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Method for diagnosing acute coronary syndrome

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Method for diagnosing acute coronary syndrome


PAPP-A complexed to proMBP/total PAPP-A. free PAPP-A/PAPP-A complexed to proMBP, or Furthermore, 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, ii) by a direct bioaffinity assay measuring only free PAPP-A. free PAPP-A is determined either i) as a calculated difference between measured total PAPP-A and measured PAPP-A complexed to proMBP, or 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), wherein
Related Terms: Acute Coronary Syndrome Papp-a Pregnancy Protein A

Inventors: Qiu-Ping Qin, Kim Pettersson
USPTO Applicaton #: #20120264139 - Class: 435 74 (USPTO) - 10/18/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Antigen-antibody Binding, Specific Binding Protein Assay Or Specific Ligand-receptor Binding Assay >To Identify An Enzyme Or Isoenzyme

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The Patent Description & Claims data below is from USPTO Patent Application 20120264139, Method for diagnosing acute coronary syndrome.

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FIELD OF THE INVENTION

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.

BACKGROUND 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.

OBJECT AND

SUMMARY

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

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.

FIG. 5 shows comparison by gel filtration of 4 ACS serum samples with two first-trimester serum samples on a Superose™ 6 precision column (PC3.2/30). PAPP-A was detected by the assay T. The ACS PAPP-A eluted as a single peak at the position where apoferritin (481 kDa) was eluted.

FIG. 6 shows comparison by gel filtration of 1 ACS serum sample (solid characters) with 1 first-trimester serum sample (open charactes) on a Superose™ 6 precision column (PC3.2/30). PAPP-A was detected by the assay T.

FIG. 7A shows PAPP-A in 3 normal serum samples (denoted as S1, S2 and S3) before and after adsorption treatment with mabA1. FIG. 7B shows PAPP-A in 2 ACS serum samples (denoted as ACS1 and ACS2) before and after adsorption treatment with mabA1.

FIG. 8A shows PAPP-A in 3 normal serum samples (denoted as S1, S2 and S3) before and after adsorption treatment with mabA11. FIG. 8B shows PAPP-A in 2 ACS serum samples (denoted as ACS1 and ACS2) before and after adsorption treatment with mabA11. PAPP-A levels were measured by the assay T.

FIG. 9 shows gel filtration of 4 ACS serum samples (denoted as S1, S2, S3 and S4) and a first-trimester serum sample on a Superose™ 6 precision column (PC3.2/30). Fractions were analysed using the assay C.

FIG. 10 illustrates box-and-whisker plots showing distributions of PAPP-A concentrations and delta values (defined as the difference between the PAPP-A values obtained by the assay T and the assay C) in normal subjects. Plot 1, PAPP-A concentrations determined by the assay T. Plot 2, PAPP-A concentrations determined by the assay C. Plot 3, Delta values derived from PAPP-A concentrations measured by the two assays. The boxes indicate the 25th-75th percentiles; the whiskers indicating the 5th and 95th percentiles. All values above the 95th percentile and below the 5th percentile are plotted separately as •. The horizontal lines indicate the medians; and the dashed boxes indicate the means.

FIG. 11 shows application of delta values (lines with open circles) in ACS patients as compared to the use of total PAPP-A concentrations (lines with solid circles). Delta values and the relevant decision limit are normalized according to the 97.5% upper reference limit for total PAPP-A concentrations. The dotted line indicates the decision limit for both the delta values and total PAPP-A concentrations.

DETAILED DESCRIPTION

OF THE INVENTION

The term “free PAPP-A” shall be interpreted to include any PAPP-A that is not complexed to the proform of major basic protein (proMBP). Thus, “free PAPP-A” will include absolutely free PAPP-A as well as PAPP-A bound to any substance except for proMBP.

The term “binder” shall be interpreted to especially include antibodies and their fragments (optionally genetically engineered), aptamers and protein scaffold derived binders, such as affibodies or fluorobodies. However, the term “binder” is not restricted to the aforementioned examples. Any binder useful in a bioaffinity assay shall be understood to be covered by the definition.

