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J Biomed Biotechnol. 2006; 2006: 56087. Published online 2006 August 8. doi: 10.1155/JBB/2006/56087. | PMCID: PMC1559920 |
Specific Immunoassays for Placental Alkaline Phosphatase As a Tumor Marker Sérvio T. Stinghen,1 Juliana F. Moura,1 Patrícia Zancanella,1 Giovanna A. Rodrigues,1 Mara A. Pianovski,2 Enzo Lalli,3 Dodie L. Arnold,4 João C. Minozzo,5 Luis G. Callefe,1 Raul C. Ribeiro,6 and Bonald C. Figueiredo1, 7* 1Centro de Genética Molecular e Pesquisa do Câncer em Crianças (CEGEMPAC), Rua Agostinho Leão Júnior, 400 Alto da Glória, Curitiba, PR, CEP 80030-110, Brazil 2Division of Pediatric Hematology and Oncology, Department of Pediatrics, Federal University of Paraná, Curitiba, PR, CEP 80060-000, Brazil 3Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 6097, 06560 Valbonne Sophia Antipolis, France 4Meharry Medical College, Nashville, TN 37208, USA 5Center for Research and Production of Immunoglobulins (CPPI), Rua Targino da Silva s/n, Piraquara, PR, CEP 83302-160, Brazil 6St. Jude Children's Research Hospital, Department of Hematology and Oncology and International Outreach Program, 332 North Lauderdale, Memphis, TN 38105, USA 7Research Institute Pelé Pequeno Príncipe (IPPP), Avenida Silva Jardim, 1632 Água Verda, Curitiba, PR, CEP 80250-200, Brazil Received December 23, 2005; Revised June 1, 2006; Accepted June 6, 2006. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Human placental (hPLAP) and germ cell (PLAP-like) alkaline
phosphatases are polymorphic and heat-stable enzymes. This study
was designed to develop specific immunoassays for quantifying
hPLAP and PLAP-like enzyme activity (EA) in sera of cancer
patients, pregnant women, or smokers. Polyclonal sheep anti-hPLAP
antibodies were purified by affinity chromatography with whole
hPLAP protein (ICA-PLAP assay) or a synthetic peptide (aa 57–71)
of hPLAP (ICA-PEP assay); the working range was 0.1–11 U/L
and cutoff value was 0.2 U/L EA for nonsmokers. The intra-
and interassay coefficients of variation were 3.7%–6.5%
(ICA-PLAP assay) and 9.0%–9.9% (ICA-PEP assay). An
insignificant cross-reactivity was noted for high levels of
unheated intestinal alkaline phosphatase in ICA-PEP assay. A
positive correlation between the regression of tumor size and EA
was noted in a child with embryonal carcinoma. It can be concluded
that ICA-PEP assay is more specific than ICA-PLAP, which is still
useful to detect other PLAP/PLAP-like phenotypes. Human alkaline phosphatases (ALP) are dimeric enzymes anchored by
glycosylphosphatidylinositol to the cell membrane. Specific
phospholipases can cleave ALP from the cell membrane, producing
free ALP in the serum [ 1– 3]. ALP consists of a family of four
isozymes: (1) placental ALP (PLAP; or hPLAP in the case of human
placental ALP), and (2) germ cell ALP (PLAP-like or GCAP), which
are both stable at 65°C and present 98% homology; as well as (3) intestinal ALP (IAP),
and (4) nonspecific tissue ALP (TNAP) or liver/bone/kidney ALP,
which are not stable at 65°C and present 88% and
56% homology with PLAP, respectively [ 4– 6]. PLAP is encoded by a highly polymorphic gene of which 3 common
alleles and at least 18 rare alleles exist; giving origin to
48 known phenotypes [ 7– 9]. The PLAP-like enzyme is encoded
by 4 alleles with at least 10 known phenotypes [ 9– 12].
