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Immunoassays developed for pregnancy-associated plasma protein-A (PAPP-A) in pregnancy may not recognize PAPP-A in acute coronary syndromes.

Pregnancy-associated plasma protein-A (PAPP-A) [6] was initially identified as a high-molecular-mass component in human late-pregnancy serum (1). The placenta is the main source of circulating PAPP-A in pregnancy. In uncomplicated pregnancies, PAPP-A concentrations in maternal serum increase with gestational age until term (2); however, in Down syndrome pregnancies, PAPP-A concentrations in the first trimester are markedly decreased (3). PAPP-A is currently an established biochemical marker commonly used for Down syndrome screening in the first trimester (4). In addition, low first-trimester PAPP-A concentrations are also associated with other adverse pregnancy outcomes, such as stillbirth and preterm delivery (5, 6).

PAPP-A has been shown to be present in unstable coronary atherosclerotic plaques (7), and increased circulating PAPP-A concentrations are associated with acute coronary syndromes (ACS) (7,8). Moreover, PAPP-A is also a strong independent stratifier for patients with ACS (9,10). Thus, PAPP-A is directly involved in the pathophysiology of ACS and may function to promote atherogenesis. PAPP-A isolated from pregnancy serum is a disulfide-bound 2:2 complex with the proform of eosinophil major basic protein (proMBP) in which each PAPP-A subunit is connected to a proMBP subunit by 2 disulfide bonds (11). However, PAPP-A in ACS lacks the proMBP subunit (12). Because of the difference in subunit composition between PAPP-A in pregnancy and PAPP-A in ACS, the relevant molecular conformations also differ substantially from one another (13). This fact is likely to have implications in clinical practice, as the antibodies currently used in immunoassays for PAPP-A in ACS are all raised by immunization with PAPP-A isolated from pregnancy serum (14,15). In addition, there have been controversial reports on whether serum PAPP-A is a useful marker for ACS (9,10,16,17). The inconsistency among laboratories could result from the use of different PAPP-A assays involving different antibodies. The purpose of this study was to investigate the effect of antibody selection on the utility of PAPP-A assays for the measurement of PAPP-A in pregnancy and/or ACS, and whether immunoassays developed for PAPP-A in pregnancy are also suitable for PAPP-A in ACS.

Materials and Methods

REAGENTS

The ITC-TEKES [Eu.sup.3+] fluorescent chelate of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6, bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine, biotin isothiocyanate, streptavidin-coated strips, and assay buffer (buffer solution, red) were obtained from Innotrac Diagnostics Oy. Wash solution was prepared as described previously (18). Assay buffer supplemented with 0.01 g/L denatured mouse IgG and 0.02 g/L native mouse IgG is referred to as diluent A. Low-fluorescence 12-well Maxisorp microtitration strips (ultraviolet-quenched) were purchased from NUNC. NAP-5[TM] and NAP-10[TM] columns were from Amersham Biosciences. Bovine serum albumin was purchased from Intergen. All other chemicals used were of analytical grade.

Sixteen monoclonal antibodies (mAbs)--mAbs A1, A2, A3, A4, A5, A6, A7, A8, A9, A10, A11, A12, A13, A14, A15, and A16--specific for pregnancy serum PAPP-A were from HyTest Oy. Six other mAbs--mAbs B1, B2, B3, B4, B5, and B6-also specific for pregnancy serum PAPP-A, were provided as a gift by Dr. Michael Christiansen from State Serum Institute.

Highly purified recombinant human PAPP-A was provided by Dr. Xuezhong Qin from J.L. Pettis Memorial Veterans' Medical Center (Loma Linda, CA) (19). Recombinant full-length human proMBP was produced with a plasmid also from Dr. Xuezhong Qin. The plasmid was transiently transfected into human embryonic kidney 293T cells by means of a FuGENE 6 transfection reagent from Roche. The cells were grown in DMEM supplemented with 100 mL/L fetal bovine serum. After a 4-day incubation in the medium, the culture supernatants were harvested, cleared by centrifugation, and stored at -20[degrees]C until use.

