| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Articles |
1
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, Canada M5G 1X5.
2
Department of Laboratory Medicine and Pathobiology,
University of Toronto, Toronto, Ontario, Canada M5G 1L5.
a Address correspondence to this author at: Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5. Fax 416-586-8628; e-mail ediamandis{at}mtsinai.on.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Methods: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients.
Results: Our assay has an excellent detection limit (6
ng/L) and precision (>90%). Recovery studies indicated that hK2 binds
to serum protease inhibitors. All sera from healthy males had
measurable hK2 concentrations (median, 402 ng/L). Almost all female
sera had undetectable hK2. Serum hK2 and PSA in males correlated
positively (r = 0.44), but hK2 was present at
concentrations ~2.5-fold lower than PSA. The PSA/hK2 ratio in male
sera was 0.1–34, with a median of 2.6. In seminal plasma, this ratio
was 100–500. More than 94% of immunoreactive hK2 in serum was in the
free form (~30 kDa); traces of hK2 complexed to
1-antichymotrypsin were present.
Conclusions: The limit of detection of the method for hK2 measurement described here (~20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.© 1999 American Association for Clinical Chemistry
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Recently, interest has emerged in a second member of the kallikrein family, human glandular kallikrein (hK2), which has an 80% amino acid sequence identity with PSA (5). Like PSA, hK2 is expressed predominantly in prostatic tissue and is up-regulated in response to androgenic stimulation (6). It recently was reported that the function of hK2 is to proteolytically activate PSA following its secretion into the ductal system of the prostate gland(7)(8)(9). Thus, hK2 appears to play a physiological role in the regulation of PSA activity.
Recent studies have reported hK2 overexpression in prostatic tumors(10). Furthermore, like PSA (11), hK2 is increased in the sera of prostate cancer patients (12). These findings demonstrate the potential of hK2 as an additional marker of prostate cancer. The recent development of recombinant hK2 and hK2-specific antibodies has generated interest in immunoassays for hK2. The purpose of this study was to design an ultrasensitive immunoassay for hK2 with minimal cross-reactivity from PSA.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
western blot analysis
Western blots were performed to confirm antibody specificity. All
necessary equipment for Western blot analysis was obtained from Novex.
Recombinant hK2 (Hybritech) and PSA purified from seminal plasma (a
gift from Dr. Tom Stamey, Stanford University, Stanford, CA) were
subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis under non-reducing conditions on 4–12%
Tris-glycine polyacrylamide gels. Separated proteins were transferred
electrophoretically to nitrocellulose membranes. After the membranes
were blocked overnight at 4 °C with 50 g/L nonfat dry milk in
Tris-buffered saline-Tween buffer (20 mmol/L Tris-HCl, pH 7.6, 137
mmol/L NaCl, 1.0 g/L Tween 20, ), the membranes were cut into
strips and incubated with the PSA and hK2 antibodies, followed by
horseradish peroxidase-conjugated sheep anti-mouse secondary antibody
(Amersham). Biotinylated molecular mass markers were visualized by
reacting with streptavidin-horseradish peroxidase, simultaneously with
the secondary antibody. The blots were incubated for 1 min with
enhanced chemiluminescence reagents (ECL; Amersham) as specified by the
manufacturer, and exposed to x-ray film for detection of immunoreactive
protein bands.
calibrators
Calibration solutions for both hK2 and PSA immunoassays were
prepared in a 50 mmol/L Tris buffer, pH 7.80, containing 60 g/L of
bovine serum albumin (BSA). PSA calibrators at concentrations of 0, 1,
5, 20, 100, 500, and 2000 ng/L were prepared using highly purified PSA
isolated from seminal plasma. The hK2 calibrators were prepared using
recombinant hK2 at concentrations of 0, 5, 20, 100, 500, and 2000 ng/L.
