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Articles |
1
Department of Clinical Biochemistry, Statens Serum Institut, 5 Artillerivej, Copenhagen DK 2300 S, Denmark.
2
Department of Molecular and Structural Biology,
University of Aarhus, 8000 Åarhus, Denmark.
3
Department of Pathology, University of Innsbruck,
A 6020 Innsbruck, Austria.
a Author for correspondence. Fax 45-3-2683878; e-mail mic{at}ssi.dk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: We developed an ELISA for the ProMBP/Ang complexes, using a monoclonal antibody against ProMBP for capture and a chicken anti-human Ang antiserum for detection. Calibrators were standardized with WHO IRP 78/610 for pregnancy proteins in the assay range 0.95–15.6 mIU/L.
Results: The concentrations of ProMBP/Ang complexes in serum of nonpregnant blood donors (n = 79) were log-normally distributed with a central 95th interval of 985-3655 mIU/L. In pregnancy, mean serum concentrations were increased from week 7, and the concentrations reached term concentrations in week 18. ProMBP/Ang complexes eluted in gel filtration as a broad peak with a molecular mass of ~230 kDa. The concentration of ProMBP/Ang/C3dg increased during blood coagulation, suggesting that the ProMBP/Ang/C3dg complex may be a marker of complement activation.
Conclusions: ProMBP/Ang complexes are present in serum from nonpregnant persons as well as pregnant women, and the direct assays described here will make it possible to study the biochemistry and the clinical significance of different ProMBP complexes in pathological conditions and pregnancy.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal serum contains MBP immunoreactivity (15), and in patients with hypereosinophilia (16), the activity is increased. Furthermore, amniotic fluid contains high MBP immunoreactivity (17). The synthesis of ProMBP is known to occur in cells other than placental X cells and eosinophil precursor cells (18), but whether ProMBP or MBP is released into the circulation from these sources is unknown. An increase in the serum concentration of ProMBP in pregnant women has been suggested to precede the onset of labor (19)(20), but this finding is not constant. The maternal serum concentration of PAPP-A/ProMBP is reduced in the first trimester when the fetus is carrying chromosomal trisomy 21 (21). Recently, the total concentration of ProMBP in the maternal circulation has been found to be a marker of fetal chromosomal disease (22).
Methods of varying analytical precision and sensitivity have been available for the analysis of ProMBP/PAPP-A complexes (21)(23) because PAPP-A is an established first-trimester maternal serum marker for Down syndrome, and many of the assays developed determine PAPP-A/ProMBP and other ProMBP-containing complexes simultaneously as well as other pregnancy proteins (23)(24)(25). However, direct assays for the other complexes have not been available, so that information concerning reference intervals, normal physiology, and variations in pathological conditions has been scarce.
In this study, we developed and characterized a sandwich immunoassay for ProMBP/Ang complexes and developed assays for ProMBP/C3dg complexes and the total concentration of ProMBP complexes to study their relationship with ProMBP/Ang complexes. In addition, we characterized the antigen detected by the ProMBP/Ang assay, established reference intervals for nonpregnant adults, and describe development through gestation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Reagents
Antibodies.
A monoclonal antibody, 234-10, raised against the
complex between PAPP-A and ProMBP and reactive with the ProMBP part of
the complex (26), was used as the capture antibody. Antibody
234-10 also reacts with MBP purified from eosinophil granules
(Christiansen et al., unpublished results). A polyclonal
chicken antiserum was raised against human Ang (Calbiochem 176870;
Calbiochem-Novabiochem) and used for detection. Briefly, three
hens were immunized weekly, five times each, with 15 µg of Ang in
Freund’s incomplete adjuvant. After 5 weeks, the titer, tested in
direct ELISA with 0.5 µg of Ang per well, reached a maximum, and the
antiserum was used in the experiments. The antiserum was tested against
1-chymotrypsin, plasminogen, antithrombin III,
1-antitrypsin, haptoglobin, complement C3, and
purified PAPP-A/ProMBP. A slight cross-reaction was found, most
prominent with 1-antithrombin III, with a
cross-reactivity <0.3% in direct ELISA, when wells were coated with
0.5 mg/L protein in carbonate buffer, pH 9.6. A polyclonal rabbit
antiserum against highly purified PAPP-A/ProMBP was produced using
standard procedures. Anti-chicken immunoglobulin conjugated with
horseradish peroxidase (HRP) was obtained from Sigma (cat. no. A9046).
