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Proteomics and Protein Markers |
1 Department of Biotechnology, University of Turku, Turku, Finland.
2 Department of Medicine, University Central Hospital of Turku, Turku, Finland.
3 HyTest Ltd., Turku, Finland.
4 Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland.
aAddress correspondence to this author at: Department of Biotechnology, University of Turku, Tykistökatu 6A, Turku 20520, Finland. Fax 358-2-3338050; e-mail qiqin{at}utu.fi.
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Abstract |
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Methods: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a SuperoseTM 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients.
Results: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of 
700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of 530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A.
Conclusions: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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During pregnancy, the vast majority of PAPP-A is synthesized in the placental syncytiotrophoblast, whereas all proMBP is synthesized in extravillous cytotrophoblasts (6). Analyses of cloned cDNA demonstrated that the PAPP-A subunit is a 1547-residue polypeptide (7) containing an elongated zinc-binding motif, three Lin-notch repeats, and five short consensus repeats (8).
Recently, PAPP-A has been found to be a protease specific for insulin-like growth factor-binding protein 4 (IGFBP-4) as well as for IGFBP-5 in vitro (9)(10). IGF-I and -II play an important role in promoting cell differentiation and proliferation in a variety of biological systems, mediated mainly through the type 1 IGF receptor. The biological activities of IGF-I and -II are modulated by six homologous high-affinity IGFBPs (11). Cleavage of IGFBP-4 and -5 by PAPP-A causes release of bound IGF and presumably increases IGF bioavailability for interactions with membrane receptors. In the PAPP-A/proMBP complex, proMBP functions as an inhibitor of PAPP-A proteolytic activity (5). The physiologic function of PAPP-A in regulating IGF signaling in vivo remains to be identified.
Decreased serum concentrations of PAPP-A are associated with Down syndrome pregnancies (12) and are now commonly used for screening in the first trimester (13). Only recently, it has been shown that PAPP-A is present in unstable atherosclerotic (coronary and carotid) plaques (14)(15) and that its circulating concentrations are increased in patients with acute coronary syndrome (ACS) (14)(16). At present, little is known about the role of PAPP-A in the plaques. Increased bioavailability of IGFs through PAPP-A-mediated IGFBP-4/5 proteolysis could play a crucial role in the progression of both coronary atherosclerosis and restenosis (14)(17). We have previously shown that occurrence of PAPP-A in the circulation is an independent prognostic stratifier in patients with unstable coronary artery disease (18). The results support, at least indirectly, the view that PAPP-A (either alone or via IGFs) is a mediator of the adverse events that promote atherogenesis.
The measurability of PAPP-A in the circulation is closely associated with its molecular structure. All of the assays used to date for PAPP-A measurement are based on the antibodies specific for the PAPP-A subunit (14)(19)(20)(21). From a methodologic point of view, the fact that PAPP-A in pregnancy occurs in a complexed form raises the question of whether other molecular forms may exist, e.g., in ACS. The possible alterations in molecular structure could affect assay performance and, consequently, the information obtained from PAPP-A measurements. The purpose of this study, therefore, was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy.
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Materials and Methods |
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Eleven monoclonal antibodies (mabs) denoted as A1, -2, -3, -4, -5, -6, -7, -8, -9, -10, and -11, specific for the PAPP-A/proMBP complex, were obtained from HyTest Ltd., Finland. Other six mabs, denoted as B1, -2, -3, -4, -5, and -6, also specific for the PAPP-A/proMBP complex, were gifts of Dr. Michael Christiansen from State Serum Institute (Copenhagen, Denmark).