The recombinant hK2 was a gift from Dr. R. Wolfert, Hybritech Inc., San
Diego, CA. The concentration of purified hK2 was assigned by total
protein analysis.
hK2 immunoassay
A two-step sandwich ELISA was used for hK2 analysis. The
hK2 capture antibody was immobilized onto polystyrene microtitration
wells (Dynatech Laboratories) at a concentration of 300 ng per 100-µL
well in coating buffer (50 mmol/L Tris, pH 7.8). This incubation was
performed overnight. Samples were applied undiluted at a volume of 100
µL simultaneously with 50 µL of assay buffer (50 mmol/L Tris, pH
7.8, 100 mL/L goat serum, 60 g/L BSA, 50 mL/L mouse serum, 10 g/L
bovine immunoglobulin, 5 g/L Tween 20, 500 mmol/L KCl) and incubated at
room temperature for 1 h. After the wells were washed with wash
buffer (150 mmol/L NaCl, 50 mmol/L Tris, 1 mmol/L
NaN3, 0.5 g/L Tween 20), they were incubated with
100 µL of biotinylated detection antibody diluted to a concentration
of 500 ng per 100-µL well in assay buffer for 1 h. The wells
were washed, and alkaline phosphatase-labeled streptavidin (Jackson
Immunoresearch) diluted to a concentration of 5 ng per 100-µL well in
a 60 g/L BSA solution was added for 15 min. Diflunisal phosphate
(10 mmol/L stock in 10 mmol/L NaOH, prepared in house) diluted 10-fold
in substrate buffer (100 mmol/L Tris, pH 9.1, 150 mmol/L NaCl, 1 mmol/L
MgCl2, 7.5 mmol/L NaN3) was
added for 10 min following washing. Developing solution (1 mol/L Tris,
400 mmol/L NaOH, 3 mmol/L EDTA, 2 mmol/L Tb3+),
was then added to the diflunisal phosphate solution, and the resulting
fluorescence was measured on a Cyberfluor 615 Immunoanalyzer (Nordion
International). More details on these time-resolved immunofluorometric
procedures can be found elsewhere (13)(14).
psa immunoassay
PSA measurements were performed with an ELISA-type assay described
in detail elsewhere (14). The procedure and reagents were
identical to the hK2 assay with the following modifications. The
coating antibody for the PSA assay was coded 8301. This assay was a
one-step sandwich assay in which 50 µL of biotinylated antibody 8311,
diluted to a concentration of 250 ng per 100-µL well, was incubated
simultaneously with 100 µL of sample. The detection limit of this
assay was determined previously to be 1 ng/L (14).
detection limit
The detection limit of the hK2 immunoassay was determined by
analyzing 12 replicates of the zero hK2 calibrator. The hK2
concentration that corresponded to the fluorescence of the zero
calibrator plus 2 SD was determined to be the detection limit of the
assay.
cross-reactivity
To check for PSA cross-reactivity in the hK2 assay, the hK2 assay
was performed with PSA diluted to 1, 5, 20, 100, 500, 2000, 10 000,
and 100 000 ng/L in a 60 g/L BSA solution. In addition, various
concentrations of PSA were incubated simultaneously with a 100-fold
lower concentration of hK2 to test for negative cross-reactivity
(defined as the ability of PSA to interfere with the hK2 measurement).
analytical recovery
To evaluate the recovery of hK2, small volumes of purified
recombinant hK2 or sera containing a known amount of hK2 were added to
various sample matrices to a final concentration of 100 and 2000 ng/L.
The matrices used were three female serum samples, three male serum
samples, and a 60 g/L BSA solution (as a control). Recovery of hK2 was
measured in quadruplicate 30 min and 24 h after the addition of
hK2.
linearity of dilution
The hK2 assay was evaluated for linearity at a range of 5–2000
ng/L by assaying, in quadruplicate, various specimens that had been
serially diluted in a 60 g/L BSA solution. Male serum samples with
endogenous hK2 concentrations, in addition to male and female serum
samples that had been supplemented with a known concentration of
recombinant hK2 to give a final concentration ~2000 ng/L, were
diluted 2-, 4-, 8-, 16-, and 32-fold and analyzed for hK2.
precision
The within-run and day-to-day assay precision was evaluated by
analyzing 12 replicates of three male serum samples with hK2
concentrations of 75, 150, and 300 ng/L and one female serum sample
supplemented with serum containing a known amount of hK2 to a final
concentration of 1000 ng/L.
analysis of serum samples
hK2 in serum.