PLAP and PLAP-like enzymes may form heterodimers [ 13]. PLAP is synthesized in the placental syncytiotrophoblast starting
after the 12th week of pregnancy [ 14] and is probably
involved in transplacental IgG transport [ 15, 16]. The
PLAP-like enzyme is primarily synthesized in testis, cervix, and
thymus. Trace amounts are synthesized in placenta and lung tissues
[ 17, 18]. In healthy, nonsmoking adults PLAP and PLAP-like
enzyme activities represent less than 1% of all ALP
[ 19, 20]. Smoking causes elevated serum concentrations of the
PLAP-like enzyme, which may diminish the value of PLAP-like enzyme
as a tumor marker. Serum concentrations of PLAP-like enzyme return
to the normal range after 1-2 months of smoking cessation
[ 21– 23]. Ectopic expression of PLAP is associated with cancer of ovary,
testis, lung, and colorectal tract [ 14]. Ectopic expression
of the PLAP-like enzyme is associated with testicular cancers:
intratubular germ cell neoplasia, unclassified (IGCNU), seminoma,
embryonal carcinoma, and choriocarcinoma [ 23– 25]. Roelofs
et al [ 26] using RT-PCR have shown that seminoma and IGCNU
express predominantly the PLAP-like enzyme, while embryonal
carcinomas express variable amounts of PLAP and PLAP-like enzymes. Due to their high homology, PLAP and PLAP-like enzymes cannot be
easily distinguished using polyclonal or monoclonal antibodies.
However, the specific monoclonal antibodies C2 and 17E3 may
recognize only PLAP [ 27, 28]. In general, the enzyme activity
is usually considered to include both PLAP and PLAP-like. Elevated serum concentrations of PLAP and PLAP-like enzymes were
found in 25%–65.5% of patients with ovarian cancer
[ 19, 29– 35], and 22%–89% of patients
with testicular neoplasms [ 23, 36– 42]. Variable
values of PLAP and PLAP-like enzyme activity or serum
concentrations may depend upon the method and antibody used for
detection, as well as upon cancer type or staging. Seminomas can
have a similar molecular profile to embryonal carcinoma;
explaining aberrant immune profiles [ 43]. Testing of serum samples from ovarian cancer patients demonstrated
that testing for PLAP/PLAP-like enzyme activity is less sensitive
than testing for CA 125 (71%–85%) and alpha-fetoprotein
(AFP) (88%) [ 44]; however, testing for PLAP/PLAP-like
enzyme activity was considered to be the best survival indicator
[ 35], and more specific (PLAP/PLAP-like; 95%) than CA 125
(71%) [ 31]. Weissbach et al [ 23] have demonstrated
that serum PLAP/PLAP-like was more sensitive (56%) than other
tumor markers, such as HCG (35%) and LDH (34%), in
the evaluation of seminoma patients. Polyclonal antibodies against PLAP and PLAP-like enzymes may
cross-react with IAP isozyme. In contrast, monoclonal antibodies
may not detect all ALP phenotypes if the epitope is absent (eg,
because of polymorphism, or if it has been cleaved by
phospholipases) [ 45, 46]. To eliminate both of these latter problems, this research was
designed to develop and test two sensitive and specific
immunoassays for PLAP and PLAP-like enzyme activities using two
sheep anti-hPLAP polyclonal antibodies purified by affinity
chromatography. Animal immunization One adult sheep was immunized with hPLAP (Sigma-Aldrich, St Louis,
Mo, USA) through seven subcutaneous injections at 14-day
intervals. The emulsion for the first injection was prepared using
2 mg of hPLAP dissolved in 1 mL of 0.05 M phosphate
