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Enzymes and Protein Markers |
2-macroglobulin
kan Stenman1
Departments of
1
Clinical Chemistry,
2
Urology, and
3
Pathology, Helsinki University Central Hospital, FIN-00290, Helsinki, Finland.
a Author for correspondence. Fax 358-0-4714804; e-mail wmzhang{at}helsinki.fi.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin (A2M) in vitro; however, PSA
complexed with A2M (PSA-A2M) is not detected by
conventional immunoassays for PSA because it is encapsulated by the
A2M. In this study, we show that denaturation of
PSA-A2M at high pH renders PSA immunoreactive. Part of the
complexed PSA is released in free form and part remains bound to
denatured A2M. These forms can be measured by a
conventional immunoassay for PSA. This finding enabled us to design a
dissociation assay for the detection of PSA-A2M, which was
based on the removal of immunoreactive PSA in serum by
immunoadsorption, denaturation of PSA-A2M at high pH, and
measurement of the released PSA immunoreactivity by a conventional PSA
immunoassay. This PSA-A2M assay was calibrated with
PSA-A2M formed in vitro. The detection limit of the assay
was 0.14 µg/L. Inter- and intraassay coefficients variation were
4–9% and 8–14%, respectively. When purified PSA was incubated with
A2M, the loss of PSA immunoreactivity was highly correlated
with the PSA-A2M formed, as measured by the dissociation
assay for PSA-A2M (r = 0.99; P
<0.0001). The concentration of PSA-A2M in serum correlated
with that of total PSA both in prostate cancer (PCa) and benign
prostatic hyperplasia (BPH); however, the ratio of PSA-A2M
in relation to total PSA was significantly higher in BPH than in PCa
(P <0.0003). ROC curve analysis suggested that measurement
of the ratio of PSA-A2M to total PSA in serum improves the
diagnostic accuracy for PCa compared with assays for total PSA only. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2-macroglobulin
(A2M), pregnancy zone protein,
1-antichymotrypsin (ACT), 1-protease
inhibitor (API), and protein C inhibitor (6)(7)(8)(9). In serum, most of the immunoreactive PSA occurs in complex with ACT (PSA-ACT); the rest is either free or in complex with API [PSA-API; Zhang et al., manuscript submitted, and Refs. (10)(11)]. Five major antigenic regions have been identified on the PSA molecule, only one of which is covered by ACT in PSA-ACT (12). The PSA-ACT and PSA-API complexes are readily detected by specific sandwich assays or by conventional PSA immunoassays [Zhang et al., manuscript submitted, and Refs. (8)(9)(10)(11)(12)(13)(14)]. Specific measurement of complexed and free PSA in serum improves the diagnostic accuracy for prostate cancer (PCa) compared with assays of total PSA only [Zhang et al., manuscript submitted, and Refs. (10)(13)(14)].
A2M is a tetramer assembled from pairwise
disulfide-bridged 180-kDa subunits, each subunit containing a bait
region, which is susceptible to cleavage by most proteases, and a
reactive internal ß-cysteinyl-
-glutamyl thiol ester
(15)(16). When A2M interacts with a
protease, the bait region of A2M is proteolytically
cleaved, causing activation of the thiol ester and covalent binding of
the protease to A2M, mainly through an 
-Lys--Glu bond
(16)(17)(18). Simultaneously, the conformation of
A2M is changed from an electrophoretically "slow"
S-form to a "fast" F-form, and the protease is entrapped within the
A2M molecule (15)(16). One
tetrameric A2M can bind two protease molecules
(17). The encapsulation of proteases by A2M
sterically hinders access of high-molecular weight substance such as
high-molecular weight inhibitors or antibodies to the enzymes
(19). Thus, the PSA-A2M complex is not detected
by conventional PSA immunoassays (8)(11). When
PSA-A2M is denatured with sodium dodecyl sulfate (SDS), PSA
epitopes are exposed, rendering it reactive with PSA antibodies
(6)(20). PSA-A2M has been detected
qualitatively in male serum with high concentrations of PSA by
immunoblotting after SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
(20).