Calibrators were prepared by diluting a filtered (through a 0.22 µm pore size filter) pool of 10 third-trimester pregnancy sera in a buffer containing 60 g/L bovine serum albumin, 50 mmol/L Tris-HCl (pH 7.75), 150 mmol/L NaCl, and 0.5 g/L NaN3 and were calibrated against the pooled third-trimester pregnancy serum-derived WHO IRP 78/610 for pregnancy-associated proteins (WHO International Laboratory for Biological Standards, Statens Serum Institut, Copenhagen, Denmark). PAPP-A/proMBP concentrations were reported in mIU/L. The calibrators were stored at –20 °C until use.
serum samples
Samples were obtained from eight patients, four males [mean (SD) age, 63 ((13)) years] and four females [72 ( (16)) years] with typical ST-elevation MI, at admission; at 1, 2, 4, 6, 24, 48 h afterward; and at discharge. In addition, three first-trimester serum samples (gestational ages, 9, 10, and 11 weeks) and one third-trimester serum sample (gestational age, 33 weeks) were included. All serum samples were obtained after written informed consent. The local ethics committee had accepted the study protocol. All samples were stored at –20 °C (pregnancy samples) or –70 °C (MI samples) before use.
labeling of antibodies with lanthanide chelate and biotin
Intrinsically fluorescent europium chelate was used for labeling the antibodies (23). Labeling reactions were performed as reported previously (19). Briefly, antibody was labeled overnight (16–20 h) at ambient temperature with a 100-fold molar excess of chelate in 50 mmol/L sodium carbonate buffer (pH 9.6). The labeled antibodies were separated from excess free chelate and aggregated proteins on a Superdex 200 HR 10/30 gel-filtration column (Pharmacia Biotech) with an elution buffer of 50 mmol/L Tris-HCl (pH 7.75), 150 mmol/L NaCl, 0.5 g/L NaN3. The flow rate was 15 mL/h, and 0.45-mL fractions were collected. The fractions containing labeled antibody were pooled, and the degree of labeling was determined with a europium calibration solution. The labeling degrees of the antibodies were between 5 and 15 Eu3+ chelates per IgG molecule.
Antibodies were biotinylated by incubation with a 50-fold molar excess of biotin isothiocyanate in 50 mmol/L sodium carbonate buffer (pH 9.6) at ambient temperature for 3 h. The biotinylated antibody was separated from free biotinylation reagent by passing the reaction mixture through NAP-5 and NAP-10 columns with 50 mmol/L Tris-HCl (pH 7.75), 150 mmol/L NaCl, 0.5 g/L NaN3 as elution buffer. Bovine serum albumin was added to a final concentration of 1 g/L to biotinylated and Eu3+-labeled antibodies, and the solutions were filtered through a 0.22 µm pore size filter and stored at 4 °C.
epitope mapping
An epitope map provided information on the gross specificity of each mab. This included information on (a) whether the mab reacted with an epitope accessible in the free form of PAPP-A as well as in the complexed form of PAPP-A, (b) whether the mab reacted with a proMBP epitope on the PAPP-A/proMBP complex, (c) whether the mab reacted with an epitope on the free form of PAPP-A only, and (d) whether the mab reacted with only the complexed form of PAPP-A, in which the epitope included part of the PAPP-A subunit and part of the proMBP subunit. All antibodies against the PAPP-A/proMBP complex were tested in pairs, with each used as a capture or a detection antibody. A one-step sandwich assay format was used together with a 100 mIU/L PAPP-A calibrator and a blank solution. The procedure used was similar to that described earlier (24). Briefly, 10 µL of PAPP-A calibrator or blank solution and 100 ng of Eu3+-labeled antibody in 20 µL of assay buffer were added, in triplicate, to wells directly coated with 0.4 µg of antibody. Subsequent incubation was performed at 37 °C for 10 min and 60 min with shaking (900 rpm; iEMS Incubator/Shaker; Labsystems Oy). After that, the wells were washed six times and dried with a stream of hot dry air for 5 min; the fluorescence was then measured with a VictorTM 1420 multilabel counter (Perkin-Elmer Life Sciences, Wallac Oy). In addition to results from the above combination studies, we also considered some previously published data (25) when we drew the schematic epitope map.
immunoassays
We developed two immunoassays. One, denoted as assay T and configured with biotinylated mab A1 and europium-labeled mab B4, was used for measurement of total PAPP-A; the other, denoted as assay C and configured with biotinylated mab A1 and europium-labeled mab A11, was used for measurement of PAPP-A complexed with proMBP. These two assays were performed in a conventional microplate assay format with the iEMS Incubator/Shaker. The biotinylated mab A1 was first immobilized on the surface of streptavidin-coated microtiter wells by incubation of 300 ng of biotinylated mab A1 in 50 µL of DELFIA assay buffer per well for 60 min at ambient temperature with slow shaking. Unbound biotinylated antibody was removed by washing the wells.