Serum samples from 61 males without prostate
cancer, ages 51–88 years, and 33 unselected females were obtained from
the clinical laboratories of Mount Sinai Hospital, Toronto, Canada.
Each sample was analyzed in duplicate for hK2 and PSA.
Characterization of hK2 in serum by gel filtration.
hK2 and
PSA from two male serum samples were analyzed by gel filtration on a
Hewlett-Packard 1100 HPLC system (Hewlett-Packard). A TSK-GEL
silica-based column (TosoHaas) was used, and the mobile phase consisted
of 100 mmol/L Na2SO4 and
100 mmol/L NaH2PO4, pH 6.8.
The isocratic runs were maintained at a flow rate of 0.5 mL/min. Column
calibration was achieved with a molecular mass calibration solution
containing thyroglobulin (670 kDa), IgG (158 kDa), ovalbumin (44 kDa),
myoglobin (17 kDa), and cyanocobalamin (1.4 kDa; Bio-Rad Laboratories).
Fractions were collected with the Pharmacia FRAC-100 fraction collector
and analyzed in duplicate with both the hK2 and PSA immunoassays.
hK2 concentrations in serum of prostatectomized patients.
hK2
and PSA were measured in serial sera of six patients with
histologically confirmed prostate cancer who were treated with radical
prostatectomy. Four of the patients showed clinical signs of cancer
relapse, whereas two remained relapse free.
Seminal plasma.
Seminal plasma samples were obtained from
subjects undergoing investigations for infertility. These samples were
diluted with a 60 g/L BSA solution and then analyzed for both hK2 and
PSA.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) demonstrated that the hK2-specific capture antibody
(Hybritech) used in the hK2 assay recognizes hK2. This antibody
previously was confirmed to have no cross-reactivity with recombinant
PSA (data not shown). The coating antibody for the PSA immunoassay
(coded 8301) was specific for PSA and showed no detectable
cross-reactivity with hK2. The detection antibody (coded 8311), used
for both the hK2 and the PSA immunoassays, reacted strongly with both
hK2 and PSA.
|
calibration curve and detection limit
A typical calibration curve for the proposed hK2 assay is shown in
Fig. 2
. The analytical detection limit of the hK2 assay was estimated
as the dose that was equivalent to the fluorescence of the mean of 12
replicates of the zero calibrator plus 2 SD. The hK2 concentration that
corresponded to this signal was calculated to be 6 ng/L.
|
cross-reactivity
The hK2 assay exhibited no detectable positive PSA
cross-reactivity up to a PSA concentration of 2000 ng/L PSA (Table 1
). The cross-reactivity did not exceed 0.2% up to a
concentration of 100 000 ng/L PSA. No negative cross-reactivity was
apparent when PSA was present in a mixture with hK2 but at a 100-fold
higher concentration. Similarly, the PSA assay described previously(14) had no detectable cross-reactivity from hK2 even at hK2
concentrations as high as 100 000 ng/L (data not shown).
|
analytical recovery
To evaluate the recovery of hK2 in serum, we added recombinant hK2
diluted in a 60 g/L BSA solution to human sera and BSA (for control
purposes) to final concentrations of 200 and 1000 ng/L. The recoveries
are listed in Table 2
. As expected, the recovery of hK2 from the BSA solution was
complete, at 97–110% recovery. The recovery of hK2 from female sera
was 25–36% after 30 min and dropped to 13–26% after 24 h.