In this study, we developed a sensitive and quantitative dissociation assay for PSA-A2M in serum, characterized PSA-A2M formed in vitro and endogenous PSA-A2M in serum, and measured the concentrations of PSA-A2M in serum from patients with PCa and benign prostatic hyperplasia (BPH). The dissociation assay for PSA-A2M is based on the removal of immunoreactive PSA, i.e., PSA-ACT, PSA-API, and free PSA, by immunoadsorption, denaturation of PSA-A2M at high pH, and measurement of the PSA thus rendered immunoreactive.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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reagents
The Superdex-200 was from Pharmacia Biotech, the
3,3'-diaminobenzidine tetrahydrochloride was from Sigma Chemical Co.,
and the PVDF membrane (Immobilon P) was from Millipore. The
streptavidin-conjugated magnetic beads were from Promega. The NaOH was
from Reagena LTD, and the HCl was from Merck. The
sulfosuccinimidyl-6-(biotinamido)hexanoate was purchased from
Pierce. The trifluoroacetic acid was from Fluka. The DELFIA assay
buffer and enhancement solution used in immunoassays were from Wallac.
proteins
The major isoenzyme of PSA was purified from human seminal fluid
(21); the A2M was purified from plasma as
described (22). Molecular mass protein markers were from
Amersham. The bovine serum albumin (BSA) was from Sigma.
antibodies
A monoclonal antibody (MAb) to PSA (5E4) was produced by standard
procedures (Leinonen et al., unpublished results); H117 and H50 were
kind gifts from B. Dowell (Abbott Diagnostics, Abbott Park, IL).
Polyclonal antibodies to PSA and A2M and a
peroxidase-conjugated swine anti-rabbit IgG immunoglobulin were from
Dakopatts. MAb 5E4 and the polyclonal antibody to PSA were biotinylated
according to the manufacturer's (Pierce) instructions. MAb H50 and the
polyclonal IgG to A2M were labeled with Eu3+ as
described (10).
immunoassays
Total PSA was determined by a time-resolved immunofluorometric
assay (IFMA) using MAb H117 as the capture antibody and MAb H50 labeled
with Eu3+ as tracer. The calibrators were prepared
from pure PSA and standardized as described (21). A sandwich
assay utilizing the polyclonal antibody to PSA as the capture antibody
and a polyclonal antibody to A2M labeled with
Eu3+ as tracer was performed as described (10)
and termed "A2M antibody-based PSA-A2M
assay" in this study. The IFMA for A2M was performed as
described (8).
gel filtration
Samples were loaded on a Superdex-200 column (60 x 1.6 cm)
and eluted with Tris-buffered saline (TBS; 50 mmol/L Tris-HCl buffer,
pH 7.4, containing 150 mmol/L NaCl and 8 mmol/L NaN3).
The flow rate was 15 mL/h, and 2-mL fractions were collected. The
column was roughly calibrated by measuring the absorbance at 280 nm in
the fractions to identify the elution volumes of human IgG (150 kDa)
and human albumin (68 kDa).
electrophoresis and immunoblotting
SDS-PAGE was performed under reducing conditions (23)
in 10 x 10 cm, 2 mm thick, 3–16% gradient polyacrylamide gels.
After electrophoresis proteins were transferred eletrophoretically to
immobilon P and incubated with polyclonal antibodies to PSA or
A2M (24). Bound antibodies were detected with
peroxidase-conjugated swine anti-rabbit IgG immunoglobulin, using 3,
3'-diaminobenzidine tetrahydrochloride as the substrate.
complex formation between psa and a2m
Purified PSA (60 µg) was incubated with 12 mg of
A2M in 800 µL of TBS containing 50 g/L BSA (1:10
molar ratio) at 37 °C. Aliquots of 100 µL were taken at time
intervals of 0, 1, 3, 6, 8, 24, 48, and 72 h. The aliquot taken at
72 h was subjected to gel filtration. The fractions obtained and
the aliquots were further analyzed by immunoassays for PSA,
A2M, and PSA-A2M and by immunoblotting.
immunoadsorption of psa
Five micrograms of biotinylated MAb 5E4 (to PSA) was incubated
with 100 µg of streptavidin-conjugated magnetic beads at 25 °C.
