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Articles |
1-Protease Inhibitor in Serum
Departments of
1
Clinical Chemistry,
2
Urology, and
3
Pathology, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland.
a Author for correspondence. Fax 00358-09-4714804; e-mail ulf-hakan.stenman{at}huch.fi
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antichymotrypsin (ACT) is the major form in
serum, and the proportion of PSA-ACT is higher in prostate cancer (PCa)
than in benign prostatic hyperplasia (BPH). PSA also forms a complex
with 1-protease inhibitor (API) in vitro, and the
PSA-ACT complex has been detected in serum from patients with prostate
cancer. The aim of the present study was to develop a quantitative
method for the determination of PSA-API and to determine the serum
concentrations in patients with PCa and BPH. Methods: The assay for PSA-API utilizes a monoclonal antibody to PSA as capture and a polyclonal antibody to API labeled with a Eu-chelate as a tracer. For calibrators, PSA-API formed in vitro was used. Serum samples were obtained before treatment from 82 patients with PCa, from 66 patients with BPH, and from 22 healthy females.
Results: The concentrations of PSA-API are proportional to the concentrations of total PSA. PSA-API comprises 1.0–7.9% (median, 2.4%) of total immunoreactive PSA in PCa and 1.3–12.2% (median, 3.6%) in BPH patients with serum PSA concentrations >4 µg/L. In patients with 4–20 µg/L total PSA, the proportion of PSA-API serum is significantly higher in BPH (median, 4.1%) than in PCa (median, 3.2%; P = 0.02).
Conclusions: The proportion of PSA-API in serum is lower in patients with PCa than in those with BPH. These results suggest that PSA-API is a potential adjunct to total and free PSA in the diagnosis of prostate cancer.© 1999 American Association for Clinical Chemistry
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin,
1-antichymotrypsin (ACT), and protein C
inhibitor in vitro (7)(8)(9), and the major immunoreactive form
of PSA in serum is a complex between PSA and ACT (PSA-ACT), whereas a
minor fraction is free (10)(11)(12)(13). Increased PSA concentrations in serum are suggestive of prostate cancer (PCa), but increased PSA is also seen in serum from patients with nonmalignant prostatic diseases such as benign prostatic hyperplasia (BPH) or prostatitis (14)(15)(16), which limits the clinical utility of PSA for the screening of PCa. We previously have shown that the proportion of PSA-ACT to total PSA in serum is higher in PCa than in BPH (10)(12) and that a reduction in the number of false-positive results caused by BPH can be achieved by measurement of the proportion of PSA-ACT or free PSA in serum(10)(12)(13).
A small fraction of PSA is bound to 1-protease
inhibitor (API) in serum with high PSA concentrations (10),
and it has recently been shown that PSA forms a complex with API
(PSA-API) in vitro (17). We have now developed a
quantitative time-resolved immunofluorometric assay (IFMA) for PSA-API
and standardized it by using purified PSA-API formed in vitro. This
assay enabled us to determine the PSA-API concentrations in sera from
patients with PCa and BPH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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materials
Reagents.
Superdex-200 (60 x 1.6 cm) and Resource Q (6
mL) columns, Phenyl-Sepharose (high performance), and CNBr-activated
Sepharose 4B were obtained from Pharmacia Biotech. The PVDF membrane
(Immobilon P) was from Millipore. Heparin was obtained from Leiras. The
DELFIA assay buffer, and washing and enhancement solutions used in IFMA
were from Wallac (Turku, Finland).
Proteins.
Intact PSA (isoenzyme B) was purified from seminal
fluid as described previously (18). For the standardization
of IFMAs for PSA-API and PSA-ACT, the respective complexes were
prepared in vitro (see below). The purified ACT was from Athens
Research and Technology. API was purified from plasma as described
previously (19). The protein molecular mass markers were
from Pharmacia. The bovine serum albumin (BSA) was from Sigma.
Antibodies.
A monoclonal antibody (MAb) to PSA (6C11) was
produced by standard procedures. This antibody recognizes equally PSA
complexed with ACT and free PSA (Leinonen et al., unpublished results).
The polyclonal antibodies to ACT and API, the peroxidase-conjugated
swine anti-rabbit IgG, and the rabbit anti-mouse IgG antibodies were
from Dakopatts. The specificity of the polyclonal antibodies was tested
by immunodiffusion (10). The polyclonal antibodies to API
and ACT were labeled with a Eu-chelate (10).
experimental procedures
IFMAs for free and total PSA.
