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Articles |
1
Department of Clinical Chemistry, Lund University, University Hospital Malmö, S-205 02 Malmö, Sweden.
2
Department of Biotechnology, Turku University, 20520
Turku, Finland.
a Author for correspondence. Fax 46-40-33-70-43; e-mail charlotte.becker{at}klkemi.mas.lu.se
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: In the assay, critical PSA epitopes were blocked with four monoclonal antibodies (MAbs) specific for PSA. Subsequently, hK2 was captured using a MAb against hK2 (5% cross-reactivity with PSA), and after washing, hK2 was detected by a europium-labeled MAb with identical affinity for hK2 and PSA.
Results: The analytical detection limit was <10 ng/L,
and functional sensitivity was 30 ng/L. Cross-reaction with PSA was
<0.01%. Between-assay imprecision was 3.1% for 1600 ng/L hK2 and
4.8% for 160 ng/L hK2; corresponding values for within-assay precision
were 1.9% and 4.5%, respectively. Complexes of
hK2-
1-antichymotrypsin (ACT) were detected in vitro with
-6% bias compared with the free form of hK2. Gel filtration of
patient samples showed that hK2 correlated in size mainly with free
hK2; only 4–19% corresponded to hK2 possibly complexed with ACT or
protein C inhibitor.
Conclusions: Our assay had extremely low cross-reactivity with PSA, provided a very low detection limit, and allowed close to equimolar detection of the free and complexed forms of hK2. Moreover, we found that free hK2 is the predominant immunoreactive form of hK2 in serum.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of PSA in serum can be useful for the detection and
monitoring of PCa; unfortunately, slightly increased PSA concentrations
are also present in benign prostatic hyperplasia (BPH) (20).
The ability to discriminate between BPH and PCa has been improved by
determining the ratio of free PSA (PSA-F) to total PSA (PSA-T), which
roughly corresponds to the sum of PSA-F and PSA complexed with
1-antichymotrypsin (ACT) (21)(22). Nonetheless, more sensitive and
specific PCa diagnostic and prognostic tools are sorely needed.
Many researchers have begun to focus on hK2 as a potential serum marker
for PCa, and it has been suggested that expression of hK2 is higher in
adenocarcinomas of the prostate than in healthy prostate
epithelium (23). hK2 recovered from seminal plasma
has been characterized and is present mainly in complex with protein C
inhibitor (7). Recombinant production of hK2 (24)(25)(26)(27) has made it possible to further assess this protein
and its ability to form complexes with other protease inhibitors, such
as 2-antiplasmin, ACT, antithrombin III,
2-macroglobulin, C1-inactivator, and
plasminogen activator inhibitor-1 (28)(29)(30).
hK2 cross-reacts with only a few monoclonal antibodies (MAbs) against PSA, and the design of a specific immunoassay for hK2 with a detection limit of 100 ng/L and 0.7% cross-reactivity with PSA (31) was based on knowledge of the cross-reactivity of various anti-PSA MAbs with rhK2 (25). An assay with improved performance (detection limit of 50 ng/L and 0.1% cross-reactivity) was later used to study the utility of hK2 analysis in distinguishing between BPH and PCa (32)(33)(34). These studies showed that analysis of hK2 in combination with either PSA-F or both PSA-T and PSA-F, compared with determination of the percentage of PSA-F, improved the possibility to discriminate between BPH and PCa. However, a considerable number of patients in the groups studied (48% of BPH patients and 26% of clinically localized PCa patients) (34) had hK2 concentrations below the functional sensitivity of the assay. Very recently, two more assays for hK2 have been published (35)(36); they both have very low detection limits but show very different results regarding the concentrations of hK2 measured in healthy men. Our objective was to develop a sensitive and specific assay for hK2, with an equimolar response ratio between the free and complexed forms of hK2 and then to apply the method to further characterize the immunoreactivity in serum.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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immunization protocol for production of MAb 6H10
Two BALB/c mice were immunized by intraperitoneal injections with
34 µg of rhK2 emulsified in Freund’s complete adjuvant. Two booster
doses (36 and 30 µg of rhK2, respectively) were given at 3-week
intervals; a third and last booster dose was given to one of the mice
(5 µg of rhK2) after 1 additional week and to the other mouse (26
µg of rhK2) after 4 additional weeks. Blood from each mouse was
tested for reactivity against hK2 at 1 and 2 months after immunization.