After 30 min, unbound antibodies were removed by washing the beads with
assay buffer. The beads (100 µg) were incubated with 200 µL of
PSA-A2M formed in vitro or male serum at 25 °C.
After 30 min, the beads were collected with a magnet, and the
supernatants were analyzed for PSA and PSA-A2M.
denaturation of psa-a2m
PSA-A2M formed in vitro was purified by gel
filtration and then diluted in TBS containing 50 g/L BSA or pooled
female serum without PSA immunoreactivity. Diluted PSA-A2M
was incubated with NaOH at a final concentration range of 0–100 mmol/L
at 25 °C. Aliquots withdrawn at 0, 30, 60, and 180 min were measured
for pH by an electronic pH meter (PW4920; Phlips) and for PSA by the
PSA IFMA. As a control, purified PSA diluted in TBS containing 50 g/L
BSA was treated in the same way. PSA-A2M incubated with
NaOH was further separated by gel filtration, and fractions obtained
were analyzed by the PSA IFMA and the A2M antibody-based
PSA-A2M assay. In addition, 10 sera with high PSA (>20
µg/L) from patients with PCa were subjected to immunoadsorption,
treatment with NaOH, separation by gel filtration, and analysis by the
PSA IFMA and the A2M antibody-based PSA-A2M
assay.
dissociation assay for psa-a2m in serum
Serum samples or calibrators dissolved in pooled female serum (200
µL) were subjected to immunoadsorption, after which 180 µL of the
adsorbed calibrators or samples were incubated with 20 µL of NaOH
(0.60 mol/L) at 25 °C. After 30 min, 340 µL of assay buffer
containing 20 µL of HCl (0.60 mol/L) was added to neutralize the
solution. Duplicates of 225 µL, corresponding to the 75 µL of
original serum or calibrators, were assayed for total PSA by IFMA. The
intra- or interassay coefficients of variation (CVs) were determined by
measuring five serum samples with PSA concentrations from 2 to 10
µg/L 10 times within the same or different analytical runs.
standardization of the dissociation assay for
psa-a2m
The loss of PSA immunoreactivity during the in vitro complexation
with A2M was assessed by gel filtration and used to
estimate the PSA content in the PSA-A2M calibrator. The
calibrators were prepared by dilution of purified PSA-A2M
formed in vitro in a pooled female serum without PSA immunoreactivity
to concentrations of 0, 0.1, 1, 4, 20, and 100 µg/L.
stability of psa-a2m in serum
Purified PSA-A2M formed in vitro was added to
pooled female serum at five different concentrations from 1 to 100
µg/L and stored at 4 or 25 °C for 0, 24, 72, and 168 h. The
concentrations of PSA and PSA-A2M were measured by the PSA
IFMA and by the dissociation assay for PSA-A2M,
respectively. Alternatively, purified PSA-A2M was added
into 46 individual female sera at a final concentration of 25 µg/L
and stored for 2 h at 25 °C. The recovery of
PSA-A2M was analyzed by the dissociation assay for
PSA-A2M. We also measured the pH of the individual sera
after the addition of NaOH.
statistical analysis
The detection limit of the dissociation assay for
PSA-A2M was defined as the concentration giving a
fluorescence signal equal to that of a female serum pool plus 2 SD
(calculated from 20 duplicates). The difference in the concentrations
and proportions of PSA-A2M between serum from patients with
PCa and BPH was tested by the Wilcoxon rank-sum test. The diagnostic
accuracy of the various PSA assays were compared by ROC curves analysis
as described (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Gel filtration of the aliquot obtained at the 72-h time point
showed that very little PSA immunoreactivity (<1% of added PSA) was
detected in the fractions containing A2M, although ~40%
of the added PSA had complexed with A2M (Fig. 2
). In the A2M antibody-based PSA-A2M
assay, a low response was observed in the fractions containing
A2M (Fig. 2
). However, a similar response was also detected
after gel filtration of pure A2M or female serum without
PSA immunoreactivity (not shown). The fractions containing
A2M obtained by gel filtration were further subjected to
immunoadsorption with biotinylated mono- and polyclonal PSA antibodies
and streptavidin-conjugated magnetic beads. Only trace amounts of
immunoreactive PSA (<1%) were adsorbed to the magnetic beads. Elution
of the adsorbed material with trifluoroacetic acid and immunoblotting
with polyclonal anti-PSA or anti-A2M antibodies showed that
the immunoreactive PSA was in the free form and that no A2M
immunoreactivity was associated with this PSA (not shown).
