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Articles |
1
Departments of Clinical Biochemistry and Immunology, Statens Seruminstitut, Artillerivej 5, DK-2300 S, Copenhagen, Denmark.
2
Department of Molecular Biology, University of Arhus,
Arhus, Denmark.
3
Department of Biotechnology, University of Turku, Turku,
Finland.
a Author for correspondence. Fax +45 32 68 38 78; e-mail qiqin{at}utu.fi
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Maternal serum screening is a method for assessing the risk for a DS
fetus according to maternal age and serum concentrations of
feto-placental proteins or hormones. At present, screening programs
that utilize age in combination with the measurements of maternal serum
human chorionic gonadotropin (hCG) and 
-fetoprotein, with or without
unconjugated estriol, in the second trimester of pregnancy are
routinely carried out in antenatal care units in many countries
(7). This approach detects ~65–75% of Down-affected
pregnancies, with a false-positive rate of ~5%
(7)(8). Serum screening for DS before week 15
of pregnancy is still at the experimental stage. However, several
markers such as pregnancy-associated plasma protein A (PAPP-A)
(8)(9)(10)(11)(12)(13), free ß-hCG (8)(9)(13),
dimeric inhibin (13)(14), pregnancy-specific
glycoprotein 1 (Schwangerschaftsprotein 1; SP1)
(11)(15)(16), and unconjugated
estriol (13)(17) have been shown to be useful
in maternal serum screening for DS in the first trimester. PAPP-A seems
to be one of the most promising markers
(13)(18).
PAPP-A is a placenta-derived glycoprotein present in circulation at term as a covalent complex with equimolar amounts of the proform of eosinophil major basic protein (proMBP), which is also synthesized in the placenta during pregnancy (19). In healthy pregnancies, the PAPP-A concentration in maternal serum increases with gestational age until delivery. However, a noticeably reduced concentration of maternal serum PAPP-A in the first trimester has recently been found to be associated with fetal DS (8)(9)(10)(11)(12)(13). Consequently, measurement of serum PAPP-A may be useful in first-trimester maternal serum screening for DS.
Currently, PAPP-A concentrations in sera are measured by isotopic and nonisotopic immunoassays that use polyclonal antibodies, either raised in-house (20)(21) or commercially (A230, from Dako A/S) (8)(11)(12)(22)(23). Unfortunately, these polyclonal antisera are not specific for PAPP-A. First, the polyclonal anti-PAPP-A antibodies raised thus far are, in fact, anti-PAPP-A/proMBP antibodies, because PAPP-A is isolated from serum as the PAPP-A/proMBP complex. Importantly, proMBP antigen is present in excess of PAPP-A/proMBP complex, thus reflecting the presence of non-PAPP-A-containing proMBP complexes—complexes that do not exist in a constant molar ratio to PAPP-A/proMBP complexes (24). Second, polyclonal antiserum has also been shown to react with haptoglobin (25) and SP1 (22). Therefore, the concentrations of PAPP-A measured with use of these antibodies may be severely biased by cross-reactivity.
Here we describe four double-monoclonal time-resolved immunofluorometric assays (TrIFMAs) developed for PAPP-A/proMBP complex determination, based on newly produced monoclonal antibodies (26). We present an evaluation of the performance of these serum assays in first-trimester discrimination between normal pregnancies and DS-affected pregnancies and compare the results with that of a polyclonal version of TrIFMA for PAPP-A (12). Furthermore, serum concentrations of the complex in nonpregnant women and in men are reported.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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reagents
Monoclonal antibodies.
The monoclonal antibodies used in
this study were Hyb234–2, Hyb234–3, Hyb234–4, Hyb234–5, and
Hyb234–6, all raised against PAPP-A/proMBP complex purified from term
serum and all type
IgG1.2
Under both native and denatured, reduced conditions, Western
blots have demonstrated that these antibodies react with the PAPP-A
part of the PAPP-A/proMBP complex and not the proMBP part
(26).
Calibrators and controls.