Peroxidase-conjugated rabbit anti-human complement C3d was obtained
from Dako A/S (cat. no. P387; Dako).
Calibrators and controls.
Calibrators were produced from a
40-week pregnancy serum pool diluted in dilution buffer [0.5 mol/L
NaCl, 2.7 mmol/L KCl, 1.5 mmol/L
KH2PO4, 6.5 mmol/L
Na2HPO4, 10 mL/L Triton
X-100 (pH 7.2), and 10 g/L bovine serum albumin (cat. no. A4503;
Sigma)], and calibrated against the third-trimester pregnancy pooled
serum-derived WHO IRP 78/610 for pregnancy-associated proteins (WHO
International Laboratory for Biological Standards, Statens Serum
Institut, Copenhagen, Denmark) (27). The contents of the
ampoule were dissolved in 750 µL of distilled water to give a
concentration of 100 IU/L as defined for schwangeschafts protein 1 and
recommended for other pregnancy proteins (27). No
weight-based standard is available for ProMBP complexes, but data from
two other studies (9)(27) made it possible to
calculate an approximate conversion factor between IU and mass units
under the reasonable assumption that the only quantitatively important
ProMBP complexes are PAPP-A/ProMBP and ProMBP/Ang complexes. The total
concentration of ProMBP monomer (molecular mass ~39 kDa) in
gestational week 25 (~100 IU/L) has been found to be ~375 nmol/L
(14.25 mg/L) (9), and the concentration of the PAPP-A/ProMBP
complex (molecular mass ~480 kDa) is ~45 mg/L at term
(27); therefore, an estimate of 1 IU of ProMBP/Ang
(molecular mass ~200 kDa) is ~0.25 mg. A mass unit of total ProMBP
complexes cannot be given because that depends on the distribution of
ProMBP on complexes containing PAPP-A or Ang. Three control samples,
representing low, medium, and high concentrations of ProMBP/Ang
complexes, 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 serum
calibrator.
Proteins.
Haptoglobin (Calbiochem 372022;
Calbiochem-Novabiochem), complement C3 (cat. no. C-2910; Sigma),
1-chymotrypsin (Calbiochem 178196),
plasminogen (Calbiochem 528180), antithrombin III (Calbiochem 169756),
and 1-antitrypsin (Calbiochem 178251) were
obtained commercially. PAPP-A/ProMBP was purified from term pregnancy
serum as described previously (8).
Procedures
Enzyme immunoassay for ProMBP/Ang complexes.
Maxisorp
polystyrene microtiter plates (Nunc) were coated with 500 ng of
anti-ProMBP, 234-10, in 100 µL of coating buffer [15 mmol/L
Na2CO3, 35 mmol/L
NaHCO3 (pH 9.6)] overnight at 4 °C. Plates
were washed once in wash buffer [2.5 mmol/L
NaH2PO4, 7.5 mmol/L
Na2HPO4, 145 mmol/L NaCl
(pH 7.2), 0.5 mL/L Tween 20], and then 100 µL of calibrators
(ranging from 0.98 to 31.25 mIU/L), controls, or appropriately diluted
samples (in dilution buffer) were added and incubated for 2 h at
room temperature with slow shaking. Serum samples from nonpregnant
persons were diluted 1:500, samples from pregnant women in the first
trimester were diluted 1:2000, and samples from pregnant women in
second trimester were diluted 1:10 000. After washing, 100 µL of
chicken antiserum against human Ang, SSI 233, was diluted 1:300 in
dilution buffer and incubated for 1 h at room temperature with
slow shaking. After the wells were washed again, bound antibody was
detected by incubation with rabbit anti-chicken immunoglobulin-HRP
diluted 1:2000 in dilution buffer for 1 h at room temperature.
Color was developed using 4 g/L o-phenylenediamine in
citrate buffer, pH 5.0, with 3 mL/L
H2O2 (Merck). The reaction
was stopped with 200 µL of 2 mol/L
H2SO4, and the absorbance
at 490 nm was measured using a VICTORTM (Wallac,
EG&G Life Sciences) spectrophotometer; after subtraction of the
absorbance at 620 nm, the calibrators were analyzed with a spline
algorithm on logarithmically transformed data, and a calibration
curve was produced. The calibration curve was used to calculate
sample concentrations. All samples were analyzed in duplicate. When the
CV between two measurements on the same sample exceeded 10%,
the sample was rerun.