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Characterization of PSA-A2M by immunoblotting.
When the fractions containing A2M obtained by gel
filtration were analyzed by SDS-PAGE and immunoblotting, two bands with
molecular masses of 300–400 and 100–200 kDa and which reacted with
PSA and A2M antibodies were observed (Fig. 3
). The band at 300–400 kDa apparently was dimeric
A2M complexed with PSA, and the band of 100–200 was
probably monomeric A2M complexed with PSA. Several bands of
molecular mass <100 kDa reacted only with antibodies to
A2M, suggesting that they were fragments of A2M
(Fig. 3B
). In addition, a weak 30-kDa band, which reacted with PSA
antibody, was also observed (Fig. 3A
), indicating that a minor portion
of PSA was released from PSA-A2M by treatment with SDS.
Endogenous PSA-A2M in serum displayed similar bands that
reacted with antibodies to PSA and A2M (not shown).
Adsorption with PSA antibody did not remove the PSA or A2M
immunoreactivity from the PSA-A2M fractions (not shown).
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treatment of psa-a2m with NaOH
Denaturation of PSA-A2M in TBS buffer.
When
purified PSA-A2M dissolved in TBS buffer containing
50 g/L BSA was incubated with NaOH for 30 min at 25 °C, PSA
immunoreactivity was measured by the PSA IFMA after neutralization with
HCl. The recovery of PSA immunoreactivity increased with increasing pH
up to 11.5 (Fig. 4
), corresponding to a NaOH concentration of 70 mmol/L in a TBS
buffer-based matrix. The recovery of PSA immunoreactivity was ~30%
of the calculated PSA content in PSA-A2M. Incubation of
PSA-A2M in TBS at pH 11.5 for up to 3 h at 25 °C
marginally affected the recovery of PSA immunoreactivity (not shown).
Incubation of purified PSA in TBS with increasing concentrations of
NaOH tended to reduce PSA immunoreactivity; however, the loss was
remarkable only above pH 12.5, which corresponded to a NaOH
concentration of 85 mmol/L (Fig. 4
). When PSA-A2M treated
in TBS at pH 11.5 was subjected to gel filtration, two components were
detected by the PSA IFMA (Fig. 5
). A high-molecular weight peak (~400 kDa) comprised about
30% of the released PSA immunoreactivity, whereas the rest eluted as a
30-kDa component (Fig. 5
). The 400-kDa component was also detected by
the A2M antibody-based PSA-A2M assay (not
shown), suggesting that it represented PSA bound to denaturated
A2M. The 30-kDa peak apparently consisted of free PSA
released from PSA-A2M (Fig. 5
). When added to fresh female
serum, the released PSA was able to form a complex with A2M
and ACT, indicating that it was enzymatically active (not shown).
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Denaturation of PSA-A2M in serum.
When
purified PSA-A2M diluted in pooled female serum or when
immunoadsorbed serum from PCa patients (n = 10) was incubated at
high pH, the appearance of PSA immunoreactivity was detected by the PSA
IFMA. Maximal recovery was obtained at pH 11.4, corresponding to a NaOH
concentration of 60 mmol/L in this matrix. Gel filtration of the
NaOH-treated serum revealed a high-molecular mass component (~400
kDa) and a 30-kDa component, which contained ~30% and 70% of the
recovered PSA immunoreactivity, respectively (not shown). The smaller
component was detected only by the PSA IFMA, whereas the 400-kDa
component was also detected by the A2M antibody-based
PSA-A2M assay (not shown). Thus, PSA-A2M formed
in vitro and endogenous PSA-A2M in serum showed similar
patterns after denaturation at high pH.
dissociation assay for psa-a2m
Immunoadsorption of PSA in serum.