Calibrators were prepared from a pregnancy serum pool as described previously (20) and calibrated against the pooled third-trimester pregnancy serum-derived WHO IRP 78/610 for pregnancy-associated proteins (WHO International Laboratory for Biological Standards, State Serum Institute). Concentrations of PAPP-A are expressed in mIU/L. The calibrators were stored at -20[degrees]C until use.

SAMPLES

We obtained serum samples with increased PAPP-A concentrations from 4 patients [3 men; mean (SD) age, 59 (7) years; 1 woman; age, 34 years] with typical ST-segment elevation myocardial infarction. The time elapsed between the onset of chest pain and blood drawing for patients 1, 2, 3, and 4 was 1.7, 0.6, 4.0, and 3.4 h, respectively. We obtained atherosclerotic plaques from 15 patients undergoing carotid endarterectomy and from 6 patients undergoing surgical treatment for abdominal aortic aneurysm. All samples were obtained after written informed consent had been received. The study protocol was approved by the Ethics Committees of the University of Helsinki and University of Turku. The samples were stored at -20[degrees]C (plaque samples) or at -70[degrees]C (myocardial infarction samples) before use.

PAPP-A from the atherosclerotic plaques was extracted into 0.05 mol/L Tris buffer (pH 7.8), and its concentration was determined by the previously described immunoassay (20). Thereafter, we prepared a pool of plaque PAPP-A extracts that contained 100 mIU/L PAPP-A, which we used in the antibody combination experiments. We have characterized PAPP-A extracted from atherosclerotic plaques and have found that plaque PAPP-A is also not complexed with proMBP. In this study, plaque PAPP-A was used to help identify antibodies whose binding to PAPP-A requires the presence of proMBP.

LABELING OF ANTIBODIES WITH LANTHANIDE CHELATE AND BIOTIN

Europium ([Eu.sup.3+]) chelate labeling and antibody biotinylation were performed as reported previously (21). Bovine serum albumin was added to a final concentration of 1 g/L to biotinylated and [Eu.sup.3+]-labeled antibodies, and the solutions were filtered through a 0.22 [micro]m pore size filter and stored at 4[degrees]C.

ANTIBODY COMBINATIONS

All antibodies were tested in pairs with each used as a capture or a detection antibody. A 1-step sandwich assay format was used with 100 mIU/L of either pregnancy serum PAPP-A or plaque PAPP-A and a blank solution. The procedure used was similar to that described earlier (12,22).

IMMUNOASSAYS

All immunoassays involved in this study were developed with a 2-site sandwich assay format. The capture antibodies were biotinylated, and detection antibodies were labeled with [Eu.sup.3+]. The assays were performed in a conventional 96-well (8 x 12) microplate format as follows: We first immobilized the capture antibody on the surface of streptavidin-coated microtiter wells by incubating biotinylated mAb (300 ng in 50 [micro]L of assay buffer per well) for 60 min at room temperature with slow shaking. Unbound biotinylated antibody was removed from the wells by washing. A 10-[micro]L volume of calibrator or sample and 200 ng of the [Eu.sup.3+]-labeled detection antibody in 20 [micro]L of diluent A were then added, in triplicate, to the wells that contained the immobilized capture antibody. The wells were incubated for 60 min at 37[degrees]C without shaking on the iEMS Incubator/ Shaker (Labsystems Oy) and washed 6 times. After that, the wells were dried for 5 min, and the time-resolved [Eu.sup.3+] fluorescence was measured directly from the dry surface with a Victor[TM] multilabel counter. The concentrations of unknown samples were obtained by calibrating their fluorescence signals against a calibration curve derived from the calibrator wells by the MultiCalc immunoassay prognm (Perkin-Elmer Life Sciences) with a spline algorithm on logarithmically transformed data.

GEL-FILTRATION CHROMATOGRAPHY AND ANALYSIS OF FRACTIONS

A third-trimester serum pool was fractionated by gel-filtration chromatography as reported previously (12). Briefly, the fractionation was carried out on a Superose[TM] 6 PC 3.2/30 column (Amersham Biosciences) equilibrated and eluted with 50 mmol/L sodium phosphate buffer (pH 7.0) containing 0.15 mol/L NaCl and 0.2 g/L Na[N.sub.3]. A 100-[micro]L sample (serum diluted 2-fold in elution buffer and filtered through 0.22 [micro]m pore-size filter) was loaded. After 0.9 mL of buffer had eluted from the column, 25-[micro]L fractions were collected. The fractions were then diluted 51-fold with 50 mmol/L Tris buffer (pH 7.8) containing 0.15 mol/L NaCl, 0.5 g/L Na[N.sub.3], and 5 g/L bovine serum albumin, and analyzed with the immunoassays studied.

