HK1208264B - Means and methods applying sflt-1/plgf or endoglin/plgf ratio to rule-out onset of preeclampsia within a certain time period - Google Patents

Description

Means and methods for using sFlt-1/PlGF or endoglin/PlGF ratios to rule out onset of pre-eclampsia within a time period

The present invention relates to the field of diagnostic assays for the prenatal diagnosis of preeclampsia. In particular, it relates to a method for diagnosing whether a pregnant subject is not at risk of preeclampsia within a short time window (window of time), comprising: a) determining the amount of at least one angiogenic biomarker selected from the group consisting of sFlt-1, endoglin, and PlGF in a sample from the subject; and b) comparing said amount to a reference, whereby the subject is diagnosed not to be at risk of developing preeclampsia within a short period of time if said amount is the same or decreased as compared to the reference in the case of sFlt-1 and endoglin and if said amount is the same or increased as compared to the reference in the case of P1GF, wherein said reference allows for making a diagnosis with a negative predictive value of at least about 98%. Devices and kits for performing the method are also contemplated.

Pregnancy can be complicated in different ways, which is associated on the one hand with pregnancy-related mortality of the pregnant female and on the other hand with increased morbidity and mortality of the newborn. Maternal mortality in a proportion of 14.5 per 100,000 live births is more common in pregnant females over the age of 39 and can be caused by bleeding, thrombotic pulmonary embolism, infection, cardiomyopathy and cardiovascular and non-cardiovascular disorders as well as hypertensive disorders in which preeclampsia is most common (Berg 2010, Obstetrics and gynecomogy: 116: 1302-.

Preeclampsia relates to about 2% to 8% of all pregnancies and is the major contributor to maternal and fetal mortality worldwide (Duley 2009, Semin period: 33: 130-37.) preeclampsia is generally defined as pregnancy related or induced hypertension and proteinuria that occurs after week 20 of pregnancy-hypertension is defined in this context as blood pressure at 140mmHg (systolic pressure) and/or 90mmHg (diastolic pressure) or higher at two independent measurements, where the two measurements are taken at least 6 hours apart-proteinuria is indicated by 300mg or more of protein in a24 hour urine sampleund Geburvshlfe e.V.,2008, 8.8. NICECLCICAL guiding Hypertension in pregnancy the management of hypertensive disorders in during pregnancy,2010 8.2011 (revised reprint 1.2011)).

The pathogenesis of preeclampsia is essentially unknown. However, it is thought to be caused by a disruption in placental function associated with impaired helical arterial remodeling. Flow defects that occur during the development of pre-eclampsia are associated with ischemia that ultimately results in the release into the circulation of anti-angiogenic factors such as sFlt-1 and endoglin.

To date, the only treatment for preeclampsia has been to terminate pregnancy by early vaginal or caesarean delivery. As discussed above, in the case of preeclampsia, maternal risk and fetal viability are significantly impaired prior to week 34 of pregnancy. Therefore, attempts should be made to delay delivery and thereby improve survival of the neonate.

Early and reliable diagnosis of preeclampsia, particularly the exclusion of preeclampsia occurring as early as the 20 th to 40 th week of pregnancy determines the clinical management of the disease. It will be appreciated that pregnant females with preeclampsia require special care, such as close monitoring, supportive care, and in the case of progression to severe preeclampsia need to be hospitalized in a specialized hospital with Maternal Fetal Intensive Care Unit (MFICU). In particular, early onset preeclampsia is extremely challenging for clinicians in view of the severe side effects and adverse outcomes associated therewith. Furthermore, the early and reliable diagnosis of preeclampsia and the prediction of preeclampsia determine the planning of preventive or therapeutic intervention studies (Ohkuchi 2011, Hypertension 58: 859-866). On the other hand, patients belonging to such risk groups should require less special care and in most cases may not be hospitalized (set as outpatient), wherein the risk group may be excluded from having an increased risk of preeclampsia within a certain time window.

Doppler ultrasonography has been applied to identify patients with abnormal uterine perfusion, and it has been suggested that those identified by Doppler ultrasonography showing abnormal perfusion are at risk of developing preeclampsia, eclampsia and/or HELLP syndrome (Stepan 2007, Hypertension,49: 818-. However, doppler ultrasonography has the disadvantage of requiring highly specialized medical personnel to perform and evaluate the results.

Recently, angiogenic and anti-angiogenic factors have been proposed as indicators of preeclampsia. In particular, placental growth factor (PlGF), endoglin, and soluble fsm-like tyrosine kinase 1(sFlt-1) have been reported to be altered in patients with pre-eclampsia. In addition to reporting individual factors and their changes in healthy individuals and patients with or at risk of developing preeclampsia (see, e.g., Rana 2007, Hypertension 50: 137-.