Similar results were obtained for male sera, with 24–31% and 22–35%
recovery after 30 min and 24 h, respectively. The low recovery may
reflect the time-dependent binding of hK2 to serum proteins, forming
complexes that are not measurable with this assay. hK2 from a male
serum sample that contained high hK2 (~25 000 ng/L) was also added
to pools of male and female sera. The recovery for these pools was
70–104%.
|
linearity of dilution
Serum samples with a hK2 concentration of 150-2000 ng/L (the
highest concentration in the calibration curve) were serially diluted
with a 60 g/L BSA solution and measured. The assay showed good
linearity: near-linear dilution curves were obtained with all samples
tested (Fig. 3
). The measured concentrations from the dilution were 80–120%
of the expected value, based on the concentration of the undiluted hK2
.
|
precision
To evaluate assay precision, 12 replicates of four serum samples
with hK2 concentrations of 75, 150, 300, and 1000 ng/L were analyzed.
The imprecision (CV) did not exceed 6% (within-run) or 9%
(between-run) for any of the replicates.
analysis of serum samples
PSA and hK2 were measured in sera from 61 males without prostate
cancer (defined as an absence of clinical symptoms and serum PSA <4
µg/L, except one patient with total PSA of 4.8 µg/L) and 33
females; the values for males are listed in Table 3
. The PSA concentration in male serum was 10–4770 ng/L, with a
mean of 1223 ng/L and a median of 1020 ng/L. All samples had detectable
hK2. The hK2 concentration in male serum was 21–3536 ng/L and
generally appeared to be ~2.5-fold less than the PSA concentration.
The mean hK2 value was 515 ng/L, and the median was 402 ng/L. A
positive correlation was observed between the hK2 and PSA
concentrations in male serum (r = 0.44; Fig. 4
). The PSA/hK2 ratios in the 61 sera were 0.1–34, with a median
of 2.6 (Table 3
). Correlation between total PSA or hK2 and patient age
indicated that both biochemical markers increase slightly with
age. Linear regression gave the following equations: PSA (ng/L) = 22
(age, years) - 322, r = 0.26, P =
0.04; hK2 (ng/L) = 7.7 (age, years) - 21, r = 0.16,
P = 0.21 (not statistically significant).
|
|
Thirty-one of 33 female sera contained hK2 concentrations below the detection limit of the assay (<6 ng/L). Two sera had hK2 concentrations of 40 and 60 ng/L. The PSA concentrations in these two sera were also the highest among the female samples, with PSA concentrations of 11 and 61 ng/L, respectively. Ten female sera had PSA concentrations between 1 and 7 ng/L, whereas PSA was undetectable in the other female samples.
The hK2 and PSA concentrations in five seminal plasma samples diluted in a 60 g/L BSA solution were determined. The PSA concentration was 0.1 x 109 to 2.3 x 109 ng/L (median, 0.8 x 109 ng/L); the hK2 concentration was 0.8 x 106 to 1.4 x 107 ng/L (median, 6 x 106 ng/L). The concentration of hK2 in seminal plasma was ~100- to 500-fold lower than the PSA concentration.
characterization of hK2 in serum by gel filtration
The elution profiles of two male sera from prostate cancer
patients (with very high PSA and hK2 concentrations) for PSA and hK2
fractionated by HPLC are shown in Figs. 5
and
6, respectively. The first serum sample (male serum 1) had a
total PSA concentration of 2 000 000 ng/L and a hK2 concentration of
25 000 ng/L (PSA/hK2 ratio = 80). The second serum sample (male
serum 2) had a total PSA concentration of 1 100 00 ng/L and a hK2
concentration of 200 000 ng/L (PSA/hK2 ratio = 5.5). Three
clearly distinguishable hK2 peaks were present in both samples (Fig. 6
), corresponding to molecular masses of ~700 kDa (first peak),
~100 kDa (second peak), and ~30 kDa (third peak). The first two
peaks, based on their molecular masses, likely correspond to hK2
complexed to 2-macroglobulin (A2M) and
1-antichymotrypsin (ACT), respectively. The
third peak is uncomplexed (free) hK2 and accounts for the vast majority
of serum immunoreactive hK2 (>95% of total immunoreactivity; see Fig. 6
, A
and C
).