For assay T, 10 µL of calibrator or sample and 200 ng of the Eu3+-labeled mab B4 in 20 µL of modified assay buffer were then added per well. The wells were incubated for 30 min at 37 °C with slow shaking and washed six times. After that, the wells were dried for 5 min, and the time-resolved europium fluorescence was measured directly from the dry surface with the Victor 1420 multilabel counter. The concentrations of unknown samples were obtained by comparing their fluorescence signals against a calibration curve derived from the calibrator wells by the MultiCalc immunoassay program (Perkin-Elmer Life Sciences, Wallac Oy) with the use of a spline algorithm on logarithmically transformed data.
For assay C, 10 µL of calibrator or sample and 20 µL of the modified assay buffer were added to each well. The wells were incubated for 30 min at 37 °C with slow shaking and washed twice. After that, 300 ng of Eu3+-labeled mab A11 in 30 µL of the modified assay buffer was added per well; the wells were then incubated for 30 min at 37 °C with slow shaking and washed six times. The subsequent steps were the same as for assay T.
gel-filtration chromatography
Both pregnancy serum specimens and ACS serum samples were fractionated by gel-filtration chromatography. This was carried out on a SuperoseTM 6 PC3.2/30 precision column [3.2 (i.d.) x 300 mm; Amersham Bioscience AB] equilibrated and eluted with 50 mmol/L sodium phosphate buffer (pH 7.0) containing 0.15 mol/L NaCl and 0.2 g/L NaN3 at a flow rate of 0.04 mL/min. We loaded 50 µL of sample (serum diluted twofold in elution buffer and filtered through 0.22 µm pore size filter). The column eluate was monitored at 280 nm, and after the initial eluate volume reached 0.6 mL, 100-µL fractions were collected. The total run time was 75 min. The column was operated at 10 °C on a SMART system (Pharmacia LKB Biotechnology) and calibrated with the following proteins: thyroglobulin (669 kDa), apoferritin (481 kDa), IgG (160 kDa), bovine serum albumin (67 kDa), chymotrypsinogen A (25 kDa), and RNase A (13.7 kDa). For one of the pregnancy samples and one of the ACS samples, fractionations were also performed with 25-µL fractions collected. In this latter case, fraction collection was started after the initial eluate volume reached 0.9 mL.
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Results |
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) was constructed according to results obtained from every possible two-site combination of the antibodies and some previously published data (25). The relationships of the location of each antibody epitope were determined on the basis of whether the binding of one antibody would allow or interfere with independent binding of another antibody. Of the 17 mabs, B1, -2, -3, and -4 were previously shown to be specific for the PAPP-A subunit of the PAPP-A/proMBP complex (5)(9)(25), whereas mabs B5 and B6 were reactive with the proMBP subunit of the PAPP-A/proMBP complex (5)(25)(26). Mab A11 was able to form sandwiches with all of the other mabs except for mabs B5 and B6, indicating that it reacts with the proMBP subunit of the PAPP-A/proMBP complex. Of the other 14 antibodies reactive with the PAPP-A subunit of the PAPP-A/proMBP complex, 2 antibodies (A10 and B4) did not share epitopes with other antibodies. Of note, epitopes located on the PAPP-A subunit of the PAPP-A/proMBP complex are also accessible on the free PAPP-A molecule, indicating that these 14 antibodies are capable of reacting with free PAPP-A.
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analytic performance of the assays for total and complexed papp-a
Mabs A1 and B4, both of which are reactive with the PAPP-A subunit of the PAPP-A/proMBP complex, were chosen for assay T because the combination gave fast assay kinetics, a low background signal, and a high specific signal. Correspondingly, mab A11, which is reactive with the proMBP subunit of the PAPP-A/proMBP complex, and mab A1 were selected for assay C.