A single ratio of sFlt-1 and PlGF has been reported as a predictive inclusion (rule-in) factor for pre-eclampsia in early pregnancy (Crispi 2008, Ultrasound Obstet Gynecol 31: 303-. Furthermore, the individual ratios of sFlt-1 and PlGF at different time points of pregnancy have been individually correlated with the risk of pre-eclampsia (DeVivo 2008, actaObstetricia et Gynecology 87: 837-; Ohkuchi 2011, supra; Kusanovic2009, J of Material-Fetal and New medical 22(11):1021- -1038; Chaiappongsa 2011, JMaternal-Fetal and New medical 24(10):1187- > 1207; Benton 2011, American journal of Obstetrics & Gynecology 205:1.e 1). Furthermore, the above-mentioned degree of ratio variation has been studied in terms of prediction of preeclampsia (Kusanovic2009, supra) or risk of emergency (emergent delivery) (Verlohren 2012, American journal of Obstltrics & Gynecology 206(1):58.e1-58.e 8).

However, the aforementioned prior art is primarily concerned with the regular diagnosis of, or prediction of, acute preeclampsia. Little is known about the exclusion of acute preeclampsia within a certain time window. It has been proposed that, depending on a certain ratio of sFLt-1 to PlGF, acute preeclampsia can be excluded on the patient's day of visit (Stepan 2010, Z GebertshNeonatol 214: 234-).

However, reliable assays for the exclusion of preeclampsia for a certain period of time in apparently healthy pregnant females are not available, but are highly desirable.

The technical problem underlying the present invention can be seen as providing means and methods for satisfying the aforementioned needs. This technical problem is solved by the embodiments characterized in the claims and hereinafter.

The present invention relates to a method for diagnosing whether a pregnant subject is not at risk for (i.e. excluding) preeclampsia within a short time window, comprising:

a) determining the amount of at least one angiogenic biomarker selected from the group consisting of sFlt-1, endoglin, and PlGF in a sample from the subject; and

b) comparing said amount to a reference, in the case of sFlt-1 and endoglin, if said amount is the same or decreased as compared to a reference, and in the case of P1GF, if said amount is the same or increased as compared to a reference, thereby diagnosing that the subject is not at risk of developing preeclampsia for a short period of time, wherein said reference allows for making a diagnosis with a negative predictive value of at least about 98%.

The method of the invention is preferably an ex vivo method. Further, it may comprise steps other than those explicitly mentioned above. For example, other steps may involve sample pre-treatment or evaluation of the results obtained by the method. The method may be performed manually or assisted by automated means. Preferably, steps (a) and/or (b) may be carried out wholly or partly with the aid of automatic means, for example using suitable robots and sensing devices for the determination in step (a), computer-implemented computational algorithms on a data processing device and/or comparison and/or diagnostic algorithms on a data processing device for step (b).

The term "preeclampsia" as used herein refers to a medical condition characterized by hypertension and proteinuria. Preeclampsia occurs in pregnant female subjects, and this hypertension is also known as pregnancy-hypertension. Preferably, the presence of pregnancy-hypertension in the individual is identified by two blood pressure measurements of 140mmHg (systolic pressure) and/or 90mmHg (diastolic pressure) or higher, wherein the two measurements are separated by at least 6 hours. Preferably, the presence of proteinuria is identified by 300mg or more of protein in a24 hour urine sample. Preeclampsia can progress to eclampsia, a life-threatening condition characterized by the appearance of tonic-clonic seizures or coma. Symptoms associated with severe preeclampsia are oliguria of less than 500ml within 24 hours, brain or visual disturbances, pulmonary edema or cyanosis, pain in the upper abdomen or right upper quadrant, impaired liver function, thrombocytopenia, fetal growth restriction. Individuals with preeclampsia involving the liver may further develop HELLP syndrome. Thus, a preferred subject of the invention at risk of developing preeclampsia may also be at risk of developing HELLP syndrome. HELLP syndrome is associated with a high risk of adverse outcomes such as premature placental denudation, renal failure, subcapsular liver hematoma, recurrencePre-eclampsia and associated symptoms and subsequent diseases, such as HELLP syndrome or eclampsia, can be found in standard medical textbooks or Guidelines of the relevant medical societyund Geburtsilfe e.V., 8.2008, NICE Clinical guidelines hypertension in prediction the management of hypertension disorders in the prediction, 8.2010 (revised reprint in 1.2011). Preeclampsia occurs in as many as 10% of pregnancies, which are usually in the middle or late gestation. However, some pregnant females develop preeclampsia as early as the 20 th week of pregnancy.

Within weeks 20 to 34 of pregnancy, preeclampsia is also referred to as early onset preeclampsia, while preeclampsia occurring after week 34 of pregnancy is also referred to as late onset preeclampsia. It is understood that early onset preeclampsia is often associated with more severe side effects and adverse outcomes than delayed preeclampsia, which is often relatively mild.