Enzyme immunoassay for ProMBP/C3dg complexes.
The enzyme
immunoassay for ProMBP/C3dg complexes was performed as for the
ProMBP/Ang complex with the modification that calibrators of 3.9, 7.8,
31.25, 125, 500, and 1000 mIU/L were used, and the detection antibody
was HRP-conjugated rabbit anti-human complement C3d (cat. no.
P387; Dako A/S) diluted 1:2000 in dilution buffer. Detection and data
acquisition were as described above. Within the assay range, the
interassay CV was <5%. No reaction was found with haptoglobin,
complement C3, 1-antitrypsin,
1-antichymotrypsin, plasminogen, or
antithrombin III.
Enzyme immunoassay for all ProMBP complexes.
The enzyme
immunoassay for all ProMBP complexes was performed as for the other
immunoassays, except that the capture antibody was rabbit polyclonal
anti-ProMBP/PAPP-A developed at Statens Serum Institut against highly
purified ProMBP/PAPP-A. Detection was performed with anti-ProMBP
(antibody 234-10) and anti-mouse immunoglobulin-HRP conjugate as
described above. The calibrators used were 3.9, 7.8, 31.25, 125, 500,
and 1000 mIU/L. Within this range, the interassay CV was <5%.
Data management was as described above. There was no reaction
with haptoglobin, complement C3,
1-antitrypsin,
1-antichymotrypsin, plasminogen, or
antithrombin III.
Gel filtration chromatography (GFC).
GFC was carried out on a
SigmaChrom GFC-1300 column (300 x 7.5 mm; Supelco) at 25 °C
with a mobile phase of 0.1 mol/L sodium phosphate buffer, pH 7.0,
containing 0.1 mol/L NaCl; the flow rate was 0.5 mL/min.
Samples [100 µL; serum diluted fivefold in mobile phase and filtered
through 0.22 µm PTFE filter (Scientific Resources)] were injected.
The column effluent was monitored at 280 nm, and 0.5-mL fractions were
collected. The total run time was 40 min, and fractions were collected
from 8 to 28 min. The column was run on a Pharmacia-LKB HPLC system
(Pharmacia-LKB Biotechnology). Chromatographic runs were performed and
data analyzed using HPLC manager software (Pharmacia-LKB Biotechnology)
and Nelson Model 2600 Chromatography Software (Perkin-Elmer, Nelson
Systems). The column was calibrated with the High Molecular Weight Gel
Filtration Calibration Kit (Pharmacia Biotech).
Anion-exchange chromatography (AEC).
AEC was carried out on a
Poros 20 HQ column (100 x 4.6 mm; Perseptive Biosystems) at
25 °C with the following mobile phases: buffer A, 0.1 mol/L sodium
phosphate buffer containing 0.1 mol/L NaCl; and buffer B, 0.1 mol/L
sodium phosphate buffer, pH 7.0, containing 2.0 mol/L NaCl. The flow
rate was 3 mL/min. Samples [100 µL; serum diluted fivefold in buffer
A and filtered through 0.22 µm PTFE filter (Scientific Resources)]
were injected. Three min after injection, a gradient from 0% to 100%
buffer B over 7 min was applied, followed by 5 min of buffer A. The
total run time was 15 min; the column effluent was monitored at 280 nm,
and 2-mL fractions were collected. The column was run on the same
system as the GFC above.
Heparin affinity chromatography.
Heparin affinity
chromatography was performed using 10-mL
Prosep®-Heparin affinity matrix (Bioprocessing).
The matrix was equilibrated with starting buffer A (0.1 mol/L sodium
phosphate buffer, pH 7.0, containing 0.1 mol/L NaCl) and operated at a
flow rate of 1.5 mL/min. Serum (1 mL) was diluted with 1 mL of starting
buffer and injected onto the column. The column was washed with buffer
A, and elution was started at 70 min with 75% buffer B (0.1 mol/L
sodium phosphate buffer, pH 7.0, containing 2 mol/L NaCl). The total
run time was 100 min. Single fractions were obtained through the
washing phase, and the high-salt eluate was pooled and concentrated.
Statistics
Groups were compared using the Mann–Whitney U-test.
Paired samples were compared using the matched sign test. Compatibility
with a gaussian distribution was assessed by probit diagrams
and the Shapiro–Wilk test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is shown an extended calibration curve for the assay, where
each point is based on eight determinations. The precision profile (CV)
is also shown in Fig 1
. The detection limit was 0.37 mIU/L.