Calibrators were made from a
40-week-pregnancy serum pool diluted in dilution buffer [10 mmol/L
phosphate buffer, 150 mmol/L NaCl, 2.5 g/L bovine
-globulin (G 5009; Sigma Chemical Co.), and 10 g/L bovine serum
albumin (Sigma A 4503)] and calibrated against WHO IRP 78/610 for
pregnancy-associated proteins (WHO International Laboratory for
Biological Standards, Statens Seruminstitut, Copenhagen, Denmark). The
contents of the ampoule were dissolved in 750 µL of distilled water
to give a concentration of 0.1 IU/mL PAPP-A as defined. Three control
samples, representing low, medium, and high values of PAPP-A, were
prepared from a first-trimester delipidated serum pool (for the low
value) and a second-trimester delipidated serum pool (for the medium
and high values). Dilution buffer was used as the zero calibrator.
Eu
3+-labeled monoclonal
antibodies. Each monoclonal antibody (500 µg) was transferred to
labeling buffer (50 mmol/L NaHCO3, 150 mmol/L NaCl, pH 8.5)
by passage through a NAP-5TM column (Pharmacia) Then 100
µL of a 2 mg/mL solution of the Eu3+-chelate of
N1-(p-isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic
acid (Wallac Oy) in distilled water was added to the antibody solution
and the mixture was left in the dark at room temperature for 24 h.
A 100-fold molar excess of Eu3+-chelate was used in the
labeling reaction. Labeled monoclonal antibody was separated from free
Eu3+-chelate by gel filtration on a PD-10 column
(Pharmacia). The labeled monoclonal antibodies contained 7–15
Eu3+ molecules per IgG molecule.
Biotinylated monoclonal antibodies.
Monoclonal
antibodies to be biotinylated were dissolved in 50 mmol/L phosphate
buffer containing 150 mmol/L NaCl (pH 7.4) and the protein
concentration was adjusted to 1 mg/mL. Next, 20 µL of 10 mmol/L
biotin isothiocyanate (Wallac Oy) in dimethylformamide and 42 µL of
0.5 mol/L sodium carbonate buffer (pH 9.8) were added to 400 µL of
monoclonal antibody solution. The mixture was kept at room temperature
for 2 h without shaking. Biotinylated monoclonal antibodies were
separated from the chemical reactants by consecutive passage through
columns of NAP-5 and NAP-10TM (Pharmacia). The
concentrations of biotinylated monoclonal antibodies were calculated
from the absorbance at 280 nm.
procedures
Monoclonal antibody assays.
Maxisorp polystyrene
microtiter plates (Nunc) were coated with 1 µg of streptavidin (Zymed
Labs.) in 100 µL of 0.1 mol/L citric phosphate buffer (35 mmol/L
citric acid, 67 mmol/L disodium phosphate, pH 5.0) per well overnight
at room temperature. Plates were washed twice with washing buffer [5
mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05 g/L Tween 20, and 1 g/L Germall
II (Wallac Oy), pH 7.5], and blocked with 200 µL of dilution buffer
for 2 h at room temperature with slow shaking. After aspiration
and washing, the plates were covered with adhesive tape and stored at
4 °C until use. The coated plates were stable at 4 °C for at
least 1 month.
To each well of the streptavidin-coated microtiter plates was added 200 ng of biotinylated 234–3 (for TrIFMA 234–3/234–2) or 400 ng of biotinylated 234–4 (for TrIFMA 234–4/234–2 and TrIFMA 234–4/234–5) or 400 ng of biotinylated 234–5 (for TrIFMA 234–5/234–6).3 All biotinylated monoclonal antibodies were diluted in assay buffer (Wallac Oy). After 1.5 h of incubation at room temperature, the plates were washed with washing buffer. Then 100 µL of either calibrators, ranging from 3.9 to 1000 mIU/L (prepared by serial twofold dilution), or first-trimester serum samples diluted 10-fold with dilution buffer was added to each well in duplicate. Incubations were performed at room temperature for 2 h (for versions of TrIFMA 234–3/234–2 and TrIFMA 234–5/234–6) or for 3 h (for versions of TrIFMA 234–4/234–2 and TrIFMA 234–4/234–5). The plates were washed twice and incubated with 100 ng of Eu3+-labeled second monoclonal antibody (234–2, 234–5, or 234–6) in 100 µL of assay buffer per well at room temperature for 1 h (234–2), 2 h (234–5), or 3 h (234–6), with slow shaking. After the wells were washed 6 times, 200 µL of enhancement solution (Wallac Oy) was added to each well, and after 10 min the time-resolved fluorescence was counted in a Wallac 1232 Arcus DELFIA fluorometer. Data were analyzed by the Multicalc® software program (Wallac Oy), with use of a spline algorithm on logarithmically transformed data.