buffered saline (PBS; pH 7.4) and 1 mL of Freund's complete
adjuvant (Sigma-Aldrich, St Louis, Mo, USA). The remaining 6
injections were prepared using Freund's incomplete adjuvant. Optimal animal immunization was indicated by high serum
concentration of anti-hPLAP. This was measured by two different
protocols: double immunodiffusion test and indirect ELISA. Animal
sera were obtained (0.8 L) and immunoglobulins were
precipitated in a saturated ammonium sulfate solution. Purification of anti-hPLAP antibodies Specific anti-hPLAP antibodies were purified through an affinity
chromatography column. One gram of cyanogen bro-mide-activated
Sepharose (CNBr-Sepharose; Sigma-Aldrich, St Louis, Mo, USA) was
coupled to 20 mg of hPLAP according to previously described
protocols [ 47]. Aliquots of sheep immunoglobulins were dissolved in PBS and
circulated through the column at a flow rate of 20 mL/h
overnight at 4°C. Afterwards, the column was washed with
PBS until absorbance (280 nm) of the eluted solution had
returned to baseline. Recovery of the immuno-globulins bound to the
hPLAP-Sepharose column was performed by washing the column with
0.1 M glycine-HCL, 0.15 M NaCl, pH 2.8, until
an immunoglobulin peak was obtained. Finally, the column was
washed with PBS until the absorbance returned to baseline. The
eluted solution containing anti-hPLAP antibodies was dialyzed
overnight at 4°C in PBS. Purification of anti-amino acid 57–71 peptide A synthetic peptide extending from amino acid (aa) residue 57 to
71 of hPLAP, made by a technique reported by Kates and Albericio
[ 48], was generously synthesized and provided by the
Biophysics Laboratory from UNIFESP (São Paulo, SP). Selected
peptide aa 57–71 of hPLAP differs in 2 residues from PLAP-like, 3
residues from IAP, and 9 residues from TNAP [ 6]. This peptide
sequence was chosen because it contains free epitope(s) in an
unfolded loop and accessible to antibodies. The synthetic peptide (22.9 mg) was immobilized to
1.0 g of CNBr-Sepharose according to previously described
methods [ 47]. Aliquots containing purified anti-hPLAP
antibodies were circulated through the Sepharose-aa 57–71 peptide
column at a flow rate of 20 mL/h. Unbound anti-hPLAP
antibodies (PLAP-Ab) that did not recognize the aa 57–71 peptide
were eluted and separated to prepare the first immunoassay, which
was named ICA-PLAP. Anti-aa 57–71 peptide antibodies (PEP-Ab) were
eluted and collected as described above. These antibodies were
separated to prepare the second immunoassay, called ICA-PEP. Standard curve Purified hPLAP (11 U/mg) (Sigma-Aldrich, St Louis, MO, USA),
3 g/L in distilled water, was used as a control, diluted in
0.25% casein, 0.05% Tween 20, and PBS (dilution buffer)
to obtain hPLAP activities ranging from 0.17–11 U/L.
Dilution buffer was used as a zero standard solution. Specificity The purified IAP (3 U/mg) (Calzyme, San Luis Obispo, Calif, USA),
1 g/L in distilled water, was used to determine
cross-reactivity. It was diluted in dilution buffer at
concentrations of 0.05–55 U/L. IAP samples were tested with
and without heat treatment at 65°C for 10 minutes. Serum samples Subjects (smokers and nonsmokers) This study was approved
by the Ethics Committee at the Hospital of Clinics of the Federal
University of Paraná, and samples from serum or tissue were
obtained after consent from patients or the patients' parents.
