FIELD OF THE INVENTION
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This invention relates to a bioaffinity assay for quantitative determination in a sample of free PAPP-A, defined as the pregnancy associated plasma protein A (PAPP-A) that is not complexed to the proform of major basic protein (proMBP). The invention relates further to a method for diagnosing acute coronary syndrome in a person by using free PAPP-A as a marker.
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OF THE INVENTION
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Pregnancy-associated plasma protein A (PAPP-A) was first identified in the early 1970s as a high-molecular weight constituent found in human late pregnancy serum (1). The concentration in serum increases with pregnancy until term (2). PAPP-A was initially characterized as a homotetramer (1, 3), but it was later demonstrated that circulating PAPP-A in pregnancy was a disulfide-bound 500-kDa heterotetrameric 2:2 complex with the proform of eosinophil major basic protein (proMBP), denoted as PAPP-A/proMBP (4). However, pregnancy serum or plasma is also reported to contain traces (<1%) of uncomplexed PAPP-A (5).
PAPP-A and proMBP are both produced in the placenta during pregnancy but mainly in different cell types. By in situ hybridization, it has been revealed that the vast majority of PAPP-A is synthesized in the syncytiotrophoblast, and all proMBP is synthesized in extravillous cytotrophoblasts (6). Analyses from cloned cDNA demonstrate that the PAPP-A subunit is a 1547-residue polypeptide (7). It contains an elongated zinc-binding motif, three Lin-notch repeats and five short consensus repeats (8).
ProMBP is a glycosylated proteoglycan composed of a strongly acidic 90-residue propiece and a highly basic 117-residue mature form of MBP (9,10). The latter is a cytotoxic protein present in granules of the eosinophil leukoucyte (11). It is released from the eosinophil leukocyte by degranulation, and plays multiple roles in the effector functions of these cells (12). Although in eosinophils mature MBP is generated by proteolytic processing of proMBP, no evidence indicates that MBP can be generated from proMBP of the PAPP-A/proMBP complex. In terms of the role of proMBP in the PAPP-A/proMBP complex, there are studies showing that proMBP acts in vitro as a proteinase inhibitor of PAPP-A (5,13). In addition to PAPP-A, proMBP also forms covalent complex with either angiotensinogen or complement C3dg (14). But the function of proMBP in other complexes remains unknown.
Recently, PAPP-A has been found to be a protease specific for insulin-like growth factors (IGF) binding protein (IGFBP)-4 as well as for IGFBP-5 in vitro (15,16). Notably, the cleavage of IGFBP-4 is in an IGF-dependent manner, whereas the cleavage of IGFBP-5 in an IGF-independent manner. However, the physiological function of PAPP-A in vivo remains to be identified. Insulin-like growth factors-I and -II (IGF-I and IGF-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 IGF binding proteins, which bind the IGFs and block them from binding to the receptor (17). Cleavage of IGFBP-4 and -5 by PAPP-A causes release of bound IGF, thereby increasing bioavailable IGF for interactions with IGF membrane receptors.
Clinically, reduced serum levels of PAPP-A are associated with Down's syndrome (DS) pregnancies (18). As a marker, PAPP-A is now commonly used for screening for DS in the first trimester (19). Only recently, it has been shown that PAPP-A is present in unstable atherosclerotic (coronary and carotid) plaques (20,21), and that its circulating levels are elevated in patients with acute coronary syndromes (ACS) (20,22). Furthermore, occurrence of PAPP-A in the circulation is an independent prognostic stratifier in patients with coronary artery disease (23). So far little is known about the role of PAPP-A in the plaques. Nonetheless, it has been suggested that increased bioavailability of IGFs through IGFBP-4 proteolysis observed in ACS plays a crucial role in the progression of both coronary atherosclerosis and restenosis (20,24).
Technically, measurability of PAPP-A in the circulation is closely associated with PAPP-A molecule structure. Whether the molecular structure of PAPP-A found in the blood of pregnant women is the same as that found in the blood of ACS patient is particularly important. Until now there is no report dealing with this critical issue. And all the assays used to date for PAPP-A measurement in both situations are based on the antibodies specific for PAPP-A subunit of PAPP-A/proMBP complex (20,25,26,27). From a methodological point of view, this fact makes the circulating PAPP-A in pregnancy indistinguishable from that in ACS.
Here we, for the first time, provide data showing that circulating PAPP-A molecule in pregnancy is different from that in ACS. These findings have important clinical implications for earlier and more specific detection of atherosclerosis related-PAPP-A in the circulation.
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OF THE INVENTION
The object of this invention is to provide a more sensitive and specific method for diagnosing individuals at risk of acute coronary syndrome at an early stage. Particularly, the aim is to achieve a diagnosing method superior to the commonly used assay based on cardiac troponin I and to the proposed assay based on the use of total PAPP-A as a marker.
Thus, according to one aspect, this invention concerns a bioaffinity assay for quantitative determination in a sample of free PAPP-A, defined as the pregnancy associated plasma protein A (PAPP-A) that is not complexed to the proform of major basic protein (proMBP). According to the invention, free PAPP-A is determined either
i) as a calculated difference between measured total PAPP-A and measured PAPP-A complexed to proMBP, or
ii) by a direct bioaffinity assay measuring only free PAPP-A.
According to another aspect, the invention concerns a method for diagnosing an acute coronary syndrome in a person by using as marker either free PAPP-A as such or a ratio
free PAPP-A/total PAPP-A,
free PAPP-A/PAPP-A complexed to proMBP, or
PAPP-A complexed to proMBP/total PAPP-A.
According to a third aspect, the invention concerns a binder which binds the free PAPP-A but not the PAPP-A complexed to proMBP.
BRIEF DESCRIPTION OF THE DRAWINGS
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FIG. 1 shows a schematic epitope map of the PAPP-A/proMBP complex. Overlapping circles indicate no possible sandwich formation. Touching circles indicate interfering sandwich formation. Separate circles indicate independent epitopes. Mabs defining epitopes accessible only on proMBP are marked with thick circles, while mabs defining epitopes accessible on PAPP-A are marked with thin circles.
FIG. 2 shows calibration curves and imprecision profiles for assay T (assay T=assay for total PAPP-A) configured with two PAPP-A subunit-specific monoclonal antibodies (A1/B4) and assay C (assay C=assay for PAPP-A complexed to proMBP) made from a proMBP subunit-specific monoclonal antibody for detection and a PAPP-A subunit-specific monoclonal antibody for capture (A1/A11). Four replicates were used for each concentration. Curves with filled characters relate to counts and curves with open characters relate to concentration CV.
FIG. 3 shows gel filtration of a first-trimester serum sample on a Superose™ 6 precision column (PC3.2/30). PAPP-A was detected by assay T, and by assay C. The PAPP-A/proMBP eluted as a single peak at the position where thyroglobulin (669 kDa) was eluted.
FIG. 4 shows serum kinetics of PAPP-A for patients with ACS. PAPP-A was detected by the assay T, and by the assay C.