Polyclonal TrIFMA for PAPP-A.
This assay was performed
as described in detail elsewhere (12). Briefly, 100 µL
of serum samples diluted 10-fold in dilution buffer was added to each
well of microtiter plates that had been coated with polyclonal antibody
A230 (Dako; lot 025). The calibrators were the same preparations as
used in the monoclonal TrIFMAs. The calibrators and samples were
incubated at room temperature for 3 h with slow shaking. After
washing twice, 100 ng of Eu3+-labeled A230 was dispensed
into each well. The plates were incubated at 4 °C overnight, then
transferred to room temperature for 30 min and washed 6 times. After
addition of 200 µL of enhancement solution (Wallac Oy) and 10 min of
slow shaking, the time-resolved fluorescence was measured at 613 nm
with the Wallac 1232 Arcus fluorometer; the data were analyzed with a
spline algorithm on logarithmically transformed data.
Gel filtration.
The gel-filtration studies were carried
out on a 1.6 cm x 49 cm column of Sephacryl HR-400 (Pharmacia).
Elution buffer was 50 mmol/L phosphate buffer, 150 mmol/L NaCl, pH 7.2.
The column was operated at room temperature with a flow rate of 1.0
mL/min, and 1.0-mL fractions were collected. A sample of 0.5 mL of a
9th week pregnancy serum pool was loaded on the column. For molecular
mass estimation the column was calibrated with catalase (232 kDa),
ferritin (440 kDa), and thyroglobulin (669 kDa), all from Pharmacia
Biotech. PAPP-A concentrations in the fractions were measured by both
the monoclonal TrIFMAs and the polyclonal TrIFMA.
statistics
Groups were compared by Mann–Whitney U-test or matched
signed rank test. The observed (empirical) median values for PAPP-A
serum concentrations were determined from the control samples in each
of the gestational weeks 4–13. The observed medians, in combination
with the number of samples at each gestational week, were subsequently
log-linearly regressed to produce the number-weighted, log-linear
regressed medians of unaffected samples. Multiples of the medians
(MoMs) were calculated by using regressed medians for both unaffected
samples and DS-affected samples. Test accuracy was analyzed by
receiver-operator characteristics (ROC) curves (27). Area
calculation and comparison of ROC curves was performed by using
GraphROC program for Windows (vers. 2.0; Veli Kairisto and Allan Poola,
http://WWW.netti.fi/~ maxiw).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The working range of assays was from 3.9 to 1000 mIU/L for
assays 234–4/234–2 and 234–5/234–6, from 1.95 to 1000 mIU/L for
assay 234–3/234–2, and from 0.95 to 1000 mIU/L for assay
234–4/234–5—the range being defined as the concentrations where the
intraassay CVs were <10%.
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Sensitivity (detection limit).
Sensitivity was estimated
by two methods: by calculating the concentration of PAPP-A giving a
signal equivalent to that for the 0 calibrator + 2SD (theoretical
sensitivity); and by calculating the lowest concentration giving
acceptable intraassay variation (CV <10%; functional sensitivity).
Table 1
shows these data for the four versions of TrIFMAs.
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Reproducibility.
Intra- and interassay variation was
examined by analyzing 6 serum samples with low, medium, and high
concentrations of PAPP-A. Results are shown in Table 2
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Parallelism.
Parallelism of the 4 versions of TrIFMAs
was examined on 3 serum samples with low, medium, and high PAPP-A
content. Agreement between the expected values and the measured values
was good (data not shown).
Recovery.
Recovery was examined by analyzing serum
samples containing added PAPP-A calibrators. As shown in Table 3
the analytical recoveries of the 4 monoclonal assays were
satisfactory.
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comparisons of five assays for papp-a/prombp complex
There was an excellent linear correlation between each of
monoclonal assays (r >96%) and between the monoclonal and
polyclonal assays (r >90%). Difference plots show that
PAPP-A/proMBP complex concentrations determined by TrIFMA
234–3/234–2 and TrIFMA 234–4/234–2 are very similar to each
other, as are those determined by TrIFMA 234–4/234–5 and TrIFMA
234–5/234–6. However, as shown in Fig. 2
, differences were seen, especially in the samples from early
pregnancy, between the concentrations obtained by PcAb/PcAb vs those
by TrIFMA 234–3/234–2 and between those by TrIFMA 234–4/234–5 vs
TrIFMA 234–4/234–2.