The phrase "not at risk of developing preeclampsia" refers to a pregnant subject that will not develop preeclampsia within a later predicted time window with a statistically significant increased probability as compared to a pregnant subject at risk of developing preeclampsia or as compared to the incidence of preeclampsia (prevalence) within a population comprising the subject to be analyzed.

The term "subject" as used herein refers to an animal, preferably a mammal, more preferably a human. The subject of the invention should be a pregnant subject, i.e. a pregnant female. Preferably, the subject of the invention will not have significant preeclampsia, eclampsia or HELLP syndrome. Preferably, the subject of the invention is a subject who has been identified as having abnormal uterine perfusion, more preferably, may be a subject who has been identified by other diagnostic techniques as being at risk of developing preeclampsia, eclampsia and/or HELLP syndrome. Preferably, transabdominal Doppler ultrasonography has been performed (see, e.g., Albaiges 2000, ObstetGynecol 96: 559-. In particular, the Pulsatility Index (PI) of the uterine artery can be measured, the mean value of these two arteries (mPI-UtA) should be calculated and can be compared to a threshold that allows to distinguish between normal and abnormal results.

More preferably, the pregnant subject of the invention is between about week 20 and about week 40 of gestation, preferably between about week 24 and about week 40 of gestation. Thus, depending on the gestational week at which the subject is sampled, the preeclampsia to be excluded by the methods of the present invention may be early onset preeclampsia or late onset preeclampsia.

The methods of the invention may be used in routine screening methods for apparently healthy pregnant subjects. However, the pregnant subjects contemplated by the present invention may also be assigned to a risk group with a higher incidence of preeclampsia. Pregnant subjects suffering from obesity, hypertension, autoimmune diseases (such as lupus erythematosus), thrombophilia (thrombophilia) or diabetes mellitus generally have an increased probability of developing preeclampsia. This applies equally to subjects with preeclampsia, eclampsia and/or HELLP syndrome in a previous pregnancy. In addition, older females who are first pregnant also show a tendency to develop preeclampsia. However, the probability of developing preeclampsia decreases with the number of pregnancies.

The term "diagnosing" as used herein means assessing whether a subject is not at risk for developing preeclampsia for a short period of time. Preferably, the short period is less than 4 weeks, preferably between about 1 week and about 2 weeks. As will be understood by those skilled in the art, such an assessment is typically not 100% correct for the subject to be diagnosed. However, this term requires that, for some portion of the subjects (e.g., cohorts in a cohort study), the negative predictive value shown elsewhere herein is correct for the assessment. The risk of developing or not developing preeclampsia at a later time window may be diagnosed by a test, such as the method of the invention, whose performance is described in terms of false positive/negative and true positive/negative assessments with generalized statistics. A high negative predictive value indicates a high confidence level of negative assessments made by the diagnostic test. Negative predictive value can be expressed as the number of true negative results divided by the sum of true negative results and false negative results (i.e., all negative results determined by the diagnostic test). Basically, negative predictive values can be calculated based on the sensitivity and specificity of the diagnostic test and the incidence of the disease or condition in a cohort. Specifically, the negative predictive value is [ (specificity) (1-incidence) ]/[ (specificity) (1-incidence) + (1-sensitivity) (incidence) ]. Morbidity predictions can be obtained from cohort studies, while case-control studies can yield the sensitivity and/or specificity of the test. In particular, the negative predictive value of a diagnosis determined by the method of the invention should be at least about 98%, more preferably at least about 99% and most preferably 100%. Additional details regarding Statistics are described in standard textbooks (e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983), or elsewhere herein.

The term "sample" refers to a sample of a bodily fluid, a sample of isolated cells, or a sample from a tissue or organ. Samples of body fluids may be obtained by well-known techniques and preferably comprise samples of blood, plasma, serum or urine, more preferably samples of blood, plasma or serum. Tissue or organ samples may be obtained from any tissue or organ by, for example, biopsy. Isolated cells may be obtained from body fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, the cell, tissue or organ sample is obtained from those cells, tissues or organs that express or produce the peptides mentioned herein.