Control serum specimens were obtained from 100 healthy adults: 93
serum samples from blood donors representing negative controls
(19–57 years of age; 39 females and 54 males; 44 nonsmokers and
49 smokers), and 7 serum samples from 7 pregnant women
representing positive controls (24–35 years of age; at 12–37
weeks of gestation). Social smokers (1-2 cigarettes per week) or
subjects who smoke less than 1 cigarette per day were not included
in this study. A child with embryonal carcinoma (case report) An 11.2-year-old girl was admitted to the hospital complaining of
abdominal pain for the last 2 months. On admission she presented
with an enlarged abdominal volume due to a palpable mass in the
lower third of the abdomen. Her height was 141 cm and body
weight was 26 kg; 5 kg less than 2 months before
admission. There was no record of cancer among her relatives. A CT
scan revealed an 11 × 12 × 16 cm extragonadal mass
located between the bladder and the rectum, extending up to the
mesogastrium, at the level of L4. Histological evaluation and
immunohistochemistry of tumor specimens obtained from the biopsy
were positive for CKAE1/AE3 (multifocal) and Ki67 (90%), and
was negative for AFP, LCA, and CD30 markers, suggesting it to be
an embryonal carcinoma at Stage III. Serum samples for
PLAP/PLAP-like enzyme activity were collected before, during, and
after chemotherapy. Placental analysis (positive control) A tissue sample from a term placenta was obtained and used as a
positive control. All samples (sera and tissue) were stored at
−80°C until assay. Soluble tissue extracts were obtained
as previously described [ 21]. Immunoassays: ICA-PLAP and ICA-PEP A 96-well Nunc MaxiSorp microplate (Nalge Nunc International,
Roskilde, Denmark) was coated overnight at 4°C with
100 μL of a 1.6 μg/mL solution of anti-hPLAP
antibodies (ICA-PLAP assay), or an 18.4 μg/mL solution
of anti-aa 57–71 peptide antibodies (ICA-PEP assay) in
0.05 M carbonate buffer, pH 9.6. Afterwards, the wells
were washed twice with wash buffer (0.05% Tween 20 in
saline). Each well was filled with 100 μL of blocking
buffer (2% casein in PBS) and the plate was incubated for 1
hour at 37°C. After washing twice with wash buffer,
100 μL of a standard solution containing hPLAP
(0–11 U/L, range of the standard curve) in dilution buffer
(0.25% casein, 0.05% Tween 20, PBS), and 100 μL
of experimental serum sample, or of homogenized tissue
supernatant, were added to the wells. After incubation
for 1 hour at 37°C, the plate was washed 5 times with
wash buffer and 100 μL p-nitrophenyl phosphate substrate
was added. After incubation for 5 hours (ICA-PLAP assay) or
overnight (ICA-PEP assay) at 37°C, the enzymatic reaction
was stopped through the addition of 50 μL of 3 M
NaOH. The absorbance (405/620 nm) was measured using a
microplate reader (Spectra, Tecan, Zurich, Switzerland). All
standards and samples were tested in duplicate. The positive
control for all assays was a pool of sera taken from 5 pregnant
women and 1 placental extract. Positive samples were retested
after heat treatment at 65°C for 10 minutes in order to
inactivate IAP. Production, purification, and titration of antibodies Serum from hPLAP-immunized sheep was tested by immunodiffusion in
the presence of hPLAP (1 mg/mL), and the results were positive
up to 1 : 2 dilutions. After treatment the animal was given one
extra injection of hPLAP. Serum titers were reanalyzed by indirect
ELISA 2 weeks later, when adequate immunization was revealed by
titers of 1 : 256000. Purified polyclonal antibodies by Sepharose-hPLAP had a final
concentration of 1.6 g/L, whereas purified polyclonal
antibodies by Sepharose-aa 57–71 peptide had a final
concentration of 0.92 g/L. Immunoassay characteristics The standard curve was linear between 0.1 U/L and 11 U/L
(), and the detection limit (defined as the
mean +4 SD of the absorbance of the zero standard for ALP) was
0.1 U/L, for both ICA-PLAP and ICA-PEP assays. The intra- and interassay variability (n = 12) was assessed by
measuring 2 pregnant serum pools with a mean PLAP activity of
5.5 and 0.75 U/L for ICA-PLAP, and 4.8 and
0.98 U/L for ICA-PEP. The mean intra- and interassay CV were
6.5% and 9.9%, respectively, for the ICA-PLAP assay, and
3.7% and 9%, respectively, for the ICA-PEP assay. Cross-reactions in the ICA-PLAP assay were noted at IAP
concentrations of 55 U/L and 27.5 U/L, exhibiting
activity of 1.22 U/L and 0.75 U/L, respectively. At
IAP concentrations 55 U/L, the activity for the ICA-PEP assay
was 0.4 U/L. No cross-reactivity was noted for heated IAP in
either assay; showing that IAP is very sensitive to high
temperature. Results from serum blood donors Average hPLAP/PLAP-like activity for nonsmoker blood donors was
0.06 ± 0.12 U/L (mean ± SD), using the ICA-PLAP
assay, and 0.01 ± 0.04 U/L (mean ± SD) with the
ICA-PEP assay. Smokers had hPLAP/PLAP-like enzyme
activities ranging from 0-1.72 U/L, with a mean
activity of 0.36 ± 0.41 U/L (mean ± SD), as
determined by the ICA-PLAP assay, and activities from
0-1.65 U/L, with a mean activity of 0.25 ± 0.36 U/L
(mean ± SD), using the ICA-PEP assay.