Total and free PSA were
determined simultaneously with a dual-label IFMA (DELFIA Prostatus
free/total PSA kit; Wallac) as described previously (20).
The assay was standardized against purified PSA (18). The
antibodies used in the IFMA for total PSA reacted equally with free PSA
and PSA-ACT (20)(21).
IFMA for PSA-API and PSA-ACT.
The solid-phase antibody of the
DELFIA Prostatus free/total PSA kit (Wallac) was used. The coated
microtitration wells were preincubated with heparin at a concentration
of 50 kIU/L at 4 °C for 12 h to reduce the nonspecific
background. Calibrators were prepared by dilution of PSA-API or PSA-ACT
formed in vitro (see below) to concentrations of 0.1, 0.5, 2, 10, and
50 µg/L in assay buffer, pH 5.0, containing 50 g/L BSA. The
concentration of PSA-API or PSA-ACT was expressed according to the mass
of PSA, disregarding the mass of API or ACT in the complex. The PSA
content in the complexes was determined by the IFMA for total PSA. For
serum assays, 25 µL of samples or calibrators in duplicates and 200
µL of assay buffer were pipetted into the microtitration wells. For
assays of chromatographic fractions, a sample volume of 200 µL was
used. Before the assay, 100 µL of assay buffer with a 10-fold
concentration of BSA and bovine serum globulin was added to each 1-mL
fraction. After incubation for 1 h at room temperature, the wells
were emptied, washed six times with wash solution with an automatic
washer (DELFIA Platewash 1296-024; Wallac), and filled with 200 µL of
assay buffer containing 100 ng of polyclonal antibody to either API or
ACT labeled with Eu3+. After further incubation
for 2 h, the wells were emptied and washed six times with wash
solution. Enhancement solution (200 µL) was then added to the wells,
and after 5 min the fluorescence was measured with a 1234 DELFIA
research fluorometer (Wallac). As a control for the nonspecific
background, 22 female sera were measured using microtitration wells
without capture antibody but otherwise identical to the PSA-API assay.
Gel filtration.
Gel filtration was performed on a 1.6 x
60 cm Superdex-200 column using 50 mmol/L Tris-HCl buffer, pH 7.4,
containing 0.15 mol/L NaCl and 8 mmol/L sodium azide (TBS). The flow
rate was 15 mL/h, and 1-mL fractions were collected. The column was
roughly calibrated by measuring the absorbance at 280 nm in the
fractions to identify IgG (150 kDa) and albumin (68 kDa) in serum.
Anion-exchange chromatography.
Anion-exchange chromatography
was performed on a 6-mL Resource Q column equilibrated with 10 mmol/L
Tris-HCl buffer containing 8 mmol/L sodium azide, pH 8.4 (buffer A).
Serum samples of 0.5 mL were diluted to 10 mL (20-fold) in buffer A.
Protein fractions were dialyzed against this buffer before
chromatography. Bound proteins were eluted with a linear gradient
composed of 60 mL of buffer A and 60 mL buffer B, which consisted of
buffer A containing 300 mmol/L NaCl.
Hydrophobic interaction chromatography.
Hydrophobic
interaction chromatography was performed on a 10-mL Phenyl-Sepharose
(high performance) column equilibrated with 50 mmol/L Tris-HCl, pH 7.0,
containing 0.8 mol/L ammonium sulfate and 8 mmol/L sodium azide. The
flow rate was 1 mL/min, and 4-mL fractions were collected. After sample
application, the column was washed with 10 bed volumes of equilibration
buffer. Bound proteins were eluted with a gradient composed of 30 mL of
water and 30 mL of 400 mL/L 2-propanol.
Immunoaffinity chromatography.
MAb 6C11 was immobilized on
CNBr-activated Sepharose 4B (2 g/L) according to the instructions of
the manufacturer. Samples containing ~2 mg of PSA were applied to a
15-mL PSA immunoaffinity column equilibrated with TBS; the column was
then washed with TBS containing 1 mol/L NaCl and 2 mL/L Tween 20. The
bound proteins were then eluted with 1 mL/L trifluoroacetic acid, pH
2.0.
Electrophoresis and immunoblotting.
Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed
under reducing conditions in 2 mm thick and 10 x 10 cm 12.5%
homogeneous polyacrylamide gels (22). Proteins were
electrophoretically transferred to an Immobilon P membrane(23). A MAb (6C11) to PSA and polyclonal antibodies to API
or ACT were used to probe the immunoreactivity of PSA and its inhibitor
complexes.