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gel filtration
As shown in Fig. 3
, PAPP-A/proMBP immunoreactivity eluted as a symmetrical peak,
slightly ahead of thyroglobulin. The elution position was that of a
globular protein with Mr of ~800 000. The
monoclonal combinations 234–3/234–2 and 234–4/234–2 gave
concentrations similar to those measured by the polyclonal TrIFMA,
whereas the combinations 234–4/234–5 and 234–5/234–6 gave much
higher values, even though the peak had the same elution position as
that seen with the polyclonal TrIFMA.
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papp-a concentrations measured
In healthy blood donors.
PAPP-A could be detected by all
4 versions of monoclonal TrIFMAs in serum from nonpregnant women and
men. After logarithmic transformation, the distributions of the data in
both groups (n = 35 each) were compatible with a gaussian
distribution, as judged by Shapiro–Wilks' test. Table 4
shows geometric means and SD of PAPP-A/proMBP concentrations
assayed by 4 versions of TrIFMAs in the serum samples from nonpregnant
donors. PAPP-A concentrations in the men's samples were slightly but
not significantly higher (assayed by TrIFMA 234–3/234–2 and TrIFMA
234–4/234–2) than in the women's samples, or were clearly and
significantly higher (assayed by TrIFMA 234–4/234–5 and TrIFMA
234–5/234–6; P <0.05) than in the women's samples.
Results similar to those obtained by TrIFMA 234–4/234–5 and TrIFMA
234–5/234–6 were reported for the PcAb/PcAb assay in men (n =
78) and women (n = 69) (12). PAPP-A values determined
by TrIFMA 234–5/234–6 differed statistically from those obtained by
other assay versions, as did those assayed by TrIFMA 234–4/234–5.
There was no statistically significant difference between the PAPP-A
values assayed by TrIFMA 234–3/234–2 and TrIFMA 234–4/234–2.
Compared with the other assay versions, PAPP-A concentrations
determined by TrIFMA 234–5/234–6 were generally higher in most blood
donor samples. In some samples, the PAPP-A concentrations determined by
TrIFMA 234–5/234–6 were much higher, exceeding 100 mIU/L in 12 of
the 70 samples. The maximal value in these samples was 810 mIU/L,
equivalent to the median of serum PAPP-A in gestation weeks 10–11.
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In unaffected pregnancies.
The empirical median
values and regressed medians of PAPP-A assayed by each version of
TrIFMA in unaffected pregnancies for gestational ages from week 4 to 13
are shown in Table 5
. The PAPP-A concentration measured by the polyclonal TrIFMA was
higher than that measured by monoclonal TrIFMA 234–3/234–2 and
TrIFMA 234–4/234–2 until gestational week 11, and higher than that
measured by TriFMA 234–4/234–5 and TrIFMA 234–5/234–6 until
gestational week 8. As pregnancy advanced, the relative differences
became smaller. The concentrations measured by TrIFMA 234–3/234–2
were very similar to those obtained by TrIFMA 234–4/234–2, but
different from those seen with TrIFMA 234–4/234–5 and TrIFMA
234–5/234–6. The PAPP-A value determined by TrIFMA 234–3/234–2
and TrIFMA 234–4/234–2 was lower than that by TrIFMA 234–4/234–5
and TrIFMA 234–5/234–6, especially in gestational weeks 4–10, where
the difference was almost twofold.
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In DS pregnancies.
Data for DS-affected pregnancies,
expressed as MoMs, are given in Table 6
. MoM values obtained by use of the monoclonal TrIFMAs were
significantly lower than those obtained by the polyclonal TrIFMA
(P <0.01; Mann–Whitney U-test). In the 26 DS
pregnancies (gestational weeks 7–12), the median PAPP-A MoM values
determined by TrIFMA 234–3/234–2, 234–4/234–2, 234–4/234–5,
and 234–5/234–6 were 0.35, 0.37, 0.42, and 0.44, respectively,
whereas the median MoM determined by the polyclonal assay was 0.56. No
matter which assay version was used, however, the resulting median MoMs
for DS-affected pregnancies were all significantly lower than the
median MoM values for the unaffected pregnancies at 7–12 weeks of
gestation (P <0.01; Mann–Whitney U-test). The
distributions of the log MoM values in both the healthy control group
and the DS-affected group were compatible with a gaussian distribution
as judged from normal plots. Two samples had a very high MoM value in
assay combination 234–5/234–6(4.73 and 7.24), whereas the other
combinations gave MoMs <1.0 for the same samples.