|
|
As expected, fractionation of PSA produced two major peaks, which
represent PSA complexed to ACT (~100 kDa, first peak) and uncomplexed
PSA (~33 kDa, second peak). PSA-ACT represents the major PSA
immunoreactive form (Fig. 5
).
hK2 concentrations in prostatectomized patients
Serial serum specimens from six prostatectomized males were
analyzed for PSA and hK2. The PSA and hK2 concentrations over time in
four prostatectomized males who showed clinical signs of prostate
cancer relapse are shown in Fig. 7
. The change in hK2 concentration over the course of disease
progression was proportional to that of PSA. PSA and hK2 were
undetectable in all sera from the two prostatectomized patients who
remained relapse-free over the monitoring period of ~5 years. The
approximate PSA/hK2 ratios in the four patients of Fig. 7
were 6, 23,
15, and 3.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
hK2, also known as glandular kallikrein, and human kallikrein 3 (hK3, also known as PSA) are the products of androgen-regulated genes that are expressed primarily in the human prostate gland (16). It recently was reported that hK2 converts pro-PSA, an inactive zymogen, to mature, enzymatically active PSA (7)(8)(9), thus establishing a physiological connection between hK2 and PSA. PSA and hK2 have high sequence homology (80% identity at the amino acid level) and the same molecular mass (~30 kDa). It is challenging to ascertain whether purified preparations of PSA or hK2 from seminal plasma (which contains relatively large amounts of both proteins) are devoid of cross-contamination. Thus, only recombinant preparations are reliable reagents for antibody production, standardization, and cross-reactivity studies. Recombinant hK2 has been produced by a number of groups, and monoclonal antibodies have been raised(17)(18)(19). Surprisingly, many investigators have found that it is possible to generate monoclonal antibodies against hK2 that have little or no cross-reactivity with PSA, opening the possibility for development of assays that are highly specific for hK2 [Ref.(20) and see below]. Furthermore, investigators have found that many PSA assays already reported in the literature do not cross-react with hK2; an assay developed in our laboratory is one example (14).
Recently, many investigators have begun to focus on hK2 as a potential prostatic marker that could have utility, alone or in combination with PSA, for the diagnosis and monitoring of prostatic disease. Assays for hK2 that do not cross-react with PSA have already been reported by others, and preliminary clinical data have been published(12)(21)(22). The method reported here is an important extension of the previous studies, primarily because of its improved detection limit (6 ng/L); this assay thus can provide clinical insight not easily obtainable with previously developed assays, which have detection limits of ~100 ng/L.
The hK2 assay developed here displays minimal cross-reactivity with PSA
(
0.2%; Table 1
), enabling hK2 to be measured accurately in the
presence of a large excess of PSA. Because of the absence of
cross-reactivity in both assays, this hK2 assay and the previously
described ultrasensitive PSA assay (14) provide reliable
tools for monitoring independently, and with very high sensitivity, the
concentrations of PSA and hK2 in biological fluids.
The recovery of recombinant hK2 added to female and male sera after a 30-min or 24-h incubation is low (<35%) and is similar to the recovery of PSA in serum (14). Low recovery was also reported by Piironen et al. (21) and may be the result of the binding and masking of hK2 by A2M, as is also suggested by the data of Mikolajczyk et al. (17). However, when hK2 from a male serum sample with a high concentration of hK2 was added to sera from healthy males and females, the recovery was much improved (70–104%), which also is in accord with the results reported for hK2 by Piironen et al. (21) and those reported for PSA(14). These data suggest that like PSA(15)(23), hK2 binds to proteinase inhibitors in serum. Free hK2 may represent a fraction that cannot bind to serum proteinase inhibitors. Similarly to PSA, free hK2 may represent either a proenzyme or an enzymatically inactive molecular form.