Immunoadsorption of PCa
serum with the PSA antibody removed >99.9% of PSA immunoreactivity if
the concentration of PSA in the serum was <500 µg/L. After the beads
were washed with assay buffer, the PSA immunoreactivity was eluted from
the beads with 1 mL/L trifluoroacetic acid and subjected to SDS-PAGE
and immunoblotting. The extracted PSA consisted of complexed PSA (90
kDa) and free PSA. No A2M immunoreactivity was detected
(not shown).
Characteristics of the assay procedure.
The dissociation assay
for PSA-A2M included immunoadsorption, treatment at pH
11.4, neutralization, and determination of the released PSA
immunoreactivity. The quantification range of PSA-A2M was
0–100 µg/L (Fig. 6
), and the detection limit was 0.14 µg/L. The values in Fig. 6
represent PSA and disregard the content of A2M. The
intraassay CV was 4–9%, and the interassay CV was 8–14% in samples
with PSA concentrations in the range of 2–10 µg/L.
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Validation of the dissociation assay for PSA-A2M.
When purified PSA was incubated with A2M at 37 °C, the
concentrations of PSA-A2M measured by the assay for
PSA-A2M increased with time, whereas the concentration of
free PSA decreased (Fig. 1
). The loss of free PSA (x)
correlated with the concentration of PSA-A2M formed
(y; Fig. 7
). The equation for the line in Fig. 7
is: y =
-7.81 + 1.07x; r = 0.99).
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Recovery of PSA-A2M added to female serum.
Purified PSA-A2M formed in vitro was added to pooled female
serum to give concentrations of 1–100 µg/L. After storage for 7 days
at 4 °C, the mean recovery was 97% (range, 94–101%); after
storage at 25 °C, it was 95% (range, 90–98%). When purified
PSA-A2M was added to 46 individual female sera and
incubated for 2 h at 25 °C, the median recovery of
PSA-A2M was 96% (95% confidence interval, 92–97%). No
PSA immunoreactivity was detected in serum before denaturation.
Addition of NaOH to 46 female sera to a final concentration of 60
mmol/L increased the pH to 11.40 ± 0.13 (mean ± SD).
determination of psa-a2m and total psa in serum from
healthy females and pca and bph patients
The concentration of PSA-A2M was below the
detection limit of the assay in 44 (96%) of the 46 female sera. Two
samples had apparent PSA-A2M concentrations of 0.18 and
0.43 µg/L. The female serum used as a matrix for the calibration had
no detectable PSA immunoreactivity, as determined by the IFMA for PSA.
The median concentration of total PSA was 13.5 µg/L (range, 0.4–432
µg/L) in sera from 73 patients with PCa and 4.9 µg/L (range, 1–73
µg/L) in sera from 58 BPH patients (P = 0.001). In
patients with PCa, the concentrations of PSA-A2M ranged
from 0 to 49 µg/L (median, 1.2 µg/L); in BPH patients, it ranged
from 0 to 14 µg/L (median, 0.7 µg/L; P <0.001). The
PSA-A2M concentrations correlated with those of total PSA
both in PCa and BPH (Fig. 8
). The ratio of PSA-A2M in relation to total PSA was
higher in BPH (median, 17%; range, 0–60%) than in PCa (median, 12%;
range, 0–63%; P <0.001; Fig. 9
). In samples with PSA concentrations of 4–10 µg/L, the
median ratio of PSA-A2M to total PSA in serum was also
significantly higher in BPH (19.5%; n = 28) than in PCa (14.5%;
n = 23; P = 0.002). ROC curve analysis of samples
with PSA concentrations in the range 4–10 µg/L showed that the area
under the curve was 0.78 for the ratio of PSA-A2M to total
PSA, whereas for total PSA, the area under the curve was 0.66 (Fig. 10
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatment at high pH causes partial dissociation of PSA-A2M. Both the free PSA released and that remaining covalently bound to denatured A2M can be detected by a conventional PSA immunoassay. The recovery of PSA immunoreactivity after denaturation is ~30% of the calculated PSA content in PSA-A2M. Because the denaturation at pH 11.5 does not cause any considerable loss of the immunoreactivity of free PSA, the low recovery is probably attributable to the reduced immunoreactivity of the PSA bound to denatured A2M. The recovery of PSA immunoreactivity is to some extent dependent on the final pH during the treatment of PSA-A2M with NaOH. In different sera, the final pH may vary because of differences in buffering capacity. However, treatment of 46 female sera with NaOH produced very little variation in the final pH, and there was little variation in the recovery of PSA immunoreactivity of PSA-A2M added to these sera. Thus, differences in the buffering capacity of serum had a negligible effect on the recovery of PSA from PSA-A2M.