The term "sFlt-1" as used herein refers to a polypeptide which is a soluble form of fms-like tyrosine kinase 1. This polypeptide is also known in the art as soluble VEGF receptor 1(sVEGF R1) (see, e.g., Sunderji 2010, Am J ObstetGynecol 202:40e 1-7). It was identified in conditioned medium of human umbilical vein endothelial cells. The endogenous sFlt1 receptor is chromatographically and immunologically similar to recombinant human sFlt1 and binds [125I ] VEGF with reasonably high affinity. Human sFlt1 was shown to form a VEGF stable complex with the extracellular domain of KDR/Flk-1 in vitro. Preferably, sFlt1 refers to human sFlt1 as described in Kendall1996, Biochem Biophs Res Commun 226(2), 324-; the amino acid sequence of sFlt1 is also found, for example, in Genebank accession numbers P17948, GI:125361 (human sFlt-1) and BAA24499.1, GI:2809071 (mouse sFlt-1) (Genebank is available from NCBI, USA at www.ncbi.nlm.nih.gov/entrez). The term also encompasses variants of the aforementioned human sFlt-1 polypeptides. Such variants have at least the same basic biological and immunological properties as the previously described sFlt-1 polypeptides. In particular, these variants have the same basic biological and immunological properties if they can be detected by the same specific assays mentioned in the present specification, for example by ELISA assays using polyclonal or monoclonal antibodies specifically recognizing the sFlt-1 polypeptide. Furthermore, it is understood that variants mentioned in the present invention will have an amino acid sequence which differs by at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of the variant has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98% or 99% identity to the full length of a particular sFlt-1 polypeptide, preferably human sFlt-1, respectively. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, wherein a fragment of the amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) for optimal alignment as compared to the reference sequence (which does not comprise additions or deletions). The percentage was calculated by: the number of matched positions is determined by determining the number of positions at which the same amino acid residue is present in both sequences, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The optimal alignment of sequences for comparison can be performed by the local homology algorithm disclosed in Smith 1981, Add.APL.Math.2:482, by the homology alignment algorithm of Needleman 1970, J.mol.biol.48:443, by the method of searching for similarity of Pearson1988, Proc.Natl.Acad Sci. (USA)85:2444, by computerized execution of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group (GCG),575Science Dr., Madison, GAP, BESTFIT, BLAST, FAST, PASTA and TFASTA in WI) or by visual inspection. Given that two sequences have been identified for comparison, it is preferred to use GAP and BESTFIT to determine their optimal alignment and thus the degree of identity. Preferably, default values of 5.00 for the slot weight and 0.30 for the slot length weight are used. The variants mentioned above may be allelic variants or any other species-specific homologues, paralogues or orthologues. Furthermore, variants mentioned herein include fragments or subunits of the particular sFlt-1 polypeptide or variants of the aforementioned types, provided that such fragments have the basic immunological and biological properties mentioned above. Such fragments may be, for example, degradation products of sFlt-1 polypeptides. Variants are considered to have the same basic biological and immunological properties if they can be detected by the same specific assay mentioned in the present specification, e.g., by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the sFlt-1 polypeptide. Preferred assays are described in the accompanying examples. Variants that differ due to post-translational modifications such as phosphorylation or myristylation are also included. sFlt-1 may be detected in bound or free form or as the amount of total sFlt-1 in the sample.

The term "endoglin" as used herein refers to a polypeptide having a molecular weight of 180kDa when unreduced, 95kDa after reduction and 66kDa in reduced and N-deglycosylated form, which polypeptide is capable of forming a dimer and binds to TGF- β and TGF- β receptor preferably endoglin refers to human endoglin more preferably human endoglin has the amino acid sequence shown in Genebank accession number AAC63386.1, GI:3201489 two isoforms of endoglin, S-endoglin and L-endoglin have been described, L-endoglin consists of 633 total amino acids with a cytoplasmic tail of 47 amino acids and S-endoglin consists of 600 amino acids with a cytoplasmic tail of 14 amino acids preferably endoglin used herein is soluble endoglin, which is preferably described in EP 1804836B 1. furthermore, it is understood that variants mentioned herein may have amino acid sequences which differ due to at least one amino acid substitution, deletion and/or addition, wherein such variants have at least the specified amino acid sequence modification, such as specified homology, or homology, which is detected by ELISA, or by a specific assay, such as a substitution of 80%, or addition, or by a substitution of a polypeptide, which polypeptide is capable of binding to the same or to a polypeptide.

The term "P1 GF (placental growth factor)" as used herein refers to a placenta-derived growth factor, which is a polypeptide 149 amino acids in length and highly homologous to platelet-derived growth factor-like regions of human Vascular Endothelial Growth Factor (VEGF). Like VEGF, P1GF has angiogenic activity in vitro and in vivo. For example, biochemical and functional characterization of P1GF from transfected COS-1 cells revealed that it is a glycosylated dimeric secretory protein capable of stimulating endothelial cell growth in vitro (Maqlione 1993, Oncogene 8(4): 925-31). Preferably, PlGF refers to human PlGF, more preferably, human PlGF having the amino acid sequence set forth in Genbank accession number P49763, GI: 17380553. The term encompasses variants of this particular human PlGF. Such variants have at least the same basic biological and immunological properties as the particular PIGF polypeptide. Variants are considered to have the same basic biological and immunological properties if they can be detected by the same specific assays mentioned in the present specification, e.g., by ELISA assays using polyclonal or monoclonal antibodies that specifically recognize the PIGF polypeptide. Preferred assays are described in the accompanying examples. Furthermore, it is to be understood that variants referred to herein will have an amino acid sequence that differs by at least one amino acid substitution, deletion, and/or addition, wherein the amino acid sequence of the variant is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%, respectively, identical to a particular PIGF polypeptide. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and described elsewhere herein. The variants mentioned above may be allelic variants or any other species-specific homologues, paralogues or orthologues. Furthermore, the variants mentioned herein include fragments of the particular PIGF polypeptide or variants of the aforementioned type, provided that these fragments have the basic immunological and biological properties mentioned above. Such fragments may be, for example, degradation products or splice variants of the PIGF polypeptide. Variants that differ due to post-translational modifications such as phosphorylation or myristylation are also included. PIGF may be detected in bound or free form or as the amount of total PIGF in the sample.