Serum (25 µL in 100 µL of DELFIA assay buffer) was added to
microtiter plates coated with anti-mouse antibodies, which were
subsequently incubated overnight and then washed. Thereafter, europium
(Eu)-labeled hK2 (10 ng in 200 µL of DELFIA assay buffer) and
samarium (Sm)-labeled PSA (25 ng in 200 µL of DELFIA assay buffer)
were added, and the fluorescence was measured after 90 min with a
DELFIA 1234 Plate fluorometer (39). Serum was also tested in
an assay using microtiter plates coated with hK2 (incubation for 90
min, followed by four washes) and Eu-labeled anti-mouse antibodies (20
ng in 200 µL of DELFIA assay buffer; 90-min incubation). The mice
were killed, and their spleen cells were homogenized and fused with SP
2/0 myeloma cells at a 1:1 ratio (40)(41) and
applied to microtiter plates. Supernatants from each cell population
were tested against hK2 and PSA as described previously, and some were
selected and cloned by limiting dilution (42).
We found that clone MAb 6H10 produced an antibody that reacted well with Eu-labeled hK2 but not with Sm-labeled PSA; we further subcloned MAb 6H10 and selected for production in a MiniPerm bioreactor. The MAb was subtyped with a MAb Mouse Isotyping Kit (Gibco), and the immunoglobulin concentration was measured with Eu-labeled anti-mouse antibodies. Finally, immunoglobulins were purified on a HiTrap Protein G gel column (Pharmacia) and eluted with 2 mol/L glycine-HCl (pH 2.5). Epitope characteristics were determined in sandwich assays with known MAb epitopes on hK2 and PSA (43). Affinity constants for MAb 6H10 were determined by the Scatchard method (44)(45).
immunofluorometric assay
The hK2 assay was based on a previously described assay for hK2 in
serum (31). In the first step of our new optimized assay
protocol (Fig. 1
), blocking of PSA-T was enhanced by the use of four
PSA-specific anti-PSA MAbs that do not cross-react with hK2 (27)(43): MAbs 2H11, 36, and 10, which block the
binding of MAb H50; and MAb 2E9, which blocks the binding of MAb 6H10
to PSA. Fifty microliters of calibrator or sample and the four
MAbs (1000 ng of each in 50 µL of PSA Assay Buffer) were incubated
for 1 h in streptavidin-coated microtiter wells. All calibrators
and samples were measured in duplicate.
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After the PSA-blocking step, specific capture of hK2 on the microtiter plate was accomplished with biotinylated anti-hK2 MAb 6H10, which manifests 5% cross-reactivity with PSA (200 ng of the MAb in 50 µL of PSA Assay Buffer in each well; 2-h incubation). Thereafter, the plate was washed, and detection was accomplished with Eu-labeled MAb H50 (150 ng in 200 µL of PSA Assay Buffer in each well; 1-h incubation); MAb H50 had identical affinity for PSA and hK2. This was followed by a second washing step, after which 200 µL of enhancement solution was added to each well. The plate was incubated for 5 min, and the europium fluorescence was measured. PSA-T and PSA-F were detected by the DELFIA ProstatusTM PSA F/T dual assay and MAbs H117/H50 and H117/5A10, respectively (Wallac Oy). The MAb combination H117/H50 measures PSA-F and PSA in complex with ACT in an equimolar fashion (46) and also fully cross-reacts with hK2 (25), whereas the assay for PSA-F does not cross-react with hK2. PSA-ACT and hK2-ACT complexes were analyzed with an in-house assay using anti-PSA/hK2 MAb H117 and anti-ACT MAb 241 (47).
analytical validation
Standardization and calibration.
Standardization was performed
with rhK2 (range, 10–26 800 ng/L) diluted in the calibration diluent
[50 mmol/L Tris, 150 mmol/L NaCl, 0.5 g/L NaN3,
and 75 g/L bovine serum albumin (BSA)] according to a previously
described procedure (31). Calibration was achieved with the
PSA-T assay, which provides equimolar detection of PSA and hK2, as
related elsewhere (46).
Cross-reaction with PSA.
Cross-reaction was determined by
measuring recombinant PSA (range, 0.08–165 µg/L) diluted in the
calibration diluent, as described by Piironen et al. (31).
The cross-reaction was then calculated by dividing the concentration
obtained by the hK2 assay with the concentration obtained by the PSA-T
assay.
Analytical detection limit and functional sensitivity.