The PSA IFMA used in the new dissociation assay for PSA-A2M recognizes PSA-ACT and free PSA in an equimolar fashion (8); however, it underestimates PSA remaining bound to denatured A2M. This is to be expected because proteases captured by A2M are covalently bound in a random fashion by a reactive thiol ester in A2M, which binds mainly to lysine residues in the proteases to form stable amide cross-links (16). A2M also forms base-labile ester cross-links to serine, threonine, tyrosine, or carbohydrate groups in captured proteases. The free PSA released by NaOH treatment has probably been bound by such bonds (16). The recognition of PSA in complex with A2M after denaturation was not assay-specific. Various assays using other PSA antibodies, including those specific for free PSA, measured PSA bound to denatured A2M in a similar way (not shown).
It is possible that the proportion of PSA released from and bound to denaturated A2M varies from one serum to another. This would affect the recovery of PSA immunoreactivity because the two forms of PSA measurable after the denaturation of PSA-A2M have different immunoreactivities. We observed a ratio of ~70% to 30% between free PSA released and that bound to A2M in 10 sera with high PSA concentrations that had been separated by gel filtration after treatment with NaOH. A similar ratio was also observed when PSA-A2M formed in vitro was subjected to denaturation and separation by gel filtration.
NaOH can be neutralized with HCl with little sample dilution. After treatment of PSA-A2M with NaOH and neutralization, A2M was permanently denatured, whereas PSA retained its immunoreactivity. With a sample volume of 225 µL, corresponding to 75 µL of serum or calibrators, the detection limit was 0.14 µg/L. This enabled us to reliably analyze PSA-A2M in sera with total PSA concentrations >4 µg/L.
Treatment of PSA-A2M with SDS has been used to expose PSA encapsulated by A2M (6)(20). However, SDS interferes with antibody binding. This effect can be reduced by dilution; dilution however, causes considerable reduction in assay sensitivity (26). We found that a portion of the encapsulated PSA was released by SDS. This is compatible with the observation that portions of the proteases captured by A2M are not covalently bound (16). The proportion of PSA released by SDS was much lower than that released by NaOH (not shown).
Denaturation of PSA-A2M at high pH rendered it immunoreactive in the A2M antibody-based PSA-A2M assay as well as in the conventional PSA assay. Denaturation of pure A2M at high pH also induced a small response in the A2M-antibody-based PSA-A2M assay, which probably was a result of increased nonspecific background. This effect was not present in the new immunoassay for PSA-A2M, which measured PSA released from the complex and bound to denaturated A2M.
Immunoblotting of PSA-A2M formed in vitro showed that two bands with molecular masses of 300–400 and 100–200 kDa, respectively, reacted with PSA antibody. The 300- to 400-kDa band apparently represents PSA complexed with a dimer of A2M subunits, as also observed in a recent study (27). The low-molecular mass band probably represents PSA bound to a monomeric A2M subunit (27).
Because PSA-A2M formed in vitro and endogenous PSA-A2M in serum showed similar patterns after denaturation at high pH, we used PSA-A2M formed in vitro to calibrate the PSA-A2M assay. To obtain the same buffering capacity as in serum, the calibrators were prepared by dilution of purified PSA-A2M in female serum. PSA-A2M was stable in female serum; thus it was a suitable matrix for the PSA-A2M calibrators.