According to one preferable embodiment, free PAPP-A is determined as a calculated difference between measured total PAPP-A and measured PAPP-A complexed to proMBP.

This alternative can, for example, be performed by use of two separate assays, where one aliquot of the sample is exposed to a binder which binds total PAPP-A and the binder is detected to give total PAPP-A. Another aliquot of the sample is exposed to a binder which binds only PAPP-A complexed to proMBP. The binder is detected to give PAPP-A complexed to proMBP. Finally, the amount of free PAPP-A is calculated as a difference between determined total PAPP-A and PAPP-A complexed to proMBP. The two assays can be competitive assays, or more preferably non-competitive sandwich assays, where the specific binders are either capture binders or detecting (labelled) binders.

Alternatively, free PAPP-A and PAPP-A complexed to proMBP can be measured in one single dual analyte assay. The sample can be exposed to a capture binder, which binds total PAPP-A, and to two detecting binders labelled with different labels, so that the first detecting binder labelled with the first label is directed to an epitope present in any PAPP-A molecule, where the signal of the first label is used to give total PAPP-A. The second detecting binder labelled with a second label is directed to an epitope in the proMBP subunit of the molecule, where the signal of the second label is used to give exclusively PAPP-A complexed to proMBP.

The wording “epitope in the proMBP subunit of the molecule” shall be understood to cover epitopes solely within said proMBP subunit as well as epitopes which are partly located in the proMBP subunit and partly in another part of the PAPP-A molecule. Thus, PAPP-A complexed to proMBP can also be measured specifically by binders that only react with epitopes which are partly located in the proMBP subunit and partly in another part of the PAPP-A molecule.

According to another preferable embodiment, free PAPP-A is determined by a direct bioaffinity assay measuring only free PAPP-A. This can, according to one alternative, be performed by exposing the sample to an antibody (including antibody fragments such as Fab and single chain variable (scFv) fragment) or other binder which binds the free PAPP-A but not the PAPP-A complexed to proMBP and detecting the antibody or other binder to give free PAPP-A. Such an antibody or other binder could, for example, be raised to an epitope of PAPP-A which is available only in the molecules not bound to proMBP, such as in the region of amino acids from 381 to 652. A polyclonal antibody specific for free PAPP-A can be raised by immunizing a host animal such as rabbit and sheep with free PAPP-A and an immune adjuvant. While a monoclonal antibody specific for free PAPP-A can be obtained by using hybridoma technology and the same immunogen (here the host animal for immunization is usually mouse). Additionally, an antibody or its fragments such as Fab and single-chain variable fragment (scFv) specific for free PAPP-A can be generated using phage display from either a synthetic or a naïve antibody library. Free PAPP-A can be made available from ACS plaques or from pregnancy PAPP-A that is free of proMBP or from recombinant expression of PAPP-A encoding DNA sequence.

Alternatively, the bioaffinity assay measuring only free PAPP-A could be carried out by making PAPP-A complexed to proMBP non-capable of participating in the bioaffinity reaction in which the sample is exposed to an antibody or other binder binding total PAPP-A. There are two approaches towards achieving this goal. One relates to the use of adsorption as already demonstrated in FIG. 8B, PAPP-A complexed to proMBP was removed in a preceding step by adsorption with mabA11, which then allowed measurement of free PAPP-A. The other relates to the use of blocking strategy in which access for certain PAPP-A subunit-specific antibody or other binder to its epitope is blocked due to the binding of a proMBP reactive antibody/other binder either derivatized with a special group or not. Blocking can take place in a preceding step or simultaneously with the assay. In this way, free PAPP-A can be effectively measured as well.

The invention will be illuminated by the following non-restrictive Experimental Section.



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stats Patent Info
Application #
US 20120264139 A1
Publish Date
10/18/2012
Document #
13536810
File Date
06/28/2012
USPTO Class
435/74
Other USPTO Classes
International Class
01N33/573
Drawings
11


Acute Coronary Syndrome
Papp-a
Pregnancy
Protein A


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