The
analytical detection limit, i.e., the lowest detectable hK2
concentration, was defined by measuring the inaccuracy of the zero
calibrator in 40 replicates and then calculating the dose associated
with the mean zero signal + 2 SD. Precision profiles to define
"functional" or "biological" sensitivity were constructed by
analyzing 1775 male serum samples in duplicate and determining the dose
associated with an intraassay coefficient of variation (CV) of 20% (48).
The biological sensitivity and the analytic recovery were studied by serial dilution of three sera (hK2 concentration, 293-6960 ng/L) of samples in female sera devoid of hK2.
Precision.
We determined between- and within-assay imprecision
(mean CVs) by analyzing two samples of rhK2 diluted in female sera (160
and 1620 ng/L, respectively) in 17 subsequent assays and as 20
replicates in 1 assay. We also determined between- and within-assay
imprecision when we analyzed the samples with rhK2 in duplicate because
this is the way we have chosen to analyze serum samples. In addition,
six patient sera (hK2 concentration range, 125–906 ng/L) were analyzed
as duplicates in three subsequent assays and as four replicates in one
assay.
Equimolarity.
Equimolar detection of free and ACT-complexed
hK2 was determined by incubating 20 000 ng/L purified rhK2 with and
without a 100-fold molar excess of ACT overnight at 37 °C in 0.1
mol/L phosphate buffer (pH 7.2) containing 10 g/L BSA and 0.5
mol/L NaCl. The extent of complex formation in the vial containing ACT
was determined by gel filtration and a previously reported PSA-ACT
assay that fully cross-reacts with hK2-ACT (43)(47). An equimolar assay should detect hK2
in both vials, i.e., the vial containing only free hK2 and the vial to
which ACT was added and hence contained a mixture of free hK2 and
hK2-ACT complexes, with an identical signal intensity. Bias
toward hK2-ACT was calculated by dividing the percentage of signal
obtained in the vial with hK2-ACT by the complexation degree in that
vial.
Gel filtration.
Two male serum samples and four male
EDTA-plasma samples with high PSA-T concentrations (range, 658-3176
µg/L; very likely sera from males with PCa although the clinical data
are not known) were subjected to gel filtration on a Superose 12 column
(1 x 30 cm; Pharmacia). The sample volume was 0.1 mL. The column
was eluted with phosphate buffer, pH 7.0, containing 5 g/L BSA, at a
flow rate of 200 µL/min. Fractions (0.4 mL) were collected and
individually analyzed for PSA-T, PSA-F, and hK2.
Clinical performance.
We determined the correlation of the new
assay with the assay developed by Piironen et al. (31) by
measuring 76 clinically undefined serum samples collected at the
Department of Clinical Chemistry in Malmö with both assays (hK2
concentration, 10–25 000 ng/L). The procedures followed were in
accordance with the Helsinki Declaration of 1975.
Sera collected from 29 males and 32 females, ages 10 to 59 years, who presented for routine blood sampling at the Department of Clinical Chemistry in Malmö were analyzed for concentrations of PSA-T, PSA-F, and hK2. Clinical data were not known for these samples. The procedures followed were in accordance with the Helsinki Declaration of 1975.
One hundred ninety-nine serum samples, randomly collected from males 50–66 years of age with PSA <3.0 µg/L who participated in the Göteborg screening study of 1995–1996 (Hugosson J. et al., submitted for publication) were analyzed for PSA-T, PSA-F, and hK2. All participants gave informed consent. In this screening study, males with PSA <3.0 µg/L were not subjected to further examination. Therefore, the presence or absence of PCa in this group is not known.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) (43).
linearity
The dose-response of the assay was linear in the range 10 ng/L to
the highest hK2 calibrator at 26 800 ng/L (Fig. 2
). A similar dose–response curve was obtained when PSA-specific
blocking antibodies were removed (data not shown); thus, there is
evidence that the recovery of hK2 is not influenced by the addition of
blocking antibodies.
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cross-reaction with psa
Immunological cross-reaction with PSA was <0.01% in weight units
at PSA-T concentrations of 0.08–165 µg/L.
analytical detection limit and functional sensitivity
The analytical detection limit (i.e., the signal imprecision + 2
SD of the zero calibrator) was <10 ng/L (Fig. 2
). The functional
sensitivity (defined as the concentration at which the intraassay CV
was <20%) (48) was <30 ng/L. Serial dilution of three
serum samples in female sera gave a linear response down to 30 ng/L
(Fig. 3
). Analytic recoveries of these samples were 93–134% above the
functional sensitivity value of 30 ng/L.