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Calibration curve.
A routine calibration curve for the
ProMBP/Ang assay is shown in Fig. 1
. The working assay range was
0.98–15.6 mIU/L.
Reproducibility.
The intraassay CV, shown in Fig. 1
, was calculated by measuring four samples in five consecutive ProMBP/Ang
assays. At concentrations of 1.8, 4.8, 9.6, and 12 mIU/L, the CVs were
11%, 7.5%, 7.6%, and 12%, respectively.
Parallelism.
The dilution curves for a second-trimester serum
with a high ProMBP/Ang concentration, a first-trimester serum sample
with a medium ProMBP/Ang concentration, and a normal serum pool with a
low concentration of the complex were parallel (data not shown).
Recovery.
When a serum sample with a concentration of 1.77
mIU/L was mixed with serum to reach calculated concentrations of 3.32
or 3.84 mIU/L, the mean (n = 5) recoveries were 100.7%, and
101.2%, respectively. When a serum sample of 9.74 mIU/L was mixed with
serum to reach a calculated concentration of 11.8 mIU/L, the mean
(n = 5) recovery was 98.8%.
Specificity.
The specificity of the assay was assessed
by analyzing the response obtained when measuring commercially
available purified Ang, complement C3,
1-antitrypsin,
1-chymotrypsin, antithrombin III, plasminogen,
and haptoglobin. None of these proteins gave an appreciable
response in the assay, nor did semi-purified PAPP-A/ProMBP (data not
shown).
Stability.
Serum samples were incubated at different
temperatures for up to 14 days as shown in Fig. 2
. ProMBP/Ang was remarkably stable, with a half-life at 56 °C
of 3–4 days.
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Relationship with ProMBP/C3dg complexes.
We compared
the concentrations of ProMBP/Ang with the concentration of ProMBP/C3dg
in 17 paired serum and K2EDTA-plasma samples from
pregnant women in first trimester. The concentration of ProMBP/C3dg was
significantly higher [mean (SD), 49% (31%)] in serum than in plasma
(P = 0.0003), suggesting that complement activation or
release from cells during clotting may profoundly influence the
concentration in serum. There was no significant difference between
concentration values in the paired serum and plasma samples for
ProMBP/Ang complexes and total ProMBP complexes. Thus, variations in
the extent of complement C3dg attachment to ProMBP/Ang complex does not
seem to influence the quantification of these complexes.
Concentrations measured
Healthy blood donors.
In all 79 healthy blood donors,
ProMBP/Ang was detectable in serum, with a median concentration of 1928
mIU/L (lower quartile, 1469 mIU/L; upper quartile, 2327 mIU/L). The
concentrations did not follow a gaussian distribution (Shapiro–Wilk
W = 0.9095; P <5 x
10-6) but conformed to a log-normal distribution
with a mean (log ProMBP/Ang) of 3.2783 and a SD of 0.1452
(Shapiro–Wilk W = 0.9840; P = 0.781).
There was no significant gender difference (Mann–Whitney
U-test, P = 0.961). Based on the log-normal
distribution, a central 95th interval (985–3655 mIU/L) could
be constructed. By comparison, the total concentration of ProMBP
complexes was also log-normally distributed with a central 95%
interval of 385-1531 mIU/L.
Pregnant women.
The serum concentration in pregnant women
increased log-linearly with gestational age (Fig. 3
). From gestational week 7, the serum concentration of
ProMBP/Ang was increased; the majority of pregnant women had serum
concentrations above the upper reference limit as defined above for
blood donors. A serum concentration of ~100 IU/L, the concentration
found at term, was reached in gestational week 18 (Fig. 3
). The same
development over gestational age was seen for the total concentration
of ProMBP complexes. The concentrations of ProMBP/Ang and total ProMBP
complexes correlated significantly in both blood donors and pregnant
women.
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Amniotic fluid.
In amniotic fluid obtained from
gestational age 100–133 days (gestational weeks 14–19), no
correlation was found between the concentrations of ProMBP/Ang
complexes or ProMBP complexes and gestational age. The median
ProMBP/Ang complex concentration was 11 686 mIU/L (interquartile
range, 6592–16 326 mIU/L). The median concentration of the
total ProMBP complexes was 14 680 mIU/L (interquartile range,
8422–23 410 mIU/L). Thus, the concentrations of the ProMBP/Ang
complexes and the total ProMBP complexes in amniotic fluid were
~10–15% of the serum concentrations.