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diagnostic accuracy
The diagnostic accuracy of serum PAPP-A determined by 4 monoclonal
TrIFMAs in gestational weeks 7–12 for detecting DS-affected
pregnancies was evaluated by plotting the ROC curves (see Fig. 4
). Areas under the ROC curves and their SEs are given in Table 7
. All monoclonal TrIFMAs gave an area significantly
(P <0.05) greater than that of the polyclonal TrIFMA. The
detection rates for a false-positive rate of 5% varied from 26.9% for
the polyclonal TrIFMA to 37.2% for the monoclonal TrIFMA
234–4/234–5. However, there was no statistically significant
difference in the areas under the ROC curves for any of the monoclonal
assays (P >0.16).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PAPP-A exists in pooled pregnancy serum as a heterotetrameric complex of two molecules of PAPP-A and two molecules of proMBP (19). At least 2 other proMBP complexes are present in term pregnant serum, i.e., complexes with angiotensinogen and complexes with complement 3dg and angiotensinogen (24). Nothing is currently known about the function of these complexes or their serum concentrations in DS pregnancies.
For analysis of the clinical usefulness of PAPP-A determination in screening, it is important to establish that an immunoassay determines only the concentration of PAPP-A, either free (if that form exists) or complex-bound, and not the concentrations of other serum constituents. Unfortunately, the only commercially available polyclonal antibody against PAPP-A (Dako A230) has been shown to cross-react with proMBP (19)(24), SP1 (22), and haptoglobin (25), all present in pregnancy serum in concentrations greatly exceeding the concentration of PAPP-A. Recently, an absorption procedure to make A230 free of cross-reactivity has been devised that discriminates well between DS and unaffected pregnancies (22). Absorption procedures have previously been shown to increase the specificity of polyclonal anti-PAPP-A antibodies (37), but such procedures are difficult to standardize and very time-consuming to perform. Thus, the double-monoclonal assay format is preferred.
In healthy blood donors, a low concentration of PAPP-A was detected by all 4 monoclonal versions of TrIFMA—in agreement with findings with the polyclonal TrIFMA (12). The source of this immunoreactivity is likely to be seminal fluid, Graffian follicles, corpus luteum, and testes (38). Furthermore, recent results suggest that PAPP-A mRNA is also transcribed in the brain, but whether PAPP-A synthesized there reaches the circulation is not known (39). In some samples, the 234–5/234–6 assay version found much higher concentrations of PAPP-A than did the other assays. This unusual, high value of PAPP-A is thought to be caused by cross-reactants in the sera.
In pregnant women, PAPP-A serum concentrations are found to increase with gestational age. However, absolute concentrations seem to differ widely between investigators (9)(10)(11)(12)(13), probably because of differences in the calibrators or labeled tracers and antibodies used. Compared with the findings of other groups, the medians obtained here are lower. This has no consequences for maternal screening, however, because the MoM, not the absolute concentration, is used to calculate the individual risk that a pregnancy is affected by DS. When the unaffected controls and affected samples are determined with the same assay, the MoM value obtained for each sample should be the same as that obtained by the other assays.
PAPP-A has been described to exhibit immunological heterogeneity, and several plasma proteins have been suggested as having epitopes that are cross-reactive with PAPP-A (37)(40). We think it surprising that some monoclonal assay combinations, i.e., 234/234–5 and 234–5/234–6, give much higher PAPP-A concentrations than do the polyclonal TrIFMA and the two other monoclonal combinations, given that the immunoreactivities determined by all of the assays elute as one predominant peak of Mr ~820 000. The differences in the relative size of the peak could be caused by epitope sharing with other high-Mr substances or perhaps more likely by differential reactivity of monoclonal antibodies to different glycoforms of the PAPP-A/proMBP complex (carbohydrate content 17.4% (19)). It could also be attributable to variations in attached ligands or perhaps preanalytical modifications. Whether the cause is cross-reactivity or variations in the detection of isoforms of PAPP-A/proMBP, we find it noteworthy that the discrimination between DS pregnancies and unaffected pregnancies obtained with the double monoclonal assays is very similar to each other and better than that with the polyclonal TrIFMA. The fact that the difference in concentrations determined by different assays for PAPP-A/proMBP complex depends on gestational age suggests that different isoforms of PAPP-A/proMBP complex or that other complexes containing either or both of the two proteins PAPP-A and proMBP may exist in different stages of pregnancy. This possibility is currently under investigation.