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precision
The between-assay imprecision values (mean CVs) were 3.1% and
4.8% for the high and low rhK2 controls, respectively. Corresponding
values for within-assay imprecision were 1.9% and 4.5%, respectively.
When the controls were analyzed in duplicate, the corresponding
between-assay CVs were 3.0% and 3.5% and the within-assay CVs were
1.8% and 4.0%. The between-assay CVs for the six serum samples were
0.2–5.5%, and the within-assay CVs were 0.5–7.9%.
equimolarity
According to the gel filtration profiles and the hK2-ACT
immunoassay results, the vials in which hK2 was incubated with a
100-fold molar excess of ACT contained ~55% of hK2-ACT and 45% of
free hK2. The hK2 assay bias when comparing the immunoreactivity in the
vials containing free hK2 only and the vials containing hK2-ACT
complexes as well as free hK2 was 94% at 20 000 ng/L hK2, i.e., the
assay underestimated the hK2-ACT concentration by 6%.
gel filtration
Two serum samples and four plasma samples with PSA-T
concentrations of 658-3176 µg/L were subjected to gel filtration.
Most of the immunodetectable hK2 in the plasma eluted at a position
corresponding to an approximate size of 30 kDa, i.e., similar to the
position where purified rhK2 and purified PSA-F eluted. A minor
fraction of hK2 in plasma eluted at a position corresponding to ~90
kDa, i.e., similar to a position where hK2-ACT and PSA-ACT complexes
eluted (Fig. 4
). In these six samples, 81–96% of the total hK2
immunoreactivity corresponded to the 30-kDa free hK2 and 4–19% of the
total hK2 immunoreactivity corresponded to the 90-kDa complexed form of
hK2 (Fig. 4
).
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clinical performance
We obtained a correlation coefficient (r) of 0.999 when
we compared the new assay with the assay by Piironen et al. (31)).
The median values as well as the 25th and 75th percentiles for hK2,
PSA-T, and the percentage of PSA-F are given in Table 1
. Only 1 of 32 (3%) samples from females, ages 10 to 59
years, who had presented for routine screening had a detectable
concentration (i.e., 
30 ng/L) of hK2. That sample was from a female
54 years of age and had a hK2 concentration of 35 ng/L (Table 1
). Of
the samples from males, ages 10–59 years, who had presented for
routine screening, 13 of 29 (45%) had detectable hK2 concentrations.
In males with PSA concentrations <3 µg/L, 50 to 66 years of age, and
participating in the Göteborg screening study, 129 of 199 (65%)
had detectable hK2 concentrations. The correlation coefficient
(r) for hK2 and PSA-T in the 129 males with detectable hK2
concentrations was 0.28 (Fig. 5
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously published assays for hK2 (31)(35)(36)(49) have demonstrated somewhat dissimilar results regarding the cross-reactivity and concentrations of immunodetectable forms of hK2 in serum. Piironen et al. (31) used an assay with a detection limit of 100 ng/L and <0.7% cross-reactivity with PSA. The assay described by Finlay et al. (49) determined primarily free hK2 but also hK2 complexed with ACT, and had a detection limit of 120 ng/L and <0.5% cross-reactivity with PSA. The hK2 concentrations found in sera differed considerably between these two assays: the latter method detected at least 10-fold higher concentrations of hK2. Very recently, two more assays have been reported by Black et al. (35) and Klee et al. (36). These assays have detection limits of 6 and 4 ng/L, respectively, which are very low. The reported cross-reactivities with PSA differ somewhat, however, with the assay by Black et al. showing no cross-reactivity for samples with PSA <2 µg/L but cross-reactivities of <0.2% for samples with PSA up to 100 µg/L, whereas the assay by Klee et al. had negligible cross-reactivity.