Preparation and purification of PSA-API and PSA-ACT formed in
vitro.
Pooled female EDTA plasma (100 mL) was sequentially
precipitated with ammonium sulfate at 50% and 80% of saturation. The
precipitate forming at 80% of saturation was collected by
centrifugation (15000g for 20 min at 4 °C), dissolved,
and dialyzed against TBS with three changes of dialysis buffer.
Purified PSA (10 mg) was added to the dialyzed solution at 37 °C.
After 72 h, a saturated solution of ammonium sulfate was added to
give 20% of saturation. After 1 h at 4 °C, the preparation was
clarified by centrifugation (35 000g for 20 min at 4 °C)
and applied to a 10-mL Phenyl-Sepharose column. Unbound proteins in the
flowthrough fractions were collected and applied to the PSA
immunoaffinity column (see below). Bound proteins were eluted with 1
mL/L trifluoroacetic acid, pH 2.0, and immediately neutralized by the
addition of 0.2 mL of 0.5 mol/L Tris base to each 2-mL fraction. After
dialysis against 10 mmol/L Tris-HCl buffer containing 8 mmol/L sodium
azide, pH 8.4 (buffer A), the eluted fractions were applied to the
Resource Q column and eluted as described below.
Stability of PSA-API.
Purified PSA-API formed in vitro was
dissolved in 50 mmol/L phosphate buffer containing 1 g/L BSA and stored
at 4, 25, or 37 °C. Alternatively, PSA-API was stored in different
buffers at pH 5.0, 7.4, or 8.5 (pH measured at 25 °C). Aliquots were
withdrawn at 0, 24, 72, 120, and 168 h and analyzed by IFMAs
specific for total PSA and PSA-API, respectively. Before each assay,
the pH of the samples was adjusted to 7.4 by the addition of
concentrated TBS (10-fold).
statistical analysis
The analytical detection limit was defined on the basis of the
mean plus 2 SD of the fluorescence response of 10 aliquots of assay
buffer only. The biological detection limit of the assay for PSA-API
was calculated from the mean concentration plus 2 SD in 22 female sera
devoid of PSA immunoreactivity. Differences in the concentrations of
total PSA and PSA-API, and the proportion of free PSA, PSA-ACT, and
PSA-API between PCa and BPH patients were determined by the Wilcoxon
rank-sum test. The correlation between the concentration of PSA-API
determined by various methods was examined by linear regression
analysis.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). When the mixture was applied to Phenyl-Sepharose (high
performance), the PSA complexes appeared in the flowthrough fraction,
whereas free PSA was retained (not shown). The PSA complexes were
applied to the PSA immunoaffinity column, eluted with 1 mL/L
trifluoroacetic acid, pH 2.0, and fractionated further by
anion-exchange chromatography. Three peaks (defined as I, II, and III)
with PSA immunoreactivity were observed (Fig. 1
).
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SDS-PAGE and immunoblotting of the peak fractions (I, II, and III)
obtained by anion-exchange chromatography showed that the PSA in peak I
contained a single 30-kDa band reacting only with the antibody to PSA
(Fig. 2
A, lane 4), suggesting that it was free PSA. The PSA in peak II
consisted of a major band of ~80 kDa, which reacted with antibodies
to PSA (Fig. 2A
, lane 5) and API (Fig. 2B
, lane 5), indicating that it
was the PSA-API complex. In addition, a less intense band with a
molecular mass slightly smaller than that of intact API reacted only
with the antibody to API (Fig. 2B
, lane 5), and a band with a molecular
mass of 30 kDa reacted with antibody to PSA (Fig. 2A
, lane 5). However,
free PSA was not observed when fractions of peak II were fractionated
by gel filtration (not shown). The PSA-API in peak II contained ~14%
of the added PSA (Table 1
). Peak III contained a major 90-kDa band that
reacted with antibodies to PSA (Fig. 2A
, lane 6) and ACT (Fig. 2C
, lane
6). A less intense band with a molecular mass slightly smaller than
that of intact ACT reacted only with the antibody to ACT (Fig. 2C
, lane
6), and a band with a molecular mass of 30 kDa reacted with the
antibody to PSA (Fig. 2A
, lane 6). The PSA-ACT in peak III contained
~18% of the added PSA (Table 1
). PSA-API did not react with the ACT
antibody, and PSA-ACT did not react with the API antibody (Fig. 2
, B
and C).
