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Method for diagnosing acute coronary syndrome

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20120264139 patent thumbnailZoom

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.

EXPERIMENTAL SECTION Materials and methods Reagents

ITC-TEKES Eu3+ fluorescent chelate of 4-[2-(4-isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)-amino]methyl}pyridine and biotin isothiocyanate (BITC) were obtained from Innotrac Diagnostics Oy. DELFIA assay buffer and wash solution were prepared as described previously (28). Assay buffer supplemented with 0.01% denatured mouse IgG and 0.02% native mouse IgG was referred to as modified assay buffer. Low-fluorescence 12-well Maxisorp microtitration strips (ultraviolet-quenched) were purchased from NUNC. Streptavidin-coated single wells and strips were obtained from Innotrac Diagnostics Oy. Bovine serum albumin (BSA) was purchased from Intergen. NAP-5™ and NAP-10™ columns were from Pharmacia Biotech. All other chemicals used were of analytical grade.

Six monoclonal antibodies denoted as mabB1, 2, 3, 4, 5, and 6, specific for the PAPP-A/proMBP complex, were gifts of Dr. Michael Christiansen from State Serum Institute, Denmark. Other eleven monoclonal antibodies denoted as mabA1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11, also specific for the PAPP-A/proMBP complex, were gifts of Dr. Maria Severina from HyTest Oy, Finland

Calibrators were prepared by diluting a filtrated (through a 0.22 μm pore size filter) pool of ten 3rd-trimester pregnancy sera in a buffer containing 60 g/L bovine serum albumin, 50 mmol/L Tris-HCl (pH 7.75), 15 mmol/L NaCl, and 0.5 g/L NaN3, 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). Levels of PAPP-A and proMBP were expressed in milliunits per liter. The calibrators were stored at −20° C. until use.

Serum Samples

Eight patients (4 males aged 57±5 years and 4 females aged 81±3 years) with ACS had prolonged chest pain accompanied by ST-segment elevation and abnormally increased levels of CKMB and cTn I. From these patients, serum samples were taken on admission to the Department of Cardiology, Turku University Central Hospital and at 1, 2, 4, 6, 24, 48 and 72 hours afterward. In addition, 2 first-trimester serum samples (gestational age: weeks 9 and 11) were included in this study. All the serum samples were obtained with informed consent. The procedures followed were in accordance with the Helsinki Declaration of 1975 as revised in 1996. All samples were stored at −20° C. (pregnancy samples) or −70° C. (ACS samples) before the measurement.

Labeling of Antibodies with Lanthanide Chelate and Biotin

Intrinsically fluorescent europium chelate was used for labeling of the antibodies (29). Labeling reactions were performed as reported previously (25). Briefly, antibody was labeled overnight (16-20 h) at room 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, Sweden) operated with 50 mmol/L Tris-HCl (pH 7.75), 15 mmol/L NaCl, 0.5 g/L NaN3 at 25 mL/h Fractions of 0.45 mL 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 of Eu3+ chelates per molecule of IgG.

Biotinylation of antibodies was conducted with a 50-fold molar excess of biotin-isothiocyanate in 50 mmol/L sodium carbonate buffer (pH 9.6) at room 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 (Amersham Biosciences AB) with 50 mmol/L Tris-HCl (pH 7.75), 15 mmol/L NaCl, 0.5 g/L NaN3 as eluent. BSA was added to a final concentration of 1 g/L, and the solution was filtered through a 0.22 μm pore size filter and stored at 4° C.

Epitope Mapping

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 (30). 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, Finland). 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 Victor™ 1420 multilabel counter (Perkin-Elmer Life Sciences, Wallac Oy, Finland).