The calibration curves of the two assays (Fig. 2
) were obtained with a standard material derived from a pool of third-trimester pregnancy sera. Both curves were linear over the concentrations range 1.0–300 mIU/L. For assay T, the imprecision was low, with intraassay concentration CVs <10% over the range 1.0–300 mIU/L. For assay C, the CV was >20% at 1.0 mIU/L and <15% over the range from 3.0 to 300 mIU/L. More importantly, both calibration curves were parallel to each other, indicating that the PAPP-A in the standard material is detected equally by the two assays. Throughout this study, we found that the intraassay concentration CVs in the two assays were <10% for the majority of samples, and only a few samples had intraassay CVs between 10% and 15%. This imprecision was considered adequate for the purpose of this study.
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molecular profile and immunoreactivity of papp-a in pregnancy
The pregnancy serum samples were fractionated by size-exclusion chromatography, and the fractions were analyzed by the two immunoassays, T and C. Pregnancy-related PAPP-A was detected by both assays as a single peak eluting at the same position as thyroglobulin (669 kDa; Fig. 3
). In addition, the two peaks obtained by the two assays totally overlapped with each other. These findings confirm that pregnancy-related PAPP-A is actually complexed with proMBP.
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molecular profile and immunoreactivity of papp-a in mi
Four serum samples with markedly increased PAPP-A concentrations from patients with MI were fractionated by size-exclusion chromatography, and the fractions were analyzed by assay T. In contrast to the pregnancy samples, a single peak of PAPP-A immunoreactivity eluted at the same position as apoferritin (481 kDa). Elution patterns from all four MI serum samples were the same regardless of the concentrations of PAPP-A and were clearly shifted from the pregnancy samples (669 kDa; Fig. 4
). When fractionation was conducted with a smaller fraction volume (25 µL), this difference became even more visible (Fig. 5
) and molecular mass determinations became more accurate for ACS-related PAPP-A (519–548 kDa) and for pregnancy-related PAPP-A (680–718 kDa).
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papp-a in serial samples from mi patients
Finally, PAPP-A concentrations in unfractionated serial serum samples from four other MI patients were measured by the two assays. With assay T, concentrations above the previously determined reference cutoff of 5.68 mIU/L (16) were observed in all cases (Fig. 6
). Marked but variable increases in PAPP-A appeared in all patients within 3 h after the onset of chest pain. With assay C, the PAPP-A concentrations detected were <4 mIU/L in all serum samples (Fig. 6
). The results were also confirmed by an assay using another proMBP-reactive antibody, i.e., B6 (data not shown). These data indicate that the ACS-specific PAPP-A present in the circulation is undetectable by the proMBP-reactive antibody.
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Discussion |
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In ACS, PAPP-A concentrations seldom exceed 30 mIU/L, which is equivalent to 10 µg/L (16)(18). Such low concentrations make it difficult to detect ACS-related PAPP-A directly from blood samples by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and Western blot analyses. Therefore, other detection technologies with high sensitivity and specificity are needed for relevant investigations. Time-resolved fluorometry of lanthanide chelates is one of the most sensitive current detection technologies (27). Using this technology and specific antibodies, we developed two immunoassays for total PAPP-A and PAPP-A complexed with proMBP. As expected, the superior sensitivities of these assays allowed detection of total PAPP-A and proMBP-complexed PAPP-A directly from blood samples.
There are two reasons that the epitope on proMBP might be undetectable by mab A11 or mab B6: (a) the epitope can be lost either by lack of the subunit that bears the epitope or by a modification that causes changes in molecular conformation; or (b) the epitope can be blocked from binding to the antibody by covalent linking of the epitope to another macromolecular substance. Our finding that ACS-related PAPP-A is clearly smaller in molecular size than pregnancy-related PAPP-A makes modification of the PAPP-A/proMBP complex, including bridging to an additional substance, very unlikely. Furthermore, our data from the present study and additional recovery experiments (Q.P. Qin and S. Kokkala, unpublished results) using ACS sera to which complexed PAPP-A was added (recovery, 90%) do not support the possibility that the assay for complexed PAPP-A is negatively affected by possible soluble interfering factors present in ACS sera. The most probable explanation, therefore, is that ACS-related PAPP-A in the circulation is not complexed with proMBP.