Recently Espana et al. (28) measured PSA-A2M in serum, using an A2M antibody-based PSA-A2M assay consisting of a capture antibody to PSA and a tracer antibody to A2M; they found that the serum concentrations were not related to the concentrations of total PSA. With an identical assay, we previously detected very low PSA-A2M immunoreactivity in male serum with high PSA concentrations (10). In the present study, we found that the apparent immunoreactivity detected by the A2M antibody-based PSA-A2M assay is caused by A2M because it was also observed with purified A2M and female serum. A similar background problem hampers assays for PSA-ACT (10). This suggests that the apparent immunoreactivity represents a nonspecific background caused by adsorption of A2M to the solid phase. This explanation is supported by the finding that <1% of the PSA-A2M in serum and that formed in vitro could be recovered by immunoadsorption when either mono- or polyclonal anti-PSA antibodies were used.
Otto et al. (27) demonstrated recently that PSA can bind to methylamine-transformed A2M without bait region cleavage, and the PSA-methylamine-transformed-A2M complex is detectable in a dual PSA antibody-based immunoassay. However, proteases are thought to complex with A2M in vivo mainly via bait region cleavage caused by the high concentration of native A2M in blood (29). This is supported by the observation that PSA-A2M occurring in vivo is not measurable by conventional PSA immunoassays without denaturation.
The ratio of PSA-A2M to total PSA in sera from BPH patients was higher in BPH than in PCa sera, which is contrary to the behavior of PSA-ACT (10)(13). This is probably explained by differences in the mechanism of complex formation of PSA with A2M and ACT, respectively, and in their clearance from circulation. PSA forms complexes more rapidly with A2M than with ACT, and even the proteolytically cleaved or "nicked" PSA isoenzymes can bind to A2M (8). A2M-protease complexes have half-lives of only 2–5 min (16), which are much shorter than that of total PSA, i.e., 2–3 days (30)(31). Although most of the enzymatically active PSA released into circulation may be expected to form complexes with A2M, PSA-ACT predominates in plasma because of its slow clearance (32). Because enzymatically active PSA is rapidly complexed with A2M (8), most of the free PSA present in blood at the time of sampling may be assumed to have low enzyme activity. It could consist of enzymatically inactive proenzyme and nicked isoenzymes that bind only with A2M (8). Nicked PSA could gradually form complexes with A2M after sampling. The high proportion of free PSA in serum from BPH patients could therefore lead to preferential formation of PSA-A2M after sampling. Thus, the concentration of PSA-A2M may reflect the concentration of free PSA at the time of sampling. This notion is actually supported by our preliminary results, which suggest that the sum of PSA-A2M and free PSA in serum may improve the diagnostic accuracy for PCa when compared with free PSA alone. Formation of PSA-A2M in vitro is also thought to cause loss of PSA immunoreactivity during long-term storage of serum (13)(24).
Measurement of the ratio of PSA-A2M to total PSA in serum improved the diagnostic accuracy compared with total PSA alone, as evidenced by ROC curve analysis. Thus, the immunoassay for PSA-A2M has the potential to improve the clinical usefulness of the PSA determination for detection of PCa. We are presently evaluating the clinical utility of the combination of PSA-A2M and free and total PSA by analyzing samples from a screening study.
In conclusion, we have developed a quantitative assay for PSA-A2M in serum that is based on immunoadsorption of PSA and PSA-ACT, denaturation of PSA-A2M at high pH, and determination of the PSA released by a conventional PSA immunoassay. Our first results with samples from referred patients indicate that the ratio of PSA-A2M in relation to total PSA in serum is higher in BPH than in PCa and that this can be used to improve the validity of the PSA assay for detection of PCa.
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Acknowledgments |
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Footnotes |
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2-macroglobulin; ACT, 1-antichymotrypsin; API, 1-protease inhibitor; PCa, prostate cancer; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; BPH, benign prostatic hyperplasia; BSA, bovine serum albumin; MAb, monoclonal antibody; IFMA, immunofluorometric assay; and TBS, Tris-buffered saline. 