shows the distribution of serum hPLAP/PLAP-like activities from
blood donors measured by the ICA-PLAP and ICA-PEP
assays. The correlation between the ICA-PLAP and ICA-PEP results in blood
donors was high (n = 93, r = 0.94), supporting a rationale for
the use of both assays. Heat inactivation did not interfere with
hPLAP/PLAP-like activity for nonsmoker or smoker blood donors in
either assay. The hPLAP/PLAP-like enzyme activity cutoff value—defined as the
90th percentile for the ICA-PLAP assay and the 100th
percentile for the ICA-PEP assay—for nonsmokers was
0.2 U/L for both assays. Based on the present data, we
propose a normal hPLAP/PLAP-like enzyme activity cutoff value
ranging from 0–0.4 U/L for smokers, but further analyses
are necessary to confirm this threshold. Pregnant sera results Serum samples from 7 pregnant patients were measured without
heating (NI) and after heat inactivation (I). hPLAP activities
ranged from 0.1–53 U/L. The pregnant patient at the 12th
week of gestation had very low hPLAP activity. The pregnant
patient at the 18th week of gestation had low hPLAP activity
(1.58–2.5 U/L). Patients from the 27th week to the 37th
week of gestation had hPLAP activities that ranged from
31–53 U/L ( Table 1). There were no differences
identified in hPLAP activities when comparing inactivated and
noninactivated samples using either the ICA-PLAP or ICA-PEP
assays. | Table 1hPLAP activities in noninactivated (NI) and
heat-inactivated (I) pregnant serum measured by ICA-PLAP and
ICA-PEP. |
Case report The 11.2-year-old patient with ovary embryonal carcinoma was
admitted with a visible and palpable abdominal mass. Before
chemotherapy (4 cycles of Ifosfamide and cisplatin, at intervals
of 21 days), NI and I serum samples had hPLAP/PLAP-like enzyme
activity of 9.5 U/L and 10.2 U/L, respectively, in the
ICA-PLAP assay, and 9.3 U/L (NI and I serum) in the ICA-PEP
assay. On the 7th day after first cycle of chemotherapy the mass
was palpable but not visible anymore, and hPLAP/PLAP-like activity
in serum was 5.7 U/L (NI) and 5.6 U/L (I),
respectively, in the ICA-PLAP assay, and 5.7 U/L (NI) and
5.3 U/L (I) in the ICA-PEP assay. On the 14th day after
first cycle of chemotherapy the abdominal mass was not palpable
anymore and hPLAP/PLAP-like activity in serum was 0.23 U/L
(NI) and 0.25 U/L (I), respectively, in the ICA-PLAP assay,
and 0.25 U/L (NI and I) in the ICA-PEP assay. Forty-two days
after initiating chemotherapy a CT scan showed evidence of
complete tumor regression, and the hPLAP/PLAP-like enzyme activity
in serum was stable; exhibiting 0.22 U/L (NI) and
0.20 U/L (I), respectively, for ICA-PLAP, and 0.23 U/L
(NI) and 0.22 U/L (I) for ICA-PEP. hPLAP/PLAP-like enzyme
activity in serum was undetectable, 210 days following initiation
of chemotherapy (). After 4 cycles of
chemotherapy (ifosfamide/CDDP/VP16) complete tumor regression was
confirmed using imaging analysis, exploratory surgery, and
histological analysis. Immunoassays for hPLAP using monoclonal or polyclonal antibodies
generally do not distinguish PLAP from PLAP-like enzymes because
of the high homology between these isoforms [ 6]. Polyclonal
antibodies raised against PLAP may cross-react with IAP
[ 29, 49] but this can be minimized by serum heat inactivation.