Immunoassays

Two immunoassays were used in this study. One denoted as assay T, configured with the biotinylated mabA1 and europium labeled mabB4; and the other denoted as assay C, configured with the biotinylated mabA1 and europium labeled mabA11, were performed in a conventional microplate assay format with the iEMS Incubator/Shaker. For both assays, first, the biotinylated mabA1 was immobilized on to the surface of streptavidin-coated microtiter wells by incubating 300 ng of biotinylated mab A1 in 50 μL of DELFIA assay buffer per well for 60 min at RT with slow shaking. Unbound biotinylated antibody was removed by washing the wells. Then, for the assay T, 10 μL of calibrator or sample and 200 ng of the Eu3+-labeled mabB4 in 20 μl of modified assay buffer were added per well. The wells were incubated for 30 min at 37° C. with slow shaking and washed 6 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 calibrating their fluorescence signals against a calibration curve derived from the calibrator wells by the MultiCalc immunoassay program (Perkin-Elmer Life Sciences, Wallac Oy, Finland) with the use of a spline algorithm on logarithmically transformed data. For the 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 the Eu3+-labeled mabA11 in 30 μL of the modified assay buffer was added per well, and the wells were incubated for 30 min at 37° C. with slow shaking, and washed 6 times. The following steps were the same as for the assay T.

Gel Filtration Chromatography

This was carried out on a Superose™ 6 (3.2×300 mm) precision column PC3.2/30 (Pharmacia Biotechnology, Sweden) equilibrated and eluted with 50 mmol/L sodium phosphate buffer, pH 7.0, containing 0.15 mol/L NaCl, and 0.02% NaN3 at the flow rate of 0.04 mL/min. Fifty μl of sample (serum diluted twofold in elution buffer and filtered through 0.22 μm pore-size filter) was loaded. The column effluent was monitored at 280 nm, and after 0.6 ml of initial elution, 100-μL fractions were collected. The total run time was 75 min. The column was operated at 10° C. on a Pharmacia SMART system (Pharmacia Biotechnology, Sweden) and calibrated with the following proteins: Thyroglobulin (669 kDa), Apoferritin (481 kDa), Immunoglobulin G (160 kDa), Bovine serum albumin (67 kDa), Chymotrypsinogen A (25 kDa) and Ribonuclease A (13.7 Kda). Both first-trimester pregnancy serum specimens and ACS serum samples were fractionated by the gel filtration chromatography.

Adsorption of PAPP-A from Normal Serum Samples

This was performed with the use of streptavidin-coated microtitration wells and biotinylated mabA1 or biotinylated A11. Thirty μl of serum sample was added to each well in which 300 ng of bioA1 or 400 ng of bio-mabA11 was already immobilized on the surface. For bioA1, incubation was carried out at RT for 1 h with slow shaking. After that, 10 μl of treated serum taken from each well was applied to above immunoassays for PAPP-A measurement. For bioA11, incubation was carried out at RT for 1 h with slow shaking, the serum sample was then transferred to another coated well and followed with 1 h incubation. After that, transfer step and incubation was repeated until third incubation was conducted. Finally, 10 μl of treated serum was applied to above immunoassays for PAPP-A measurement.

Statistical Analysis

Statistical analysis was conducted using StatView (SAS Institute, Cary, USA).

Results Epitope Map of Pregnancy PAPP-A Defined by 17 Mabs

A schematic epitope map shown in FIG. 1 was constructed according to data obtained from every possible two-site combination of the antibodies. Relationships of the location of each antibody 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 binding to PAPP-A subunit of the PAPP-A/proMBP complex, whereas B5 and B6 were reactive with proMBP subunit of the PAPP-A/proMBP complex (5,31). MabA11 was able to form sandwiches with all the other mabs except for mabB5 and B6, indicating that it should react with proMBP subunit of the PAPP-A/proMBP complex. Of the rest 14 antibodies reactive with PAPP-A subunit of the PAPP-A/proMBP complex, two antibodies (A10 and B4) did not share their epitopes with other antibodies.

Calibration Curves

The calibration curves of the two assays shown in FIG. 2 were obtained with a standard material derived from a pool of third-trimester pregnancy sera. Both curves were linear over the concentrations ranging from 1.0 mIU/L to 300 mIU/L. For the assay T, assay imprecision was low with intra-assay concentration CVs of below 10 over the range from 1.0 mIU/L to 300 mIU/L. For the assay C, assay imprecision was over 20% at 1.0 mIU/L and below 15 over the range from 3.0 mIU/L to 300 mIU/L. More importantly, both calibration curves were parallel to each other, indicating that PAPP-A in the standard material is equally detected by the two assays.