Our finding of an approximate size of 800 kDa for the PAPP-A/proMBP complex is in agreement with the findings of others (28)(40). However, this value is much higher than the mass determined from the molecular structure of PAPP-A/proMBP, 474 kDa. The reason for this discrepancy is not clear; maybe the PAPP-A/proMBP complex has an asymmetrical structure. The proMBP part of the complex is glycosylated and carries a linear glycosaminoglycan (41).
In terms of diagnostic accuracy, at a 5% false-positive rate, the rate of DS detection was 27.1–37.2% as found by the monoclonal assays and 26.9% by the polyclonal assay, both lower than that (42%) reported by Wald et al. (13), but close to (by PcAb/PcAb and TrIFMA 234–3/234–2) or higher than (by TrIFMA 234–4/234–2, 234–4/234–5, and 234–5/234–6) that reported by Spencer et al. (8): 28%. The differences in detection rates from these two published studies are most likely the result of using different antibodies. The detection rate obtained in our study, using the same polyclonal antibodies, is 26.9%, which is in close agreement with the result of Spencer's study.
Irrespective of whether polyclonal or monoclonal TrIFMAs are used, maternal serum PAPP-A concentrations are lower in DS-affected pregnancies. This finding is in accordance with previous studies (8)(9)(10)(11)(12). Compared with polyclonal TrIFMA, monoclonal TrIFMAs seem to better discriminate between unaffected pregnancies and DS pregnancies. However, some of the monoclonal combinations are unsuitable because of high amounts of PAPP-A immunoreactivity in nonpregnant serum.
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Acknowledgments |
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Footnotes |
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2 Small amounts of monoclonal antibodies may be made available for research purposes upon written request to: Michael Christiansen, Department of Clinical Biochemistry, State Serum Institute, 5 Artillerivej, DK 2300S; fax +45 32 68 38 78; e-mail mchristiansen{at}cb.diag.ssi.dk
3 The version of monoclonal assay is expressed as: Mab1/Mab2*, where Mab1 functions as a capture antibody and Mab2*, labeled with Eu3+, serves as a detection antibody in the assay.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Q.-P. Qin, S. Kokkala, J. Lund, N. Tamm, L.-M. Voipio-Pulkki, and K. Pettersson Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy Clin. Chem., January 1, 2005; 51(1): 75 - 83. [Abstract] [Full Text] [PDF]
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H. B. Boldt, K. Kjaer-Sorensen, M. T. Overgaard, K. Weyer, C. B. Poulsen, L. Sottrup-Jensen, C. A. Conover, L. C. Giudice, and C. Oxvig The Lin12-Notch Repeats of Pregnancy-associated Plasma Protein-A Bind Calcium and Determine Its Proteolytic Specificity J. Biol. Chem., September 10, 2004; 279(37): 38525 - 38531. [Abstract] [Full Text] [PDF]
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 B.-K. Chen, K. M. Leiferman, M. R. Pittelkow, M. T. Overgaard, C. Oxvig, and C. A. Conover Localization and Regulation of Pregnancy-Associated Plasma Protein A Expression in Healing Human Skin J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4465 - 4471. [Abstract] [Full Text] [PDF]
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L. S. Laursen, M. T. Overgaard, K. Weyer, H. B. Boldt, P. Ebbesen, M. Christiansen, L. Sottrup-Jensen, L. C. Giudice, and C. Oxvig Cell Surface Targeting of Pregnancy-associated Plasma Protein A Proteolytic Activity. REVERSIBLE ADHESION IS MEDIATED BY TWO NEIGHBORING SHORT CONSENSUS REPEATS J. Biol. Chem., November 27, 2002; 277(49): 47225 - 47234. [Abstract] [Full Text] [PDF]
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I. Y. C. Sun, M. T. Overgaard, C. Oxvig, and L. C. Giudice Pregnancy-Associated Plasma Protein A Proteolytic Activity Is Associated with the Human Placental Trophoblast Cell Membrane J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5235 - 5240. [Abstract] [Full Text] [PDF]