Monoclonal antibodies may not detect hPLAP/PLAP-like enzyme
activity if the epitope is not present in the polymorphic protein
[ 7, 45, 46]. The hPLAP enzyme is more polymorphic (at least 48
phenotypes) than the PLAP-like enzyme (10 phenotypes)
[ 8, 9, 11, 12]. Two cost-effective, specific, and sensitive immunoassays were
developed for determining hPLAP/PLAP-like enzyme activity. Two
polyclonal antibodies were selected by affinity chromatography;
one PLAP-Ab that is capable of recognizing most PLAP/PLAP-like
epitopes that are not recognized by monoclonal antibodies, and a
second antibody (PEP-Ab) used in the ICA-PEP that recognizes the
aa 57–71 sequence of hPLAP. PLAP/PLAP-like activity determined by
PLAP-Ab was, as expected, inferior to PEP-Ab, suggesting that the
second antibody may present monoclonal characteristics. A
substitution of 1 amino acid in an antigen may modify the
antibody-binding activities [ 28]. The synthetic peptide used
in this study differs in 3 amino acids from IAP and 9 amino acids
from TNAP [ 6], and had 15 amino acid residues (the minimum
necessary to build an epitope that may comprise 15–22 amino acid
residues [ 50]); suggesting that PEP-Ab recognizes PLAP
specific epitope(s). The sensitivity for both ICA-PLAP and ICA-PEP assays was
0.1 U/L; similar to observations in studies that used
anti-hPLAP monoclonal antibodies [ 31, 51]. Cross- reactivity
was observed for high levels of IAP (27.5 U/L and
55 U/L) in the ICA-PLAP and ICA-PEP assays, respectively. This
interference could be abolished when IAP was inactivated by
heating. The ICA-PLAP and ICA-PEP assays were highly correlated
( r = 0.94). The ICA-PLAP assay is less specific, but more
cost-effective than ICA-PEP. Although ICA-PEP was capable of
recognizing similar PLAP/PLAP-like activity in heated and unheated
samples, ICA-PLAP would reveal other PLAP/PLAP-like phenotypes in
unheated samples. Taken together, a combined assay using each of
the antibodies may minimize the disadvantage of using monoclonal
and polyclonal antibodies. For instance, hPLAP/PLAP-like activity
in unheated serum samples from 5 nonsmokers was found slightly
above (≤ 0.6 U/L) cutoff value (0.2 U/L) only in
the ICA-PLAP assay, suggesting that these findings were due to
cross-reaction with IAP; as described in other studies
[ 29, 49]. IAP activity in serum is related to diet and blood
group status [ 52]. Our data show that the normal serum value for PLAP/PLAP-like enzyme activity in adult nonsmokers is around
0.2 U/L. A normal Gaussian distribution of hPLAP/PLAP-like
enzyme activity was not observed in serum of healthy individuals
who smoked. Such heterogeneity among these individuals and
the resulting high standard deviation in average values
have been reported in previous studies [ 39, 51]. In the
literature, PLAP activity in lung tissue was reported to
correspond to PLAP activity in placenta, while PLAP in serum of
smokers was reported as PLAP-like activity [ 21, 51]; this
discrepancy is not well understood. The proposed cutoff
corresponding to hPLAP/PLAP-like findings in serum of smokers
without cancer in this study was 0.4 U/L, however, this must
be better evaluated with serial sampling and while considering the
clinical status of the individual. PLAP/PLAP-like enzyme activity
measured in serum of smokers probably originates from the lungs
(pneumocytes), where cellular damage caused by cigarette smoke may
release PLAP-like enzymes into the blood [ 21, 39], possibly in