[FIGURE 1 OMITTED]

Results

SPECIFICITY OF INDIVIDUAL ANTIBODIES

All 22 antibodies were raised by standard hybridoma technology with pregnancy serum PAPP-A as immunogen. mAbs B5, B6, and A11 were known from previous studies to react with the proMBP subunit of the PAPP-A/ proMBP complex (12,15, 23, 24). As shown in Fig. 1, the epitopes of these 3 antibodies largely overlapped with each other because no signals were detected with combinations with these antibodies. Importantly, antibody combinations including any of the 3 antibodies were able to detect pregnancy serum-derived PAPP-A/proMBP complex but not plaque-extracted uncomplexed PAPP-A. mAbs A8, A9, and A16 behaved similarly to the above 3 proMBP-reactive antibodies in terms of reactivity with pregnancy serum PAPP-A and with plaque PAPP-A. In addition, the epitopes of mAbs A8, A9, and A16 also largely overlapped with each other, but were well separated from those of mAbs B5, B6, and A11 (Fig. 1). These results suggest that mAbs A8, A9, and A16 are likely proMBP-reactive antibodies. We also found that assays using mAb A11 and any one of mAbs A8, A9, and A16 were able to detect recombinant proMBP (Fig. 2), which confirms that mAbs A8, A9, and A16 react with proMBP. The rest of the antibodies investigated were PAPP-A subunit-reactive antibodies.

IMMUNODETECTION BY ASSAYS USING proMBPREACTIVE ANTIBODIES

The assays using proMBP-reactive antibodies, such as the ones using mAb A1 as capture and any of mAbs B5, B6, and A11 as tracer, or the assays using any of mAbs A8, A9, and A16 as capture and mAb A1 as tracer, were able to detect PAPP-A in pregnancy (Fig. 3) but were unable to detect serum PAPP-A in ACS patients (see the upper part of Table 1). The latter was detected by an assay incorporating 2 PAPP-A subunit-reactive antibodies: mAbs A1 and A5. Because the access of these 2 antibodies to the epitopes in PAPP-A, either complexed or not complexed with proMBP, was not affected, the assay was able to detect PAPP-A in pregnancy (Fig. 3) and in ACS (Table 1), but it did not detect recombinant proMBP (Fig. 2). The results obtained with the assays involving proMBP-reactive antibodies were consistent with the previous finding that PAPP-A in ACS is not co-Flexed with proMBP (12).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

ALTERATION OF A PREGNANCY PAPP-A EPITOPE IN ACS PAPP-A

The epitope recognized by mAb A4 is located in the PAPP-A subunit of PAPP-A/proMBP complex (Fig. 1). As shown in Fig. 4, the epitope was also observed in recombinant human PAPP-A that exists as a homodimer and does not contain proMBP (23). However, the location of the epitope related to mAb A4 was altered in ACS serum PAPP-A compared with that in pregnancy serum PAPP-A. This was evidenced by 2 assays involving mAb A4 (i.e., Bio-mAb A4/Eu-mAb A5 and Bio-mAb A4/EumAb B4): These 2 assays were capable of detecting PAPP-A in pregnancy (Fig. 3) but were almost incapable of detecting serum PAPP-A in ACS patients (see the lower part of Table 1). The locations of the 2 epitopes recognized by mAbs A5 and B4 did not change in ACS serum PAPP-A. The assays including either of the 2 antibodies, such as the one configured with mAbs A1 and A5 (Table 1) or the one configured with n-LAbs Al and B4 (12), performed well for the measurement of PAPP-A in pregnancy and in ACS sera. Thus, assays incorporating 2 antibodies with nonoverlapping epitopes in the PAPP-A subunit of PAPP-A/proMBP complex may still be unsuitable for the detection of serum PAPP-A in ACS patients.