When evaluating the equimolarity of our assay for free hK2 and the hK2-ACT complex, we found -6% bias toward hK2-ACT at a hK2 concentration of 20 000 ng/L and a complexation degree of 55%. Therefore, our assay can be considered to provide close to equimolar detection of free hK2 and hK2 complexed to ACT. To study the importance of an equimolar response to hK2-ACT, we also evaluated six patient samples with very high PSA-T concentrations by gel filtration and found at least two immunodetectable forms of hK2. Most of the hK2 immunoreactivity detected corresponded to the size of free hK2, whereas a small fraction eluted at a size corresponding to the hK2-ACT complex. Complexed forms of hK2 accounted for 4–19% of the total hK2 immunoreactivity in these six patient samples. Thus, we conclude that our new optimized assay does detect hK2-ACT complexes in patient sera reliably and at close to equimolarity; we also conclude that the complexed hK2 forms constitute a minor fraction of the total hK2 immunoreactivity. Black et al. (35) obtained similar gel filtration data, with >94% of hK2 occurring in the free form in serum and traces of ~100-kDa hK2 complexes present. Seemingly this assay also detected complexed forms of hK2, although the equimolarity was not investigated. The possible existence of hK2-ACT complexes has also been demonstrated by Western blots of sera from men with PCa (50).
With our assay, hK2 was undetectable (<30 µg/L) in the majority of female control sera and in 35% of males 50–66 years of age with PSA <3 µg/L. The two recently published assays (35)(36) had somewhat dissimilar results. The assay by Black et al. (35) detected a median of 402 ng/L hK2 in men without PCa, whereas the assay by Klee et al. (36) detected a median concentration of 26 ng/L in a similar but larger cohort of men. With our assay, we detected a median hK2 concentration of 36 ng/L in males 50–66 years of age with PSA <3 µg/L, which is more similar to the findings by Klee et al. The discrepancy to the study of Black et al. may be attributable to differences in mode of standardization.
Our assay provided close to equimolar detection of free hK2 and hK2 complexed to ACT, and gel filtration showed that most of the hK2 was in the free form. The assay by Klee et al. (36) measured predominantly free hK2; therefore, the similarities in hK2 concentrations detected with our assay and that of Klee et al. are in agreement. There was also a difference in the ratio of hK2 to PSA-T. Piironen et al. (31) found that 79% of tested serum samples (clinically undefined male sera with PSA-T concentrations of 1–3400 µg/L; n = 334) had hK2/PSA-T values of 0–2%, whereas Finlay et al. (49) noted that 90% of tested serum samples (healthy men and women, and men with BPH or PCa; n = 671) had >2% hK2/PSA-T and that 4% of the samples (13 of 371) had hK2/PSA-T values of >100%. With our new, almost equimolar assay, we found that the ratio of hK2 to PSA-T varied from 1% to 11% (median, 4%) in the six gel-filtered patient samples with very high PSA-T concentrations and from 0.9% to 31% in samples from males 50–66 years of age with PSA <3 µg/L (median, 3.8%). Klee et al. (36) measured hK2/PSA ratios of ~1.2–3.3% with their assay, whereas Black et al. (35) measured ~40% hK2 to PSA. In the PSA range of 0.15–2.9 µg/L, Klee et al. (36) found a mean hK2/PSA ratio of 1.9–3.3%, which is fairly similar to what we found. The authors of three previous studies (32)(33)(34) that used an improved assay for hK2 (functional sensitivity, 50 ng/L) based on the assay developed by Piironen et al. (31), obtained hK2/PSA-T values between 1% and 2% for patients with BPH and PCa. The BPH and PCa patients in these studies had considerably higher PSA concentrations than males with PSA <3 µg/L and would most likely be best represented by the men measured by Klee et al. (36), who had PSA concentrations of 4–19.9 µg/L. In this range, this group had mean hK2/PSA ratios of 1.3–1.5%, which is accordance with results from the investigations cited above.
In our investigation, a considerable number of patient samples had hK2
concentrations below the functional sensitivity of the assay. Our
preliminary data showed that with our new optimized assay, a detection
limit of 30 ng/L is sufficient to reliably detect hK2 in 65% of males
with PSA-T concentrations <3 µg/L and in 89% of males with PSA-T
3 µg/L (Becker et al., unpublished results).
In conclusion, our assay provides equimolar detection of free hK2
and hK2 in complex with ACT and has a detection limit that is
sufficiently low to reliably evaluate patients with PSA-T
concentrations 3 µg/L.
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Acknowledgments |
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Footnotes |
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1-antichymotrypsin; MAb, monoclonal antibody; and BSA, bovine serum albumin.
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References |
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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.
-1-antichymotrypsin as an indicator of prostate cancer. J Urol 1993;150:100-105.
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1-antichymotrypsin. Clin Chem 1991;37:618-625.
1-antichymotrypsin complex. Clin Chem 1995;41:1480-1488.
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), PSA-F (
), and hK2
(
).