Apart from pregnancy, PAPP-A has been found to be secreted by a variety of cultured human cells, such as fibroblasts, osteoblasts (9), ovarian granulosa cells(28), endometrial stromal cells (29), and coronary artery vascular smooth muscle cells (30). Furthermore, studies using immunohistochemistry and in situ hybridization identified in vivo PAPP-A produced in the ovary (31), in vascular plaques (14), and in healing human skin(32). However, only PAPP-A isolated from medium conditioned by human fibroblasts and produced by recombinant expression has been shown to be a 400-kDa dimer (5)(9).
PAPP-A secreted by the cells mentioned above in conditioned culture media has been shown to have proteolytic activity. However, having protease activity alone cannot be used as evidence that PAPP-A is present as a free form. In pregnancy, although >99% of PAPP-A is present in the complexed form, pregnancy serum and purified pregnancy-related PAPP-A have been shown to have protease activity (5)(9)(33). The measurable protease activity in pregnancy serum or pregnancy-related PAPP-A has been attributed to the presence of <1% of an uninhibited PAPP-A dimer or perhaps an incompletely inhibited 2:1 PAPP-A/proMBP complex (5).
Recent research has indicated that inflammation is pivotal in the development of atherosclerosis (34)(35). Inflammation contributes substantially to every stage of an atherosclerotic plaque. IGF-1 binds to the type I IGF receptor, which is present on many cell types, including vascular smooth muscle cells, endothelial cells, and macrophages, which are often found in the fibrous cap and around the lipid core of the atherosclerotic plaque (17)(35). In macrophages, IGF promotes excess LDL-cholesterol uptake; release of proinflammatory cytokines, e.g., tumor necrosis factor 
; and chemotaxis. This inflammatory environment contains many matrix-degrading metalloproteinases and is able to digest the fibrous cap that overlies the lipid-rich core, leaving the plaque vulnerable to rupture (17)(35). PAPP-A cleaves IGFBP-4 and -5 in vitro (9)(10) and may function similarly to enhance local IGF bioavailability. PAPP-A has been shown to be present in unstable atherosclerotic plaques but absent in stable ones (14). More recently, it has been demonstrated that PAPP-A production is significantly enhanced by inflammatory cytokines, such as tumor necrosis factor , in adult human fibroblasts (36). If this is also true in atherosclerotic plaques, then increased PAPP-A will further increase concentrations of local bioactive IGF, which in turn leads to formation of more foam cells and release of more proinflammatory cytokines, progressing to disruption of the plaque unless the chain of reactions is interrupted. In addition, locally increased IGF can also act on vascular endothelial cells and vascular smooth muscle cells. Under strong inflammatory influence, the ultimate outcome of IGF on these cells would be likely to promote instability of the atherosclerotic plaque (37)(38). We have found that the cumulative risk of a primary cardiac endpoint is positively associated with the concentrations of circulating PAPP-A (18). These results support the role of PAPP-A as a mediator of inflammatory reactions in the atherosclerotic plaque.