IMMUNOREACTIVITY WITH HIGHLY PURIFIED RECOMBINANT PAPP-A

We tested all of the above assays involving a proMBP-reactive antibody with the recombinant PAPP-A. As shown in Fig. 5, recombinant PAPP-A detected by the assay configured with PAPP-A subunit-specific mAbs A1 and A5 was not detected by the assays involving any of the proMBP-reactive antibodies. These results are in good agreement with those obtained with either ACS serum PAPP-A or plaque PAPP-A.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Discussion

This is the first report concerning whether immunoassays developed for PAPP-A in pregnancy are also applicable for PAPP-A in ACS. Our results show that immunoassays for PAPP-A in pregnancy are not necessarily suited for PAPP-A in ACS. Immunoassays developed for PAPP-A in pregnancy do not detect PAPP-A in ACS in a manner similar to PAPP-A in pregnancy if (a) the immunoassay uses a proMBP-reactive antibody or (b) the immunoassay uses an antibody for which the epitope conformation in pregnancy-related PAPP-A differs from that in ACS. If epitope access is considerably hindered, as seen with mAb A4 in the mAb A4/mAb A5* and mAb A4/mAb B4* assays, the PAPP-A in ACS will hardly be detected. Apparently, the first situation is easily understandable and less likely to be overlooked. The second situation may be more easily neglected when applying or developing PAPP-A assays for ACS-associated samples.

The mechanisms behind our findings may be attributable to the differences in molecular structure between PAPP-A in pregnancy and PAPP-A in ACS. PAPP-A in ACS is not complexed with proMBP (12), whereas PAPP-A in pregnancy is complexed with proMBP (11, 25). It is therefore natural that all epitopes associated with the proMBP subunit of the PAPP-A/proMBP complex, including those that involve parts of both the proMBP and PAPP-A subunits, are not found in PAPP-A not complexed with proMBP. Furthermore, formation of the PAPP-A/proMBP complex involves extensive intra-and intermolecular disulfide rearrangements (13). This likely causes a series of conformational changes that make the complexed PAPP-A molecule different from the un-complexed PAPP-A molecule, although the details of these changes remain to be elucidated. Consequently, some conformation-dependent epitopes in the PAPP-A/ proMBP complex will not appear in PAPP-A not complexed with proMBP. On the other hand, it is also possible that the locations of some epitopes relative to other epitopes in the PAPP-A/proMBP complex are not the same as in PAPP-A not complexed with proMBP. The reasons that assays developed for PAPP-A in pregnancy may not recognize PAPP-A in ACS are thus understandable.

As shown in Fig. 1, some combinations that included 2 proMBP-reactive antibodies, e.g., pairs mAb A8/mAb B5, mAb A8/mAb B6, mAb A8/mAb A11A, mAb A11/mAb A8, mAb A11 /mAb A9, and mAb A11 /mAb A16, gave positive binding signals for plaque PAPP-A. This reactivity was not attributable to plaque PAPP-A because other combinations including each of those antibodies with any PAPP-A subunit-reactive antibody failed to detect plaque PAPP-A (Fig. 1). It is likely that the reactivity resulted from proMBP in complexes with other proteins (26) because the plaque PAPP-A used in the experiments was a rather crude preparation.

Another important issue relates to standardization of the calibrators used in the assays for PAPP-A in ACS. In this study, we used pregnancy serum pool-derived calibrators for the assays for PAPP-A in ACS. The reference material we used is WHO IRP 78/610, which is also derived from a pregnancy serum pool (27). As PAPP-A is present mainly as a complexed form in pregnancy serum, the calibrators, as well as the WHO reference material, are obviously not homologous to PAPP-A in ACS. Thus, there is a strong need to apply in these assays a material that truly represents PAPP-A in ACS. The use of recombinant PAPP-A may be a solution to the problem.

It is worth noting that assays that have been successfully used to measure PAPP-A in ACS serum are also able to detect PAPP-A in pregnancy. For example, the assay used by Bayes-Genis et al. (7) was configured with a polyclonal antibody as capture and a combination of mAbs as tracer. The assay detects PAPP-A either complexed or not complexed with proMBP. However, it is possible to generate antibodies or other specific binders that react exclusively with PAPP-A in ACS. Available technologies that can be adopted in this case include display technologies and hybridoma technologies using the uncomplexed PAPP-A form. The display technology may involve the generation of a phage-display antibody library or a synthetic library based on protein or nucleic acid scaffolds and, subsequently, the selection of the right antibody or binder with the desired properties (specificity and affinity) (28, 29). Successful production of these reagents will ultimately lead to the development of immunoassays that are unique for the determination of PAPP-A in ACS. This work is currently underway at our laboratory.