Studies of healthy male and female sera with previously described hK2
assays have concluded that male and female sera have similar values,
generally <350 ng/L (12). Piironen et al. (21),
were unable to measure hK2 in 57% of the male sera. The data from
these two studies should be regarded as unreliable because the hK2
assays used did not possess the low detection limits required to
accurately measure hK2 in healthy male or female serum. We have
concluded that hK2 in serum is much higher in men compared with women
(Table 3
). All 61 males without prostatic disease had detectable hK2,
with a median concentration of 402 ng/L. In women, 31 of 33 serum
samples had undetectable (<6 ng/L) hK2 concentrations, and the two
hK2-positive samples also had detectable PSA. The ratio of PSA to hK2
in male serum was quite variable, with a median of 2.6, which is much
higher than the ratio reported earlier (21). On average, hK2
is present in male serum at concentrations ~2.5-fold than PSA. The
variability of the PSA/hK2 ratio among different serum samples should
be examined for possible clinical value. We are now investigating if
the PSA/hK2 ratio or free PSA/hK2 ratio has any value in discriminating
between benign prostatic hyperplasia and prostate cancer. Preliminary
investigations by others and our unpublished data suggest that this may
be true (24).
The correlation between hK2 and PSA in serum is good (r = 0.44), in accordance with the findings of others (21) but in some disagreement with the data of Charlesworth et al.(22), who found a weaker correlation.
Gel filtration of two male sera with high hK2 concentrations revealed
that >95% of the total hK2 immunoreactivity elutes as free hK2 at a
molecular mass of ~30 kDa. This finding is very different for PSA,
for which most immunoreactivity elutes as a PSA-ACT complex at a
molecular mass of ~100 kDa (Figs. 5
and 6
). Similar conclusions for
hK2 serum immunoreactivity were reported by Piironen et al.(21). The improved detection limit of our assay allowed the
detection of another two minor peaks with molecular masses of ~700
kDa and 100 kDa (Fig. 6
). We speculate that these peaks represent hK2
complexed to A2M and ACT, respectively. However, we do not know the
relative response of our assay to free hK2, hK2-A2M, and hK2-ACT
complexes, and therefore can draw no conclusions related to the actual
ratios of these hK2 subfractions in serum. We have, however,
indications from our recovery experiments that once hK2 is added to
serum, its immunoreactivity is substantially reduced, presumably
because of the formation of such complexes. Finlay et al.(12) reported that their hK2 assay was skewed, detecting
free hK2 with a 3.5-fold higher efficiency that hK2-ACT. The formation
of complexes between hK2 and ACT is less likely to occur than between
PSA and ACT, mainly because of the suggested trypsin-like enzymatic
activity of hK2 (18).
It has been reported that the hK2 mRNA in the prostate represents ~10–50% of that of PSA mRNA (24). In the present study, analysis of five seminal plasma specimens revealed that PSA is present at a concentration 100- to 500-fold higher than that of hK2. Our data on this agree with the data presented previously (12). Whether this finding is because of much lower production of hK2 in the prostate cells in comparison with PSA or to the degradation, inactivation, or binding of hK2 in seminal plasma requires further investigation.
Previous studies have reported increased hK2 in the serum of patients
with prostate cancer (12)(21). These findings
were verified in our study with a small number of prostate cancer
patients. Because the current major application of PSA is monitoring
prostatectomized patients, we studied the hK2 concentrations of six
patients who had serial serum samples collected over many years
postprostatectomy, an application not examined in previous reports of
hK2 detection methods. In the two patients who did not relapse over 5
years of monitoring, both PSA and hK2 remained undetectable in all
serum samples collected. The remaining four patients suffered a
relapse, with PSA rising over time, as shown in Fig. 7
. Remarkably, hK2
correlates very well with PSA; thus, they have utility for monitoring.
However, it appears from this limited data that there is no advantage
of hK2 over PSA because for the former, the concentrations are lower by
a factor of 3- to 23-fold. The variable PSA/hK2 ratios in
postprostatectomy patients was also seen in healthy subjects (Table 3
)
and may have clinical importance.