Our finding that ACS-related PAPP-A in circulation is not complexed with proMBP may have important clinical implications. In non-ACS individuals, variable concentrations of PAPP-A can be measured (16)(20)(39). The source of this immunoreactivity is not known, but it may originate from seminal fluid, follicle fluid, corpus luteum, testes, and other organs/tissues in which PAPP-A production has been reported (40). The PAPP-A concentrations determined for 80 non-ACS males 50–69 years of age varied from 1.51 to 7.59 mIU/L with a 97.5% upper reference limit of 5.68 mIU/L (16). The extent of increase in PAPP-A concentrations during ACS is widely variable but is usually <30 mIU/L (16)(18). Use of the upper reference limit as the decision limit means that a significant proportion of patients with ACS could be missed. We have previously reported (16) that the PAPP-A values in 5 of 14 MI patients were below the upper reference limit despite showing clear dynamic changes over time. Ideally, a decision limit not influenced by differences in individual basal PAPP-A concentrations should be identified. Our preliminary observations (data not shown) revealed that, in the absence of ACS, basal PAPP-A forms are detected almost equally by assay T for total PAPP-A and assay C for PAPP-A in complex with proMBP, implying that the basal immunoreactivity of PAPP-A is caused by the PAPP-A/proMBP complex. Low concentrations (<4 mIU/L) of this complexed form of PAPP-A were also detected in the MI patients in this study but exhibited no or little dynamic changes in the acute condition. Evidently, the form of PAPP-A associated with ACS is not in complex with proMBP. However, the PAPP-A assays currently in use for ACS measure total PAPP-A irrespective of whether it is complexed with proMBP. This constitutes a major limitation of PAPP-A assessment in ACS. One approach for determining ACS-related PAPP-A is to calculate the difference between the measured total PAPP-A and the measured PAPP-A complexed with proMBP. We predict that immunoassays designed to measure the circulating form of PAPP-A specifically released from unstable atherosclerotic plaques, as reported in this study, are likely to improve the clinical specificity and sensitivity of PAPP-A when used as a cardiac risk marker.
In conclusion, our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. Because early measurements of circulating PAPP-A may have diagnostic and prognostic value in patients who present with suspected ACS, these findings have important clinical implications for the design of assays to accurately measure circulating atherosclerosis-associated plasma protein A.
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Acknowledgments |
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Footnotes |
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References |
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-induced c-Jun and NF-
B activation and adhesion molecule expression. Circ Res 2002;90:1222-1230.The following articles in journals at HighWire Press have cited this article:
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  C. Gyrup, M. Christiansen, and C. Oxvig Quantification of Proteolytically Active Pregnancy-Associated Plasma Protein-A with an Assay Based on Quenched Fluorescence Clin. Chem., May 1, 2007; 53(5): 947 - 954. [Abstract] [Full Text] [PDF]
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S. Wittfooth, Q.-P. Qin, J. Lund, I. Tierala, K. Pulkki, H. Takalo, and K. Pettersson Immunofluorometric Point-of-Care Assays for the Detection of Acute Coronary Syndrome-Related Noncomplexed Pregnancy-Associated Plasma Protein A Clin. Chem., September 1, 2006; 52(9): 1794 - 1801. [Abstract] [Full Text] [PDF]
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S. Fredericks, V. Bertomeu-Gonzalez, I. Petrovic, D. W. Holt, and J. C. Kaski Comment on Immunoassays Developed for Pregnancy-Associated Plasma Protein-A (PAPP-A) in Pregnancy May Not Recognize PAPP-A in Acute Coronary Syndromes. Clin. Chem., August 1, 2006; 52(8): 1619 - 1620. [Full Text] [PDF]
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 J. C. Kaski and D. W. Holt Pregnancy-associated plasma protein-A and cardiovascular risk Eur. Heart J., July 2, 2006; 27(14): 1637 - 1639. [Full Text] [PDF]
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T. Mueller, B. Dieplinger, W. Poelz, and M. Haltmayer Increased Pregnancy-Associated Plasma Protein-A as a Marker for Peripheral Atherosclerosis: Results from the Linz Peripheral Arterial Disease Study Clin. Chem., June 1, 2006; 52(6): 1096 - 1103. [Abstract] [Full Text] [PDF]
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Q.-P. Qin, S. Kokkala, J. Lund, N. Tamm, X. Qin, M. Lepantalo, and K. Pettersson Immunoassays Developed for Pregnancy-Associated Plasma Protein-A (PAPP-A) in Pregnancy May Not Recognize PAPP-A in Acute Coronary Syndromes Clin. Chem., March 1, 2006; 52(3): 398 - 404. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) on a Superose 6 precision column (PC3.2/30).