The present study has 2 limitations. First, all results concerning PAPP-A in ACS serum involved samples from 4 patients with ST-elevation myocardial infarctions. Although the results are believed to be the same for other forms of ACS, such as non-ST-elevation myocardial infarction and unstable angina, unfortunately there were no samples representing such conditions in the study. Second, plaque extracts were not from coronary atherosclerotic plaques of ACS patients because of the difficulties encountered in obtaining such plaque samples. However, we believe that PAPP-A extracted from atherosclerotic plaques of carotid or abdominal aortic aneurysms should be the same as PAPP-A found in coronary atherosclerotic plaques. Furthermore, all major findings with plaques were confirmed with recombinant proMBP, recombinant PAPP-A, and most importantly, with pregnancy and ACS serum samples.

In conclusion, we demonstrate 2 situations in which immunoassays for PAPP-A in pregnancy do not recognize serum PAPP-A in ACS patients. Care thus should be taken when applying assays developed for PAPP-A in pregnancy to samples from ACS patients. Moreover, when developing assays for PAPP-A in ACS, emphasis should be placed on careful antibody selection and subsequent extensive testing with clinical ACS samples.

We thank Dr. Michael Christiansen from State Serum Institute (Copenhagen, Denmark) for generously providing us with antibodies relevant for this study. This study was supported financially by a grant (Project 40279) from the National Technology Agency of Finland (TEKES).

References

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QIU-PING QIN, [1] * SAARA KOKKALA, [1] JUHA LUND, [2] NATALIA TAMM, [3] XUEZHONG QIN, [4] MADRI LEPKNTALO, [5] and KIM PETTERSSON [1]

Departments of [1] Biotechnology, and [2] Medicine, University of Turku, Turku, Finland.

[3] HyTest Ltd., Turku, Finland.

[4] J.L. Pettis Memorial Veterans' Medical Center, Loma Linda, CA.

[5] Department of Vascular Surgery, Helsinki University Central Hospital, Helsinki, Finland.

[6] Nonstandard abbreviations: PAPP-A, pregnancy-associated plasma protein-A; ACS, acute coronary syndrome(s); proMBP, proform of eosinophil major basic protein; and mAb, monoclonal antibody.

* Address correspondence to this author at: Department of Biotechnology, University of Turku, Turku, Finland. Fax 358-2-3338050; e-mail gigin@utu.fi.

Received July 28, 2005; accepted December 29, 2005.

Previously published online at DOI: 10.1373/clinchem.2005.058396
Table 1. ACS serum PAPP-A determined by 9 assays.

 PAPP-A, mIU/L

 ACS ACS ACS ACS
 serum serum serum serum
Assaya sample 1 sample 2 sample 3 sample 4

mAb A1/mAb A5 * 36.5 67.4 34.5 58.8
Assays involving proMBP
 subunit-reactive mAbs
 mAb A8/mAb A1 * 2.2 3.8 1.5 8.6
 mAb A9/mAb A1 * <3.0 <3.0 <3.0 <3.0
 mAb A16/mAb A1 * <3.0 <3.0 <3.0 <3.0
 mAb A1/mAb A11 * 3.1 3.3 2.1 4.1
 mAb A1/mAb B5 * 1.2 3.6 3.1 5.7
 mAb A1/mAb B6 * 3.8 5.3 5.6 5.6
Assays involving mAb A4
 mAb A4/mAb B4 * 7.5 11.8 4.7 8.8
 mAb A4/mAb A5 * 2.7 4.4 3.7 9.2

(a) * after antibody indicates that it is labeled with europium
chelate.
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Title Annotation:Proteomics and Protein Markers
Author:Qin, Qiu-Ping; Kokkala, Saara; Lund, Juha; Tamm, Natalia; Qin, Xuezhong; Lepantalo, Mauri; Pettersso
Publication:Clinical Chemistry
Date:Mar 1, 2006
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