In conclusion, we have presented a new method that can measure hK2 accurately in the presence of a large excess of PSA. This method, in combination with a method that can measure PSA in the presence of a large excess of hK2, will allow for the measurement of either hK2 or PSA in clinical samples that contain both analytes. Because our method has a detection limit that is ~20-fold lower than previously reported assays, hK2 concentrations in the serum of healthy males could be determined. Furthermore, it is evident here that the major immunoreactive fraction in serum is free hK2. There is a significant correlation between serum hK2 and PSA, the hK2 concentration being approximately 2.5-fold lower than that of PSA. More clinical studies will be necessary to establish if the simultaneous measurement of PSA and its subfractions and hK2 in serum has any advantage over PSA measurements alone.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2-macroglobulin; and ACT, 1-antichymotrypsin. 
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1-antichymotrypsin is the major form of prostate-specific antigen in serum of patients with prostate cancer: assay of the complex improves clinical sensitivity for cancer. Cancer Res 1991;51:222-226.
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  R. K. Nam, W. W. Zhang, L. H. Klotz, J. Trachtenberg, M. A.S. Jewett, J. Sweet, A. Toi, S. Teahan, V. Venkateswaran, L. Sugar, et al. Variants of the hK2 Protein Gene (KLK2) Are Associated with Serum hK2 Levels and Predict the Presence of Prostate Cancer at Biopsy. Clin. Cancer Res., November 1, 2006; 12(21): 6452 - 6458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. H. Slagter, A. Scorilas, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, E. J. Giltay, M. Paliouras, and E. P. Diamandis Effect of Testosterone Administration on Serum and Urine Kallikrein Concentrations in Female-to-Male Transsexuals Clin. Chem., August 1, 2006; 52(8): 1546 - 1551. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. H. Slagter, L. J.G. Gooren, W. de Ronde, A. Soosaipillai, A. Scorilas, E. J. Giltay, M. Paliouras, and E. P. Diamandis Serum and Urine Tissue Kallikrein Concentrations in Male-to-Female Transsexuals Treated with Antiandrogens and Estrogens Clin. Chem., July 1, 2006; 52(7): 1356 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. V. Obiezu, S. J.C. Shan, A. Soosaipillai, L.-Y. Luo, L. Grass, G. Sotiropoulou, C. D. Petraki, P. A. Papanastasiou, M. A. Levesque, and E. P. Diamandis Human Kallikrein 4: Quantitative Study in Tissues and Evidence for Its Secretion into Biological Fluids Clin. Chem., August 1, 2005; 51(8): 1432 - 1442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Vaisanen, S. Eriksson, K. K. Ivaska, H. Lilja, M. Nurmi, and K. Pettersson Development of Sensitive Immunoassays for Free and Total Human Glandular Kallikrein 2 Clin. Chem., September 1, 2004; 50(9): 1607 - 1617. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. K. Nam, W. W. Zhang, J. Trachtenberg, E. Diamandis, A. Toi, M. Emami, M. Ho, J. Sweet, A. Evans, M. A.S. Jewett, et al. Single Nucleotide Polymorphism of the Human Kallikrein-2 Gene Highly Correlates With Serum Human Kallikrein-2 Levels and in Combination Enhances Prostate Cancer Detection J. Clin. Oncol., June 15, 2003; 21(12): 2312 - 2319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Haese, V. Vaisanen, J. A. Finlay, K. Pettersson, H. G. Rittenhouse, A. W. Partin, D. J. Bruzek, L. J. Sokoll, H. Lilja, and D. W. Chan Standardization of Two Immunoassays for Human Glandular Kallikrein 2 Clin. Chem., April 1, 2003; 49(4): 601 - 610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. G. Blijenberg, M. F. Wildhagen, C. H. Bangma, J. A. Finlay, V. Vaisanen, and F. H. Schroder Comparison of Two Assays for Human Kallikrein 2 Clin. Chem., February 1, 2003; 49(2): 243 - 247. