results shown in Fig. 3
demonstrate that each of the PSA assays measured the mixtures
of free and complexed PSA accurately. The Immuno 1 total PSA Assay
demonstrated an equimolar response, as expected. The free PSA assay
showed a decreasing response as the proportion of free PSA decreased,
whereas the complexed PSA assay gave increasing values. When the values
obtained with the free and complexed PSA assays were added and compared
with concentrations measured by the total PSA assay, the sums of the
free PSA and complexed PSA values were within 5% of the measured
concentrations of total PSA.
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preliminary measurement of complexed psa in patients with benign
and malignant CaP
We tested the clinical utility of the cPSA assay, using sera from
86 patients with biopsy confirmed CaP and from 103 patients with biopsy
confirmed benign prostate disease. Each serum sample was tested on the
Immuno 1 analyzer with the total, free, and complexed PSA assays.
Results shown in Table 1
demonstrate that the mean complexed PSA concentration in
patients with CaP was higher than in patients without cancer, and the
proportion of complexed PSA was also higher in men with CaP (95%)
compared with men without cancer (87%). The mean values for free PSA
cPSA gave values that were 107% and 106% of the mean values for total
PSA in the benign and malignant prostate disease groups, respectively.
Very similar results were obtained using regression analysis for a
comparison of measured free PSA measured cPSA, compared with measured
total PSA (data not shown).
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We also estimated the Decision Limit for cPSA. To maintain current standards of sensitivity for CaP detection, cPSA must demonstrate a sensitivity at least equivalent with total PSA. Therefore, rather than develop a Decision Limit based on healthy individuals, we estimated the highest value of cPSA that would provide equal sensitivity for cancer detection to total PSA at the accepted cutoff value of 4.0 µg/L (4 ng/mL). The sensitivity of total PSA in the CaP population in this study was 89%. To achieve the same sensitivity, the Decision Limit for cPSA was determined to be 3.75 µg/L (3.75 ng/mL).
relative proportion of complexed psa in serum of patients with
benign prostate disease and CaP
Previous studies have shown that the proportion of PSA complexed
to ACT is higher in patients with CaP compared with those with benign
prostate disease (13). We examined the proportion of
complexed PSA as a function of the total PSA concentration, and the
results are shown in Fig. 4
. Only a slight trend can be seen toward increasing proportions
of complexed PSA with increasing concentrations of total PSA. The
clustering of patients with CaP in the upper right portion of Fig. 4
suggests that cancer patients do in fact have increased cPSA values,
but no positive trend in the proportion of complexed PSA with total PSA
could be seen for the cancer or benign prostate disease patient groups.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reactivity of the MM1 antibody with PSA is similar to that reported for the 2E9 antibody, which also reacts with free and complexed PSA but does not form sandwiches with antibodies to free PSA (16). However, the 2E9 antibody differs from MM1 in two key characteristics: 2E9 binds to reduced and denatured PSA (16), whereas the MM1 antibody does not (25); and 2E9 has a lower affinity for PSA-ACT than for free PSA, whereas the MM1 antibody binds to both forms with similar very high affinity, Ka = 2 x 10 L/mol (23).
The cross-inhibition of the MM1 monoclonal antibody with other
antibodies to the E epitope of PSA forms the basis of a novel
immunoassay format for the accurate measurement of PSA in complex with
protease inhibitors. The assay described here is identical to the
current Bayer Immuno 1 PSA Assay, which measures total PSA, except that
the ME2 antibody is added in large molar excess. It is not clear at
present which complexes of PSA with protease inhibitors react in the
cPSA assay. Preliminary experiments have shown that the predominant
reactivity is with PSA complexed to ACT (data not shown), but we cannot
rule out reactivity with PSA complexed to other inhibitors, such as
-1-antitrypsin, protein C inhibitor, or inter-alpha-trypsin
inhibitor. An additional possibility is that, if MM1 and ME2 bind to
different epitopes on free PSA, then some forms of free PSA that are
not recognized by ME2 could be bound by MM1. We consider this unlikely
because the results shown in Table 1
suggest that the addition of
measured free and complexed PSA is approximately equal to the average
value for the measurement of total PSA.
We have shown that the measurement of complexed PSA using the present
assay is accurate in two ways. First, when PSA and PSA-ACT were mixed
in vitro and measured using assays for total, free, and complexed PSA,
the measured concentration of free complexed PSA was equal to the
measured concentration of total PSA for each of the mixtures (Fig. 3
).
In addition, for the serum samples from patients with and without
cancer, the measured values for free complexed PSA were approximately
equal to the measured values for total PSA (Table 1
). It is unlikely
that the small overrecovery of free complexed PSA compared with total
PSA is the result of false high measurements using the total PSA assay,
because this assay provides PSA concentrations that agree almost
exactly with PSA measured by biophysical means (24), and the
same is true for the cPSA assay (data not shown). The free PSA assay
used in these studies is a research assay that has been partially
characterized and may give false high values. In any event, the
discrepancy is slight, and taken together, these data demonstrate that
the cPSA assay measures complexed PSA accurately in vitro and in vivo.