Characterization of ProMBP/Ang COMPLEXES
GFC.
GFC was performed on a serum pool from healthy,
nonpregnant women and a pool of serum from women in the first trimester
(Fig. 4
). In both pools, the ProMBP/Ang eluted as a broad peak with a
molecular mass of ~230 kDa.
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AEC.
Both first-trimester and nonpregnant serum pools
contained ProMBP/Ang, and in both cases it was adsorbed to the AEC
column. However, the elution profile was slightly different, with the
nonpregnant ProMBP/Ang eluting earlier than the first-trimester
ProMBP/Ang (Fig. 5
).
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Heparin affinity chromatography.
In contrast to
ProMBP/PAPP-A, the ProMBP/Ang complexes did not bind to the heparin
column (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The concentrations of all ProMBP complexes and ProMBP/Ang complexes were found to be increased from gestational week 7, and the concentrations increased with gestation to reach the term concentration (100 IU/L) at gestational week 18. This temporal development is similar to that of MBP immunoreactivity, as described previously. It is also similar to that of Ang in pregnancy (28).
The gel filtration profile of ProMBP/Ang complexes (Fig. 4
), with a
very broad peak eluting at a molecular mass of ~230 kDa in both
pregnancy and non-pregnancy sera, is compatible with the previously
found stoichiometry of 2:2:2 and 2:2 for complexes with and without
complement C3dg, respectively. Similarly, the adsorption to an
anion-exchange column at a neutral pH is similar to what has been
described earlier (9). The higher concentration of salt
necessary to desorb the ProMBP/Ang complexes in pregnancy compared with
non-pregnancy serum (Fig. 5
) may reflect increased
glycosylation/sialylation in pregnancy.
Previously, the C3dg-containing ProMBP complex was found in both
pregnancy serum and plasma, but the concentrations were not compared
(9). We here document that this complex is produced during
the clotting of blood, but to establish whether this complex is only an
artifact will require careful studies. The complement C3d-containing
complex was present in plasma and amniotic fluid, but samples were not
obtained under conditions suitable for avoiding postsampling complement
activation; therefore, quantitative results cannot be reported.
However, if the ProMBP/C3dg complex is not an artifact, a functional
role of ProMBP complexes might be to act as a scavenger for split
products from the alternative pathway of complement activation, a
function that is very similar to that of
2-macroglobulin for other immunologically
active molecules (29); in that case, ProMBP/C3dg complexes
might be a tentative biochemical marker for complement activation.
Complement C3dg, which is a strong stimulant of humoral immunity
(14), may also be produced in situ, i.e., while attached to
ProMBP/Ang, from complement C3b. The complement C3dg moiety could also
serve as a ligand, facilitating the binding of the whole complex to
complement receptor 2 (30). A complex of two
complement C3dg molecules held together by
(ProMBP/Ang)2 may be expected to have such a high
affinity for the single binding site receptor complement receptor 2
that it could block normal stimulation of the humoral immune response
(31). This mechanism could be of importance in controlling
the humoral immune system during pregnancy. MBP has been found to be
able to modulate complement activation (32)(33)(34), but whether
this is also the case for ProMBP remains to be seen.
Whether ProMBP/Ang complexes constitute a part of the pregnancy-associated high-molecular weight Ang (35), HMrA, has been debated (36), but recently it has been documented that ProMBP/Ang complexes can function as renin substrate (37), so it is fair to assume that at least a part of HMrA is ProMBP/Ang complexes. How much of HMrA is ProMBP/Ang complexes remains to be established in quantitative studies. The ProMBP/Ang complexes are not pregnancy specific because the concentration in nonpregnant individuals is 1–5% of the concentration at term. Why the concentration increases during pregnancy is as yet unknown.
In conclusion, the assays described here will, for the first time, make it possible to examine the concentrations of ProMBP/Ang and ProMBP/C3dg in various clinical conditions and to perform studies on the regulation of synthesis in in vitro systems. Furthermore, it will be possible to get a better understanding of the relationship between the maternal serum concentration of ProMBP complexes and fetal chromosomal disease.
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Acknowledgments |
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Macroglobulins: structure, shape, and mechanism of proteinase complex formation. J Biol Chem 1989;264:11539-11542.
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) and precision profile (
) of the
ProMBP/Ang assay.
) and a pool from healthy, nonpregnant women (
) on a
Poros 20 HQ column.