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. P. Diamandis, A. Okui, S. Mitsui, L.-Y. Luo, A. Soosaipillai, L. Grass, T. Nakamura, D. J. C. Howarth, and N. Yamaguchi Human Kallikrein 11: A New Biomarker of Prostate and Ovarian Carcinoma Cancer Res., January 1, 2002; 62(1): 295 - 300. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. A. Finlay, J. R. Day, C. L. Evans, R. Carlson, K. Kuus-Reichel, L. S. Millar, S. D. Mikolajczyk, M. Goodmanson, G. G. Klee, and H. G. Rittenhouse Development of a Dual Monoclonal Antibody Immunoassay for Total Human Kallikrein 2 Clin. Chem., July 1, 2001; 47(7): 1218 - 1224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Yousef and E. P. Diamandis The New Human Tissue Kallikrein Gene Family: Structure, Function, and Association to Disease Endocr. Rev., April 1, 2001; 22(2): 184 - 204. [Abstract] [Full Text]
|
|
|
|
 |
|
 C. V. Obiezu, A. Scorilas, A. Magklara, M. H. Thornton, C. Y. Wang, F. Z. Stanczyk, and E. P. Diamandis Prostate-Specific Antigen and Human Glandular Kallikrein 2 Are Markedly Elevated in Urine of Patients with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., April 1, 2001; 86(4): 1558 - 1561. [Abstract] [Full Text]
|
|
|
|
|
|
K. M. Slawin, S. F. Shariat, C. Nguyen, A. K. Leventis, W. Song, M. W. Kattan, C. Y. F. Young, D. J. Tindall, and T. M. Wheeler Detection of Metastatic Prostate Cancer Using a Splice Variant-specific Reverse Transcriptase-Polymerase Chain Reaction Assay for Human Glandular Kallikrein Cancer Res., December 1, 2000; 60(24): 7142 - 7148. [Abstract] [Full Text]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
C. V. Obiezu, E. J. Giltay, A. Magklara, A. Scorilas, L. J.G. Gooren, H. Yu, D. J.C. Howarth, and E. P. Diamandis Serum and Urinary Prostate-specific Antigen and Urinary Human Glandular Kallikrein Concentrations Are Significantly Increased after Testosterone Administration in Female-to-Male Transsexuals Clin. Chem., June 1, 2000; 46(6): 859 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. M. Yousef, A. Chang, and E. P. Diamandis Identification and Characterization of KLK-L4, a New Kallikrein-like Gene That Appears to be Down-regulated in Breast Cancer Tissues J. Biol. Chem., April 14, 2000; 275(16): 11891 - 11898. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. K. Nam, E. P. Diamandis, A. Toi, J. Trachtenberg, A. Magklara, A. Scorilas, P. A. Papnastasiou, M. A. S. Jewett, and S. A. Narod Serum Human Glandular Kallikrein-2 Protease Levels Predict the Presence of Prostate Cancer Among Men With Elevated Prostate-Specific Antigen J. Clin. Oncol., March 1, 2000; 18(5): 1036 - 1036. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Becker, T. Piironen, J. Kiviniemi, H. Lilja, and K. Pettersson Sensitive and Specific Immunodetection of Human Glandular Kallikrein 2 in Serum Clin. Chem., February 1, 2000; 46(2): 198 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Magklara, A. Scorilas, W. J. Catalona, and E. P. Diamandis The Combination of Human Glandular Kallikrein and Free Prostate-specific Antigen (PSA) Enhances Discrimination Between Prostate Cancer and Benign Prostatic Hyperplasia in Patients with Moderately Increased Total PSA Clin. Chem., November 1, 1999; 45(11): 1960 - 1966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Magklara, A. Scorilas, C. Lopez-Otin, F. Vizoso, A. Ruibal, and E. P. Diamandis Human Glandular Kallikrein in Breast Milk, Amniotic Fluid, and Breast Cyst Fluid Clin. Chem., October 1, 1999; 45(10): 1774 - 1780. [Abstract] [Full Text] [PDF]
|
, PSA; 
,
hK2.