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When purified PSA-API was incubated at 4, 25, and 37 °C for 168 h, the concentration of PSA-API decreased from 50 µg/L to 48 µg/L (96%) at 4 °C, to 44 µg/L (88%) at 25 °C, and to 36 µg/L (72%) at 37 °C. When stored at pH 5.0 at 25 °C for 168 h, the concentration of PSA-API was virtually unchanged (49 µg/L, 98%), whereas it decreased to 44 µg/L (88%) at pH 7.4 and to 24 µg/L (48%) at pH 8.5. The concentrations of total PSA did not change significantly under these conditions (not shown).
ifmas for free and total psa, psa-act, and psa-api
The analytical detection limit was 0.01 µg/L for the Prostatus
PSA free/total kit (20), and the analytical detection limit
for PSA-ACT was 0.16 µg/L (9). In the concentration range
0.5–50.0 µg/L, the intra- and interassay coefficients of variation
(CVs) were 3–5% and 5–8% for the free- and total-PSA assays(20), and 4–9% and 8–12% for the PSA-ACT assay,
respectively (9).
The calibration curve of the IFMA for PSA-API was linear over the range
0–50 µg/L. The analytical detection limit was 0.1 µg/L. The intra-
and interassay CVs were 5–10% and 8–14%, respectively, determined
by repeated measurement of three serum samples with PSA-API
concentrations of 0.5, 2, and 11 µg/L 10 times in one assay or in 10
consecutive assays. Separation of PSA-API from PSA-ACT by
anion-exchange chromatography (Fig. 1
) and analysis of the fractions
showed that the cross-reaction between the IFMAs for PSA-ACT and
PSA-API was <1%.
characterization of psa-api in PCa SERUM
When sera (n = 14) from PCa patients with PSA concentrations
of 20–700 µg/L were fractionated by anion-exchange chromatography,
endogenous PSA-API showed the same chromatographic behavior as that
formed in vitro (Figs. 1
and 3
). The peak containing PSA-API was separated from the major peak
containing PSA-ACT and several minor peaks containing free PSA. The
PSA-API peak was detected by the IFMAs for total PSA and PSA-API, but
not by those for PSA-ACT (Figs. 1
and 3
) and free PSA (not shown). The
concentrations of PSA-API in sera (n = 14) determined directly by
the PSA-API IFMA (x) correlated strongly with those measured
by the IFMA for total PSA (y) in the PSA-API-containing
fractions separated by anion-exchange chromatography (Fig. 4
; r = 0.97; y =
1.3x - 2.1; P <0.0001), but the
correlation was weaker in sera (n = 11) with low concentrations
(<2 µg/L) of PSA-API (r = 0.42).
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When endogenous PSA-API in serum separated by anion-exchange chromatography was further fractionated by gel filtration on Superdex-200 (not shown), a major peak (75%) of ~80 kDa was detected by the IFMAs for total PSA and PSA-API (not shown), indicating that the endogenous PSA-API in serum had the same molecular mass of ~80 kDa as PSA-API formed in vitro. In addition, a minor peak (~25%) of 30 kDa was detected by the IFMAs for free and total PSA (not shown), suggesting that PSA-API fractions obtained by the separation of PCa serum by anion-exchange chromatography contained some free PSA.
psa-api in female serum
When female sera (n = 22) devoid of PSA immunoreactivity were
analyzed by the IFMA for PSA-API, the apparent concentration of PSA-API
was 0.12–0.57 µg/L (median, 0.24 µg/L). This is comparable to the
apparent concentrations (0.04–0.44 µg/L; median, 0.20 µg/L)
observed when PSA-API was analyzed in a control assay without PSA
antibody on the solid phase (P = 0.2). When purified
API was diluted in assay buffer at concentrations of 0.5–2.0 g/L,
which correspond to those occurring in nondiseased serum, the apparent
concentrations in the PSA-API IFMA were 0.15–0.46 µg/L (median, 0.25
µg/L). Thus the concentrations of PSA-API measured in female serum or
purified API preparations were probably attributable to nonspecific
adsorption of the huge excess of API to the solid phase. This
background was reduced by preincubation of the coated microtitration
wells with heparin, which reduced the apparent concentrations of
PSA-API in female sera to 0.04–0.25 µg/L (median, 0.1 µg/L;
P <0.0001). The biological detection limit of the assay for
PSA-API in serum estimated from the mean concentration of PSA-API in
female serum plus 2 SD was 0.21 µg/L. Values below 0.21 µg/L were
therefore considered undetectable.