Determining the amount of any peptide or polypeptide mentioned in this specification relates to measuring the amount or concentration, preferably semi-quantitatively or quantitatively. The measurement may be made directly or indirectly. Direct measurement involves measuring the amount or concentration of the peptide or polypeptide from a signal obtained from the peptide or polypeptide itself and whose intensity is directly related to the number of molecules of the peptide present in the sample. Such a signal (sometimes referred to herein as an intensity signal) may be obtained, for example, by measuring the intensity value of a particular physical or chemical property of the peptide or polypeptide. Indirect measurements include measuring signals obtained from a second component (i.e., a component that is not the peptide or polypeptide itself) or a biological readout system, such as a measurable cellular reaction, ligand, label, or enzymatic reaction product.

According to the present invention, determining the amount of a peptide or polypeptide can be achieved by all known means for determining the amount of a peptide in a sample. TheApproaches include immunoassay devices and methods that utilize labeled molecules in various sandwich, competitive, or other assay formats. The assay will produce a signal indicative of the presence or absence of the peptide or polypeptide. Furthermore, the signal intensity may preferably be directly or indirectly (e.g., inversely) related to the amount of polypeptide present in the sample. Other suitable methods include measuring a particular physical or chemical property of the peptide or polypeptide, such as its precise molecular weight or NMR spectrum. The method preferably comprises a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device (such as a mass spectrometer, an NMR analyzer, or a chromatographic device). In addition, methods include microplate ELISA-based methods, fully automated devices or robotic immunoassays (e.g., as available in Elecsys^TMObtained on an analyser), CBA (enzymatic cobalt binding assay, e.g.as can be found in Roche-Hitachi^TMObtained on an analyzer) and latex agglutination assays (e.g., as available on Roche-Hitachi)^TMObtained on an analyzer).

Preferably, determining the amount of the peptide or polypeptide comprises the steps of: (a) contacting a cell with the peptide or polypeptide for a sufficient time to elicit a cellular response, the intensity of which is indicative of the amount of the peptide or polypeptide; (b) the cellular response is measured. For measuring cellular responses, it is preferred to add the sample or treated sample to the cell culture and measure internal or external cellular responses. The cellular response may include measurable expression of a reporter gene or secretion of a substance (e.g., a peptide, polypeptide, or small molecule). The expression or substance will produce an intensity signal that correlates with the amount of the peptide or polypeptide.

Also preferably, determining the amount of the peptide or polypeptide comprises measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As discussed above, such a signal may be the intensity of the signal observed in the peptide or polypeptide specific mass spectrum or NMR spectrum at the m/z variable (variable) specific to the peptide or polypeptide.

Determining the amount of the peptide or polypeptide may preferably comprise the steps of: (a) contacting the peptide with a specific ligand; (b) preferably removing unbound ligand; (c) the amount of bound ligand is measured. The bound ligand will generate an intensity signal. Hair brushBinding includes both covalent and non-covalent binding. The ligand of the invention may be any compound, such as a peptide, polypeptide, nucleic acid, or small molecule, that binds to a peptide or polypeptide described herein. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides (e.g., receptors or binding partners for the peptides or polypeptides and fragments of receptors or binding partners comprising the binding domain of the peptides) and aptamers (e.g., nucleic acid or peptide aptamers). Methods for preparing such ligands are well known in the art. For example, commercial suppliers also provide for the identification and production of suitable antibodies or aptamers. Those skilled in the art are familiar with methods for developing derivatives of such ligands with higher affinity or specificity. For example, random mutations can be introduced into a nucleic acid, peptide, or polypeptide. These derivatives can then be tested for binding according to screening methods known in the art (e.g., phage display). Reference herein to antibodies includes polyclonal and monoclonal antibodies, and fragments thereof, such as Fv, Fab and F (ab) capable of binding an antigen or hapten₂And (3) fragment. The invention also includes single chain antibodies and humanized hybrid antibodies in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence will typically include at least the antigen binding amino acid residues of the donor, but may also include other structurally and/or functionally related amino acid residues of the donor antibody. Preferably, the ligand or agent specifically binds to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or reagent should not substantially bind (i.e., cross-react) with other peptides, polypeptides or substances present in the sample to be analyzed. Preferably, the specifically binding peptide or polypeptide should bind with an affinity that is at least 3-fold higher, more preferably at least 10-fold higher and even more preferably at least 50-fold higher than any other related peptide or polypeptide. Non-specific binding can be tolerated if it can still be clearly distinguished and measured (e.g. by its size on a western blot, or by its relatively higher abundance in the sample). Binding of the ligand can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. Other suitable techniques for assaying polypeptides or peptides are described below.