Molecular Profile and Immunoreactivity of Pregnancy PAPP-A

A first-trimester serum sample was fractionated by size-exclusion chromatography and the fractions were analyzed by the two immunoassays. Pregnancy PAPP-A revealed by the two assays as a single peak eluted at the same position in which thyroglobulin (669 kDa) was eluted (shown in FIG. 3). Furthermore, the two peaks obtained by the two assays totally overlapped with each other.

The PAPP-A concentrations measured by the assay T were slightly higher than those measured by the assay C.

PAPP-A in ACS Patients

PAPP-A levels in serial serum samples from 4 patients with ACS were measured by the two assays. Using the assay T, PAPP-A levels above the reference level of 5.68 mIU/L (22) were observed in all 4 patients at different times after the onset of chest pain (shown in FIG. 4). Although the degree of maximal increase in PAPP-A levels was varied, a marked increase in PAPP-A levels appeared in all the 4 ACS patients early within 2 h after the onset of chest pain. Using the assay C, no significant increase in the levels of PAPP-A was found in these 4 patients, in which the concentrations of PAPP-A were below 4 mIU/L for all serum samples. The results show that ACS specific PAPP-A present in the circulation is undetectable by the proMBP-reactive antibody.

Molecular Profile and Immunoreactivity of ACS PAPP-A

Four serum samples with markedly increased level of PAPP-A obtained from another 4 patients with ACS were fractionated by size-exclusion chromatography. Fractions were analyzed by the assay T. A single peak of PAPP-A immunoreactivity eluted at a position in which apoferritin (481 kDa) was found (shown in FIG. 5). Elution patterns from all 4 ACS serum samples were the same regardless of the concentrations of PAPP-A, and clearly shifted from the two pregnancy samples (669 kDa). The difference in molecular size between the pregnancy PAPP-A and ACS PAPP-A was clearly demonstrated in FIG. 5, and became much more pronounced in FIG. 6 when fractionation was conducted with a smaller fraction volume (25 μl).

PAPP-A in Normal Subjects

Serum PAPP-A levels in normal subjects (n=130, aged between 50 and 69 years) were less than 7.6 mIU/L, with a median value of 3.0 mIU/L. Such levels of circulating PAPP-A found in normal subjects were detectable not only by the assay T but also by the assay C.

FIG. 7A shows that low levels of circulating PAPP-A found in normal subjects were detectable by the assay T as well as by the assay C. In addition, adsorption treatment could be used to remove PAPP-A from the normal sera by either PAPP-A subunit-specific antibody A1 (FIG. 7A) or proMBP-specific antibody A11 (FIG. 8A).

PAPP-A in Acs Subjects

PAPP-A in ACS patients can be classified into two categories, i.e., proMBP complexed form and proMBP uncomplexed form, which together form the total PAPP-A detected by the assay T. The proMBP complexed form constitutes the basal level of PAPP-A and can be specifically detected by the assay C. The proMBP uncomplexed form relates to the ACS and can be specifically determined by delta value obtained from use of the both assays mentioned above or dual-label assay or blocking assay or, in particular, free PAPP-A specific assay.

FIG. 7B shows that PAPP-A (both proMBP-complexed and -uncomplexed forms) in ACS patients could be measured by the assay T, whereas the proMBP-complexed form (ACS-irrelevant) could be measured by the assay C. Adsorption with mabA1 effectively removed both the proMBP-complexed and -uncomplexed forms (FIG. 7B), however, adsorption with mabA11 only removed the proMBP-complexed form (FIG. 8B), thereby allowing the uncomplexed form, namely free PAPP-A to be detected.

Analysis of fractions from ACS serum samples and from pregnancy serum sample reveals that prominent signals detected by the assay C indeed coincided with that of pregnancy PAPP-A, which is known to be a complex with proMBP (see FIG. 9).



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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


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