Previous attempts to measure PSA-ACT complexes used antibodies to PSA coated onto a microplate or beads for capture, and labeled anti-ACT for the detection phase (13)(14)(17)(18)(26). These formats have been plagued with falsely increased values because of nonspecific binding of cross-reactive molecules. Several approaches have been used to combat this problem, such as the addition of heparin (16), antibodies to cathepsin G with uncoated latex particles (17), and Super Block (18). These approaches have met with limited success. The addition of an antibody specific for free PSA to the Immuno 1 total PSA Assay avoids the problems of nonspecific interference by rendering free PSA nonreactive in the immunoassay.
The cPSA assay format was used to compare the proportion of complexed
PSA in patients with benign and malignant prostatic disease. As shown
in Fig. 4
, no direct relationship between the proportion of complexed
PSA and total PSA values could be seen. Previous studies examined
similar populations and found contradictory results. Stenman et al.
(13) found that the fraction of complexed PSA increases
roughly in proportion to total PSA concentrations in men with benign
prostate disease and cancer. Subsequent studies failed to reproduce
this correlation, and showed results similar to ours
(21)(27). In all cases, however, the proportion
of PSA complexed with ACT has been found to be higher in men with CaP
compared with those who do not have cancer. In addition, in the present
study sera from all men with CaP were found to contain >77% complexed
PSA. This is in contrast with sera from men with benign prostatic
disease, where the proportion of complexed PSA ranged from 46% to
98%.
The use of the cPSA immunoassay format provides a means to directly measure the form of PSA that is known to increase in the serum of patients with CaP (13)(14). Previous data have shown that the use of complexed PSA may improve the sensitivity for CaP detection (13), and several studies have found that the ratio of PSA-ACT/total PSA showed an increase in the area under the curve by ROC analysis compared with total PSA, whereas the use of complexed PSA alone showed no improvement by ROC analysis (26)(28).
Numerous studies have shown that the discrimination of men with CaP
from those with benign prostate disease could be improved through the
implementation of a ratio of free and total PSA. This approach has
several problems. The first is that free PSA is not stable in serum,
probably as a result of complexation of free PSA with
-2-macroglobulin, which is nonreactive in current PSA assays. Free
PSA is also present in very low concentrations in serum, particularly
in the asymptomatic population, which may make accurate measurement of
serum free PSA concentrations difficult. In addition, the use of a free
PSA measurement requires that patient samples be "reflex tested" if
the total PSA value falls within a predetermined "gray zone", but
the gray zone is not well defined and has been variably suggested as
4–10 µg/L (4–10 ng/mL) (22)(29), 3–10
µg/L (3–10 ng/mL) (30), 4–25 µg/L (4–25 ng/mL)
(31), and >4 µg/L (>4 ng/mL) (32). For these
reasons, the clinical adaptation of the free-to-total PSA ratio
is complicated and may delay implementation of this approach as a
standard of care for the early detection of CaP.
Our results show that the amount of PSA in complex with protease
inhibitors increases in patients with cancer compared with patients
with benign prostate pathology (Fig. 4
). Therefore, the measurement of
cPSA could prove useful in improving the specificity of PSA testing for
CaP. Any assay aimed toward improvement of specificity in CaP screening
must maintain current levels of sensitivity. We therefore determined
the Decision Limit for the cPSA assay that would provide equivalent
sensitivity to total PSA at 4 µg/L (4 ng/mL), using this limited
patient cohort. This limit was found to be 3.75 µg/L (3.75 ng/mL); it
is to be expected that this value may be adjusted as larger studies are
conducted. Alternatively, it may be possible to choose a Decision Limit
such that both sensitivity and specificity are optimized. The
development of an automated immunoassay that measures complexed PSA
accurately will make it possible to answer such questions and to
investigate the clinical utility of the complexed fraction of PSA in
serum. Studies are currently underway to address these questions and to
compare the utility of complexed PSA measurement with that of free PSA.
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Acknowledgments |
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Footnotes |
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1 Nonstandard abbreviations: PSA, prostate-specific
antigen; CaP, prostate cancer; and ACT,
-1-antichymotrypsin. 
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-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.
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-1-antichymotrypsin in blood. Cancer 1992;70:230-234.
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-1-antichymotrypsin complex. Clin Chem 1995;41:1480-1488.
-1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.
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-1-antichymotrypsin-complexed prostate-specific antigen on an equimolar basis. J Clin Lab Anal 1996;10:155-159.
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-1-antichymotrypsin. Clin Chem 1993;39:2098-2103.