First, the binding of the ligand can be measured directly, for example by NMR or surface plasmon resonance. Secondly, if the ligand also serves as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzymatic reaction product can be measured (e.g., the amount of protease can be measured by measuring the amount of cleaved substrate, e.g., on a western blot). Alternatively, the ligand itself may exhibit enzymatic properties, and the "ligand/peptide or polypeptide" complex or ligand bound to the peptide or polypeptide, respectively, may be contacted with a suitable substrate that allows detection by the generation of an intensity signal. For the measurement of the enzymatic reaction product, it is preferred that the amount of substrate is saturated. The substrate may also be labeled with a detectable label prior to the reaction. Preferably, the sample is contacted with the substrate for a sufficient time. Sufficient time refers to the time necessary to produce a detectable, preferably measurable amount of product. Instead of measuring the amount of product, the time necessary for a given (e.g., detectable) amount of product to appear may be measured. Third, the ligand may be coupled covalently or non-covalently to a label that allows detection and measurement of the ligand. Labeling may be performed by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves the binding (covalent or non-covalent) of a second ligand to a first ligand. The second ligand should specifically bind to the first ligand. The secondary ligand may be coupled to a suitable label and/or a target (receptor) of a tertiary ligand which binds the secondary ligand. The use of second, third or even higher order ligands is often used to enhance the signal. Suitable secondary and higher ligands may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (vector laboratories, Inc.). The ligand or substance may also be "labeled" with one or more labels known in the art. Such labels may be targets for higher order ligands. Suitable tags include biotin, digoxigenin, His tag, glutathione-S-transferase, FLAG, GFP, myc tag, influenza A virus Hemagglutinin (HA), maltose binding protein, and the like. In the case of peptides or polypeptides, the label is preferably at the N-terminus and/or C-terminus. Suitable labels are any labels detectable by a suitable detection method. Typical labels include gold particles, latex beadsAcridinium ester (acridine ester), luminol, ruthenium, enzymatically active labels, radiolabels, magnetic labels (e.g., magnetic beads, including paramagnetic and superparamagnetic labels) and fluorescent labels, enzymatically active labels include, e.g., horseradish peroxidase, alkaline phosphatase, β -galactosidase, luciferase and derivatives thereof suitable detection substrates include di-amino-benzidine (DAB), 3'-5,5' -tetramethylbenzidine, NBT-BCIP (4-nitrotetrazolium chloride and 5-bromo-4-chloro-3-indolyl-phosphate, available as a ready stock solution from Roche Diagnostics), CDP-Star^TM(Amersham Biosciences)、ECF^TM(Amersham Biosciences). Suitable enzyme-substrate combinations can produce a chromogenic reaction product, fluorescence or chemiluminescence, as measured by methods known in the art (e.g., with a photographic film or suitable camera system). With respect to measuring enzymatic reactions, the criteria given above apply analogously. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, texas red, fluorescein, and Alexa dyes (e.g., Alexa 568). Other fluorescent labels are available, for example, from Molecular Probes (Oregon). Quantum dots (quantum dots) are also contemplated as fluorescent labels. Exemplary radiolabels include³⁵S、¹²⁵I、³²P、³³P, and the like. The radiolabel may be detected by any known and suitable method, such as photographic film or a phosphorescent imager. Suitable measurement methods of the invention also include, for example, precipitation (especially immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassay, electrochemiluminescence sandwich immunoassay (ECLIA), dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA), Scintillation Proximity Assay (SPA), nephelometry, turbidimetry, latex-enhanced turbidimetry or solid phase immunoassay. Other methods known in the art (e.g., gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), western blotting, and mass spectrometry) can be used alone or in combination with labeling or other detection methods described above.

The amount of peptide or polypeptide can also preferably be determined as follows: (a) contacting a solid support comprising a ligand for a peptide or polypeptide as indicated above with a sample comprising the peptide or polypeptide; (b) preferably removing unbound peptides or polypeptides and the remaining sample material; and (c) measuring the amount of peptide or polypeptide bound to the support. The ligand is preferably selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, and is preferably present in immobilized form on a solid support. Materials for preparing solid supports are well known in the art and include, inter alia, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction plates, plastic tubes, and the like. The ligand or agent may be bound to a number of different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified celluloses, polyacrylamide, agarose, and magnets. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Suitable methods for assembling/immobilizing the ligand are well known in the art and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. "suspension arrays" are also contemplated as arrays of the invention (Nolan 2002, Trends Biotechnol.20(1): 9-12). In such suspension arrays, a carrier (e.g., a microbead or microsphere) is present in suspension. The array consists of different microbeads or microspheres, possibly labeled, carrying different ligands. Methods for producing such arrays are well known, e.g. based on solid phase chemistry and photolabile protecting groups (US 5,744,305).