proportion to the duration and intensity of smoking [ 39, 53]. PLAP activity in serum samples from pregnant patients measured by
ICA-PLAP and ICA-PEP assays showed no difference between
heat-inactivated and noninactivated samples, indicating that the
circulating protein is mostly PLAP released by the placenta. As
expected, hPLAP activity increased linearly with the advance of
pregnancy, as the placental syncytiotrophoblast produces high PLAP
levels during the 27th to 37th weeks of gestation. This
relationship suggests that the monitoring of hPLAP activity could
be used as an additional tool to evaluate placenta viability. Since PLAP/PLAP-like activity could be detected at very low levels
in early stages of pregnancy and in smoking individuals, the
developed assays could also be used to detect PLAP/PLAP-like
activity in the serum of cancer patients. In fact, this
immunoassay was possible and extremely useful in the follow-up of
a young female patient with embryonal carcinoma.
Following the first cycle of chemotherapy, PLAP/PLAP-like serum
activity decreased in proportion to the size of tumor. This
suggests that the immunoassay of PLAP/PLAP-like serum activity may
be a good marker for monitoring response to chemotherapy or eventual cancer recurrence, minimizing the need for
exhaustive imaging analyses. To further validate PLAP/PLAP-like
serum activity as a tumor marker, more serum samples from patients
with cancer are necessary. As reported by other authors, increased
levels of PLAP/PLAP-like were observed in 2 patients with
recurrent testicular seminoma 2 months before detection by CT scan
[ 42]. The longest interval found between increased levels of
PLAP/PLAP-like and posterior cancer detection by imaging analysis
was 2 years in a woman with a very small ovarian cancer [ 54].
Determination of PLAP/PLAP-like activity using immunoassays could
be particularly useful for serum of patients with testicular
cancer and ovarian carcinomas, as is the case for more sensitive
markers such as CA 125 and AFP. In fact, PLAP/PLAP-like activity
seems to be more specific than CA 125 and AFP [ 31]. HCG and
AFP are used as tumor markers for nonseminomatous testicular
cancer, but PLAP/PLAP-like activity is the best tumor marker for
the follow-up of patients with seminomas [ 39]. The ICA-PLAP and ICA-PEP immunoassays could be explored to detect
hPLAP/PLAP-like activity above 0.2 U/L (cutoff value in
adults) in physiological and pathological conditions, especially
in patients with embryonal cancer or other types of cancer
reported in other studies (testis, ovary, lung, and colorectal
tract). Furthermore, it can be concluded that ICA-PEP assay is
more specific than ICA-PLAP, which is still useful to detect other
PLAP/PLAP-like phenotypes that eventually may not be detected by
ICA-PEP. Heating inactivation of samples may eliminate IAP and
other alkaline phosphatase activity and eventually also decrease
PLAP/PLAP-like activity, but this unwished effect could be
confirmed by testing unheated samples in the ICA-PEP
assay. This study was supported by Fundação Araucária-PR
(Grant #2648) and CAPES-COFECUB. Additional funding came from
the American Lebanese Syrian Associated Charities (ALSAC),
Memphis, Tenn, and from the NIH/NCMHD Grant 9T37MD001378-04
(MHIRT). We are grateful to Dr Luiz Juliano Neto, from Biophysics
Laboratory of UNIFESP (São Paulo, Brazil), for providing
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