psa-api in serum of PCa AND BPH PATIENTS
PSA-API was undetectable (<0.21 µg/L) in sera from 17 (21%)
PCa and 29 (44%) BPH patients with PSA concentrations of 4–706
µg/L. In sera with PSA-API concentrations 
0.21 µg/L, the
concentrations of PSA-API in serum increased with increasing total PSA
(Fig. 5
). The median proportion of PSA-API in relation to total PSA in
sera with PSA concentrations >4 µg/L was 2.4% (range, 1.0–7.9%)
in PCa and 3.6% (range, 1.3–12.2%; P <0.001) in BPH. In
sera with total-PSA concentrations of 4–20 µg/L, the proportion of
PSA-API was higher in BPH (median, 4.1%; Table 2
) than in PCa patients (median, 3.2%; P =
0.02). Among these patients, 5 (18%) with PCa and 8 (36%) with BPH
had a proportion of PSA-API in serum of 5–12%, which is comparable to
that of free PSA.
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free and total psa and psa-act in serum of PCa AND BPH
PATIENTS
The concentration of total PSA and the proportion of PSA-ACT were
higher in PCa patients than in BPH patients, whereas the proportion of
free to total PSA was lower (Table 2
). The differences between PCA and
BPH were statistically significant in the whole material and in the PSA
concentration range 4–20 µg/L. The median of the sum of PSA-ACT and
free PSA in individual sera was 95.3% of total PSA in PCa and 93.9%
in BPH. The median of the sum of PSA-ACT, PSA-API, and free PSA was
98.5% in PCa and 98.6% in BPH.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PSA-API in serum could be separated from free PSA and PSA-ACT by anion-exchange chromatography, and the chromatographic behavior of endogenous PSA-API in serum was indistinguishable from that of PSA-API formed in vitro. The PSA-API concentration in serum could also be determined indirectly by assay of total PSA in the PSA-API-containing fractions separated by anion-exchange chromatography. The values obtained with this method correlated with those determined directly with the specific IFMA for PSA-API. However, measurement of PSA-API with the total-PSA assay after chromatography overestimated the results by ~25% because of contamination of PSA-API with free PSA in serum.
The determination of PSA-API in female serum devoid of PSA immunoreactivity indicated that there is a nonspecific assay background. This problem also affects immunoassays for PSA-ACT(12)(21). The background in the PSA-ACT assays can be reduced by the use of heparin in the assay buffer(21). We tested this approach (not shown), but found that preincubation with heparin in the microtitration wells was more effective, reducing the median background in female serum from 0.24 µg/L to 0.10 µg/L. The background in serum was independent of the PSA antibody on the solid phase, as evidenced by a similar background when female serum was analyzed using microtitration wells without capture antibody. The ability of heparin to decrease the background noise suggests that it was caused mainly by surface charge effects, which caused nonspecific binding of the huge excess of API (or other API complexes) in serum to the solid phase(24)(25). Adsorbed API reacted with Eu-labeled anti-API antibodies. This explanation was supported by the fact that purified API at concentrations comparable to those in serum gave a similar background in the IFMA for PSA-API.
The apparent PSA-API concentration in female serum was used to establish the biological detection limit for PSA-API, and only samples with PSA-API concentration exceeding this limit were considered detectable. In these samples, the proportion of PSA-API in serum was lower in PCa than in BPH patients with serum PSA concentration between 4 to 20 µg/L. This is the reverse of the proportion of PSA-ACT, which is higher in PCa than in BPH (10)(12). The difference in the proportion of PSA-API between PCa and BPH is of potential clinical utility. Whether this finding can be used to improve the cancer specificity of the PSA test remains to be determined.
The calibrators for the PSA-API and PSA-ACT assays were prepared by the
addition of purified PSA to a plasma fraction containing API and ACT
but devoid of 2-macroglobulin. The proportions
of complexes formed between PSA and ACT or API in this plasma were
similar (~25% each). The free PSA remaining was removed by
hydrophobic interaction chromatography, after which PSA-ACT and PSA-API
were collected by immunoaffinity chromatography and then separated by
anion-exchange chromatography. Although PSA gradually dissociates from
PSA-API (17), <1% free PSA was detected by gel filtration
of the PSA-API preparation (not shown). The higher proportion of free
PSA and API observed in immunoblotting was apparently caused by the
dissociation of PSA-API by SDS and heating of the sample before
electrophoresis. The API and ACT released from PSA complexes were
cleaved, as indicated by a reduction in molecular size compared with
the intact forms. During prolonged incubation at neutral or basic pH,
PSA-API formed in vitro tended to dissociate. In serum, the PSA
released from PSA-API forms a complex with
2-macroglobulin (17). We therefore
used an artificial buffer matrix with a pH of 5 rather than female
serum as the diluent for the calibrators. In this buffer, the stability
of PSA-API was satisfactory, apparently because of the low enzyme
activity of PSA at this pH (21).