According to a preferred embodiment, the binding measurement of the ligand is performed by an analytical unit of the system disclosed herein. The measured combined amount may then be calculated by the computing device of the system disclosed herein.

The term "amount" as used herein encompasses the absolute amount of a polypeptide or peptide, the relative amount or concentration of the polypeptide or peptide, and any value or parameter associated therewith or derivable therefrom. Such values or parameters include intensity signal values from all specific physical or chemical properties, such as intensity values in a mass spectrum or NMR spectrum, obtained from the peptide by direct measurement. Furthermore, all values or parameters obtained by indirect measurements as indicated elsewhere in the specification are also encompassed, such as the level of response in response to the peptide as determined from a biological readout system or an intensity signal obtained from a specific binding ligand. It will be understood that values related to the aforementioned quantities or parameters may also be obtained by all standard mathematical operations. According to a preferred embodiment of the invention, the determination of the "amount" is performed by the system of the invention, whereby the computing device determines the "amount" on the basis of the contacting and measuring steps, which are performed by one or more analysis units of the system.

The term "comparing" as used herein encompasses comparing a measured amount of at least one angiogenic biomarker referred to herein with a reference. It is to be understood that the comparison as used herein refers to any kind of comparison made between the value of the quantity and a reference. Comparing the amount to a reference, in the case of sFlt-1 and endoglin, if the amount is the same or decreased as compared to the reference, and in the case of P1GF, if the amount is the same or increased as compared to the reference, the pregnant subject is not at risk of developing preeclampsia within a short time window ("rule out" of preeclampsia).

The comparison mentioned in step (b) of the method of the invention can be carried out manually or computer-aided. The value of the quantity and the reference may for example be compared with each other and the comparison may be automated by a computer executing an algorithm for the comparison. The computer program performing this evaluation will provide the desired assessment in the form of a suitable output. Preferably, the evaluation unit of the inventive apparatus or the computing device of the inventive system can be used to make this comparison. The computer program performing this evaluation will provide the desired assessment in the form of a suitable output. For computer-assisted comparison, the value of the measured quantity can be compared by means of a computer program with a value stored in a database corresponding to a suitable reference. The computer program may further evaluate the result of the comparison, i.e. automatically provide the desired assessment in a suitable output form. Based on the comparison of the measured amount and the reference amount, it is possible to make a desired evaluation. For example, the results of the comparison may be given as raw data (absolute or relative quantities) and in some cases as indicators, which may indicate the word, phrase, symbol or numerical form of a particular diagnosis.

The term "reference" as used herein refers to a reference amount or value that represents a cutoff for making a diagnosis with a negative predictive value of at least about 98%. Preferably, the negative predictive value envisaged in this context is about 99%, or more preferably 100%. Suitable cutoff amounts or values may preferably be determined as discussed above in terms of sensitivity, specificity and anticipation, known (e.g. from the literature) or estimated (e.g. in terms of prospective cohort studies) incidence of preeclampsia in the population of subjects to be studied. The cut-off value or amount used as a reference can be determined by a variety of techniques known in the art. Preferably, the cutoff or amount can be determined by Receiver Operating Characteristic (ROC) (see, inter alia, Zweig 1993, Clin. chem.39: 561-. ROC plots are plots of all sensitivity and specificity pairs obtained by varying the decision threshold (i.e., cutoff) continuously over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign a subject to a certain prognosis or diagnosis. ROC plots show overlap between the two distributions (results for the affected and unaffected subgroups) by plotting sensitivity over the entire range of thresholds applicable for discrimination against 1-specificity. On the y-axis is the sensitivity or true positive score, which is defined as the ratio of the number of true positive test results to the sum of the number of true positive test results and the number of false negative test results. This is also referred to as positive in the presence of a disease or disorder. It is calculated from only the affected subgroups. On the x-axis is the false positive score or 1-specificity, which is defined as the ratio of the number of false positive results to the sum of the number of true negative results and the number of false positive results. It is an index of specificity, all calculated from unaffected subgroups. Since true positive and false positive scores are calculated completely separately by using test results from two different subgroups, the ROC plot is independent of the incidence of this event in the cohort. Each point on the ROC graph represents a sensitivity and specificity pair corresponding to a particular decision threshold. Tests that are perfectly distinguishable (no overlap in the two distribution results) have ROC plots across the top left corner with a true positive score of 1.0 or 100% (perfect sensitivity) and a false positive score of 0 (perfect specificity). The theoretical plot for the test without discrimination (same distribution results for both groups) is a 45 ° diagonal from the bottom left to the top right. Most of the figures fall between these two extremes. If the ROC plots all fall below the 45 ° diagonal, it is easily corrected by reversing the "positive" criteria from "greater" to "less" and vice versa. Qualitatively, the closer the graph is to the upper left corner, the higher the overall accuracy of the test. The cutoff value can be derived from a ROC graph that allows for the proper balance of sensitivity and specificity for the diagnosis or prediction of a given event. Thus, the reference to be used in the aforementioned methods of the invention (i.e. the cut-off value that allows for distinguishing between subjects not at increased risk) is preferably generated by establishing the ROC for the cohort as described above and deriving the amount of the cut-off value therefrom. The ROC plot allows for the derivation of appropriate cut-off values depending on the desired, preselected sensitivity and specificity of the diagnostic method or corresponding confidence limits. It is understood that sensitivity and specificity are adjusted to minimize the false negative group for effective exclusion of a subject at increased risk (i.e., exclusion), while sensitivity and specificity are adjusted to minimize the false positive group for effective assessment of a subject at increased risk (i.e., inclusion). Furthermore, area under the curve (AUC) values can be derived from ROC plots, yielding an indication of the overall performance of the biomarker independent of the cutoff values. Furthermore, each point of the ROC plot represents a sensitivity and specificity pair at some cutoff value.