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L. C. Giudice, C. A. Conover, L. Bale, G. H. Faessen, K. Ilg, I. Sun, B. Imani, L.-F. Suen, J. C. Irwin, M. Christiansen, et al. Identification and Regulation of the IGFBP-4 Protease and Its Physiological Inhibitor in Human Trophoblasts and Endometrial Stroma: Evidence for Paracrine Regulation of IGF-II Bioavailability in the Placental Bed during Human Implantation J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2359 - 2366. [Abstract] [Full Text] [PDF]
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 F. M. Reis, D. D'Antona, and F. Petraglia Predictive Value of Hormone Measurements in Maternal and Fetal Complications of Pregnancy Endocr. Rev., April 1, 2002; 23(2): 230 - 257. [Abstract] [Full Text] [PDF]
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B.-K. Chen, M. T. Overgaard, L. K. Bale, Z. T. Resch, M. Christiansen, C. Oxvig, and C. A. Conover Molecular Regulation of the IGF-Binding Protein-4 Protease System in Human Fibroblasts: Identification of a Novel Inducible Inhibitor Endocrinology, April 1, 2002; 143(4): 1199 - 1205. [Abstract] [Full Text] [PDF]
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Q.-P. Qin, M. Christiansen, and K. Pettersson Point-of-Care Time-resolved Immunofluorometric Assay for Human Pregnancy-associated Plasma Protein A: Use in First-Trimester Screening for Down Syndrome Clin. Chem., March 1, 2002; 48(3): 473 - 483. [Abstract] [Full Text] [PDF]
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 A. Bayes-Genis, C. A. Conover, M. T. Overgaard, K. R. Bailey, M. Christiansen, D. R. Holmes Jr., R. Virmani, C. Oxvig, and R. S. Schwartz Pregnancy-Associated Plasma Protein A as a Marker of Acute Coronary Syndromes N. Engl. J. Med., October 4, 2001; 345(14): 1022 - 1029. [Abstract] [Full Text] [PDF]
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 A. Bayes-Genis, R. S. Schwartz, D. A. Lewis, M. T. Overgaard, M. Christiansen, C. Oxvig, K. Ashai, D. R. Holmes Jr, and C. A. Conover Insulin-Like Growth Factor Binding Protein-4 Protease Produced by Smooth Muscle Cells Increases in the Coronary Artery After Angioplasty Arterioscler Thromb Vasc Biol, March 1, 2001; 21(3): 335 - 341. [Abstract] [Full Text] [PDF]
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M. Christiansen, I. Jaliashvili, M. T. Overgaard, C. Ensinger, P. Obrist, and C. Oxvig Quantification and Characterization of Pregnancy-associated Complexes of Angiotensinogen and the Proform of Eosinophil Major Basic Protein in Serum and Amniotic Fluid Clin. Chem., August 1, 2000; 46(8): 1099 - 1105. [Abstract] [Full Text] [PDF]
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 M. T. Overgaard, C. Oxvig, M. Christiansen, J. B. Lawrence, C. A. Conover, G. J. Gleich, L. Sottrup-Jensen, and J. Haaning Messenger Ribonucleic Acid Levels of Pregnancy-Associated Plasma Protein-A and the Proform of Eosinophil Major Basic Protein: Expression in Human Reproductive and Nonreproductive Tissues Biol Reprod, October 1, 1999; 61(4): 1083 - 1089. [Abstract] [Full Text]
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 J. B. Lawrence, C. Oxvig, M. T. Overgaard, L. Sottrup-Jensen, G. J. Gleich, L. G. Hays, J. R. Yates III, and C. A. Conover The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A PNAS, March 16, 1999; 96(6): 3149 - 3153. [Abstract] [Full Text] [PDF]
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M. T. Overgaard, J. Haaning, H. B. Boldt, I. M. Olsen, L. S. Laursen, M. Christiansen, G. J. Gleich, L. Sottrup-Jensen, C. A. Conover, and C. Oxvig Expression of Recombinant Human Pregnancy-associated Plasma Protein-A and Identification of the Proform of Eosinophil Major Basic Protein as Its Physiological Inhibitor J. Biol. Chem., September 29, 2000; 275(40): 31128 - 31133. [Abstract] [Full Text] [PDF]
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) and precision profiles (•) of 4
monoclonal versions of TrIFMAs.