[Abstract]
The following articles in journals at HighWire Press have cited this article:
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  S. D Mulder, J. A Heijst, C. Mulder, F. Martens, C E. Hack, P. Scheltens, M. A Blankenstein, and R. Veerhuis CSF levels of PSA and PSA-ACT complexes in Alzheimer's disease Ann Clin Biochem, November 1, 2009; 46(6): 477 - 483. [Abstract] [Full Text] [PDF]
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 C. M. Sturgeon, M. J. Duffy, U.-H. Stenman, H. Lilja, N. Brunner, D. W. Chan, R. Babaian, R. C. Bast Jr., B. Dowell, F. J. Esteva, et al. National Academy of Clinical Biochemistry Laboratory Medicine Practice Guidelines for Use of Tumor Markers in Testicular, Prostate, Colorectal, Breast, and Ovarian Cancers Clin. Chem., December 1, 2008; 54(12): e11 - e79. [Abstract] [Full Text] [PDF]
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 H. Lilja, D. Ulmert, T. Bjork, C. Becker, A. M. Serio, J.-A. Nilsson, P.-A. Abrahamsson, A. J. Vickers, and G. Berglund Long-Term Prediction of Prostate Cancer Up to 25 Years Before Diagnosis of Prostate Cancer Using Prostate Kallikreins Measured at Age 44 to 50 Years J. Clin. Oncol., February 1, 2007; 25(4): 431 - 436. [Abstract] [Full Text] [PDF]
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D. Ulmert, C. Becker, J.-A. Nilsson, T. Piironen, T. Bjork, J. Hugosson, G. Berglund, and H. Lilja Reproducibility and Accuracy of Measurements of Free and Total Prostate-Specific Antigen in Serum vs Plasma after Long-Term Storage at -20 {degrees}C Clin. Chem., February 1, 2006; 52(2): 235 - 239. [Abstract] [Full Text] [PDF]
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T. Keller, H. Butz, M. Lein, M. Kwiatkowski, A. Semjonow, H.-J. Luboldt, P. Hammerer, C. Stephan, and K. Jung Discordance Analysis Characteristics as a New Method to Compare the Diagnostic Accuracy of Tests: Example of Complexed Versus Total Prostate-Specific Antigen Clin. Chem., March 1, 2005; 51(3): 532 - 539. [Abstract] [Full Text] [PDF]
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 S Jain, A G Bhojwani, and J K Mellon Improving the utility of prostate specific antigen (PSA) in the diagnosis of prostate cancer: the use of PSA derivatives and novel markers Postgrad. Med. J., November 1, 2002; 78(925): 646 - 650. [Abstract] [Full Text] [PDF]
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M. Martinez, F. Espana, M. Royo, J. M. Alapont, S. Navarro, A. Estelles, J. Aznar, C. D. Vera, and J. F. Jimenez-Cruz The Proportion of Prostate-specific Antigen (PSA) Complexed to {alpha}1-Antichymotrypsin Improves the Discrimination between Prostate Cancer and Benign Prostatic Hyperplasia in Men with a Total PSA of 10 to 30 {micro}g/L Clin. Chem., August 1, 2002; 48(8): 1251 - 1256. [Abstract] [Full Text] [PDF]
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 C. Stephan, K. Jung, M. Lein, P. Sinha, D. Schnorr, and S. A. Loening Molecular Forms of Prostate-specific Antigen and Human Kallikrein 2 as Promising Tools for Early Diagnosis of Prostate Cancer Cancer Epidemiol. Biomarkers Prev., November 1, 2000; 9(11): 1133 - 1147. [Abstract] [Full Text]
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K. Jung, B. Brux, M. Lein, B. Rudolph, G. Kristiansen, S. Hauptmann, D. Schnorr, S. A. Loening, and P. Sinha Molecular Forms of Prostate-specific Antigen in Malignant and Benign Prostatic Tissue: Biochemical and Diagnostic Implications Clin. Chem., January 1, 2000; 46(1): 47 - 54. [Abstract] [Full Text] [PDF]
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K. Jung, U. Elgeti, M. Lein, B. Brux, P. Sinha, B. Rudolph, S. Hauptmann, D. Schnorr, and S. A. Loening Ratio of Free or Complexed Prostate-specific Antigen (PSA) to Total PSA: Which Ratio Improves Differentiation between Benign Prostatic Hyperplasia and Prostate Cancer? Clin. Chem., January 1, 2000; 46(1): 55 - 62. [Abstract] [Full Text] [PDF]
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B. L. Croal, I. Mitchell, A. Dickie, P. A. Duff, N. P. Cohen, I. S. Ross, K. Jung, B. Brux, P. Sinha, M. Lein, et al. Complexed Prostate-specific Antigen and the ""Prostate-specific Antigen Gap"" • The authors of the Technical Brief cited above reply: Clin. Chem., November 1, 1999; 45(11): 2040 - 2041. [Full Text] [PDF]
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K. Jung, B. Brux, A. Knabich, M. Lein, P. Sinha, D. Schnorr, and S. A. Loening A Gap Between Total Prostate-specific Antigen and the Sum of Free Prostate-specific Antigen Plus {alpha}1-Antichymotrypsin-Prostate-specific Antigen in Patients with Prostate Carcinoma but not in Those with Benign Prostate Hyperplasia Clin. Chem., March 1, 1999; 45(3): 422 - 424. [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