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  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]
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 U. K. Misra, R. Deedwania, and S. V. Pizzo Binding of Activated {alpha}2-Macroglobulin to Its Cell Surface Receptor GRP78 in 1-LN Prostate Cancer Cells Regulates PAK-2-dependent Activation of LIMK J. Biol. Chem., July 15, 2005; 280(28): 26278 - 26286. [Abstract] [Full Text] [PDF]
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Y. Baumgart, A. Otto, A. Schafer, E. Usbeck, C. Cott, A. Schott, M. Tornack, A. Wenzel, A. Mossie, and G. Birkenmeier Characterization of Novel Monoclonal Antibodies for Prostate-Specific Antigen (PSA) with Potency to Recognize PSA Bound to {alpha}2-Macroglobulin Clin. Chem., January 1, 2005; 51(1): 84 - 92. [Abstract] [Full Text] [PDF]
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 C. A. Borgono, L. Grass, A. Soosaipillai, G. M. Yousef, C. D. Petraki, D. H. C. Howarth, S. Fracchioli, D. Katsaros, and E. P. Diamandis Human Kallikrein 14: A New Potential Biomarker for Ovarian and Breast Cancer Cancer Res., December 15, 2003; 63(24): 9032 - 9041. [Abstract] [Full Text] [PDF]
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S. Hutchinson, L.-Y. Luo, G. M. Yousef, A. Soosaipillai, and E. P. Diamandis Purification of Human Kallikrein 6 from Biological Fluids and Identification of its Complex with {alpha}1-Antichymotrypsin Clin. Chem., May 1, 2003; 49(5): 746 - 751. [Abstract] [Full Text] [PDF]
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C. Kapadia, A. Chang, G. Sotiropoulou, G. M. Yousef, L. Grass, A. Soosaipillai, X. Xing, D. H.C. Howarth, and E. P. Diamandis Human Kallikrein 13: Production and Purification of Recombinant Protein and Monoclonal and Polyclonal Antibodies, and Development of a Sensitive and Specific Immunofluorometric Assay Clin. Chem., January 1, 2003; 49(1): 77 - 86. [Abstract] [Full Text] [PDF]
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J. Leinonen, P. Wu, and U.-H. Stenman Epitope Mapping of Antibodies against Prostate-specific Antigen with Use of Peptide Libraries Clin. Chem., December 1, 2002; 48(12): 2208 - 2216. [Abstract] [Full Text] [PDF]
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T. Piironen, M. Nurmi, K. Irjala, O. Heinonen, H. Lilja, T. Lovgren, and K. Pettersson Measurement of Circulating Forms of Prostate-specific Antigen in Whole Blood Immediately after Venipuncture: Implications for Point-of-Care Testing Clin. Chem., April 1, 2001; 47(4): 703 - 711. [Abstract] [Full Text] [PDF]
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J. Peter, C. Unverzagt, T. N. Krogh, O. Vorm, and W. Hoesel Identification of Precursor Forms of Free Prostate-specific Antigen in Serum of Prostate Cancer Patients by Immunosorption and Mass Spectrometry Cancer Res., February 1, 2001; 61(3): 957 - 962. [Abstract] [Full Text]
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L.-Y. Luo, L. Grass, D. J.C. Howarth, P. Thibault, H. Ong, and E. P. Diamandis Immunofluorometric Assay of Human Kallikrein 10 and Its Identification in Biological Fluids and Tissues Clin. Chem., February 1, 2001; 47(2): 237 - 246. [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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U.-H. Stenman New Ultrasensitive Assays Facilitate Studies on the Role of Human Glandular Kallikrein (hK2) as a Marker for Prostatic Disease Clin. Chem., June 1, 1999; 45(6): 753 - 754. [Full Text] [PDF]
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,
PSA-A2M; 
, PSA; 
, PSA control.
, A2M measured with the
A2M IFMA after incubation with PSA; 