Because the molecular structure of API is different from that of ACT, it is possible that the PSA epitopes exposed in the PSA-API complex differ from those in PSA-ACT. The antibodies used in the DELFIA PSA kit for total PSA react equally with free PSA and PSA-ACT(20)(21). The fact that the concentration of total PSA did not change during the dissociation of PSA-API indicates that the antibodies used in the total-PSA IFMA also react equally with PSA-API (not shown). Thus, the assay for total PSA could be used to assign values to the PSA-API calibrators.
The results of this study indicate that the immunoreactive PSA in serum
consists of three major molecular forms. The median serum concentration
of the major form, PSA-ACT, was 69% of that of total PSA in BPH and
80% of that of total PSA in PCa; the median concentration of free PSA
was 26% and 14% and the median concentration of PSA-API was ~4%
and ~3% in BPH and PCa, respectively (Table 2
). The sum of the
concentrations of PSA-ACT, free PSA, and PSA-API was close to the value
of total PSA, suggesting that together they account for most, if not
all, of the immunoreactive PSA in serum. In five (18%) patients with
PCa and eight (36%) patients with BPH, the proportion of PSA-API in
sera with PSA concentrations of 4–20 µg/L was 5–12%; thus it
represents a notable fraction of the PSA immunoreactivity measured by
conventional PSA immunoassays. However, it is likely that these values
contain the nonspecific background observed in female serum. If this
background (0.1 µg/L) is subtracted from the measured values, the
median percentage of PSA-API in patient samples will decrease by
20–40% in those with the lowest PSA values, but this effect decreases
with increasing PSA values.
A PSA calibrator containing 10% free PSA and 90% PSA-ACT has been prepared (26) based on the proportion of complexed PSA in serum from PCa and BPH patients, as estimated by assay of total PSA in serum fractions obtained by gel filtration(26)(27). Because PSA-API is not resolved from PSA-ACT by gel filtration (10), the fraction of complexed PSA includes PSA-API. Thus, the proportion of PSA-ACT determined by gel filtration will be higher than that measured with a specific assay for PSA-ACT (12). A too-high value for PSA-ACT will also be obtained if free PSA is subtracted from total PSA because this value includes PSA-API. Thus, PSA-API needs to be considered in the standardization of PSA immunoassays.
In conclusion, we have developed a quantitative IFMA for PSA-API and have shown that the concentration of PSA-API in serum is correlated with the total-PSA concentration and that the proportion of PSA-API in serum is lower in PCa than in BPH. These results warrant further investigations on the clinical utility of PSA-API as an adjunct to free and total PSA in the diagnosis of PCa.
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Acknowledgments |
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Footnotes |
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1-antichymotrypsin; PCa, prostate cancer; BPH, benign prostatic hyperplasia; API, 1-protease inhibitor; IFMA, immunofluorometric assay; BSA, bovine serum albumin; MAb, monoclonal antibody; TBS, 50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 8 mmol/L sodium azide; and SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 
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P. Nurmikko, K. Pettersson, T. Piironen, J. Hugosson, and H. Lilja 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., August 1, 2001; 47(8): 1415 - 1423. [Abstract] [Full Text] [PDF]
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 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]
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 A. Paju, A. Bjartell, W.-M. Zhang, S. Nordling, A. Borgstrom, J. Hansson, and U.-H. Stenman Expression and Characterization of Trypsinogen Produced in the Human Male Genital Tract Am. J. Pathol., December 1, 2000; 157(6): 2011 - 2021. [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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J. Peter, C. Unverzagt, and W. Hoesel Analysis of Free Prostate-specific Antigen (PSA) after Chemical Release from the Complex with {alpha}1-Antichymotrypsin (PSA-ACT) Clin. Chem., April 1, 2000; 46(4): 474 - 482. [Abstract] [Full Text] [PDF]
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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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D. W. Chan and L. J. Sokoll Prostate-specific Antigen: Advances and Challenges Clin. Chem., June 1, 1999; 45(6): 755 - 756. [Full Text] [PDF]
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