The term "about" in the context of the present invention means +/-20%, +/-10%, +/-5%, +/-2%, or +/-1% from the parameter or value indicated. This also takes into account common deviations caused by measurement techniques and the like. However, the term also includes the parameter or value that is accurately indicated.

Advantageously, it has been found in the studies of the present invention that the amount of sFlt-1, endoglin or PlGF and the ratio thereof (see also below) (i.e., sFlt1/PlGF or endoglin/PlGF ratio) in pregnant subjects exhibiting no or limited clinically significant symptoms of pre-eclampsia, eclampsia or HELLP syndrome at the time the samples studied were taken can be an indicator of the exclusion of acute pre-eclampsia (i.e., the development of pre-eclampsia and/or acute HELLP syndrome within a short period of about 1 to about 2 weeks). In particular, when the aforementioned markers are used, cutoff amounts or values for individual biomarkers with high negative predictive values can be established. Furthermore, it has been found that a ratio of sFLt-1/PlGF of about 46 or even about 33 or less is a particularly valuable indicator which brings the negative predictive value of the method of the invention close to 100%. In addition, as disclosed in detail in the examples (especially example 4) and FIG. 8, a ratio of sFLt-1/PlGF of about 38 has been found to be of particular value. The method of the invention therefore operates with a particularly high level of confidence in the exclusion diagnosis to be established. Furthermore, it has been found that the method of the present invention can be reliably used not only for samples taken at the first visit of a subject, but also for samples taken at subsequent visits. Furthermore, the methods of the invention can be used to identify subjects who have been diagnosed by other diagnostic techniques, particularly by uterine doppler ultrasonography, as being at risk of developing preeclampsia, eclampsia and/or HELLP syndrome, with false positives. By applying the method of the invention to a subject who has been diagnosed by a doppler sonography study as being at risk of developing preeclampsia, eclampsia and/or HELLP syndrome, such a subject diagnosed as false positive can be effectively and reliably excluded within a short time window.

Thanks to the invention it is possible to more reliably exclude the risk of acute preeclampsia. Furthermore, when the method of the invention is used as an aid in diagnosis, time-consuming, expensive and cumbersome diagnostic measures, such as current scoring systems or doppler sonography studies, which require well-trained medical staff, can be avoided. In this context, it is noted that, unlike for example doppler sonography studies, the method of the invention can even be carried out automatically or by medical support personnel without highly specialized medical personnel. Healthcare management would greatly benefit from the methods of the present invention because the need for intensive and special care (such as close monitoring and hospitalization) required by pregnant females who have, or are at risk of developing, preeclampsia can be better assessed and considered for the purposes of healthcare management.

It is to be understood that the definitions and explanations of the terms made above and below apply correspondingly to all embodiments described in the present description and in the appended claims.

In a preferred embodiment of the method of the invention, the method comprises determining in said step a) the amount of the biomarkers sFlt-1 or endoglin and PlGF in the sample of said subject, and comparing in step c) the ratio with a reference value, whereby said subject is diagnosed not to be at risk of developing preeclampsia within a short period of time, if said ratio is the same or decreased compared to said reference value. More preferably, the method comprises, before said step b), a further step of: determining the amount of sFlt-1 or endoglin and PlGF from the sample of step a) to calculate the ratio.

The term "calculating a ratio" as referred to herein relates to calculating the ratio of the amount of sFlt-1 or endoglin to the amount of PlGF by dividing by this amount or by any other equivalent mathematical calculation that relates the amount of sFlt-1 or endoglin to the amount of P1 GF. Preferably, the ratio is calculated by dividing the amount of sFlt-1 or endoglin by the amount of PlGF, i.e., the ratio is preferably the amount of sFlt-1 divided by the amount of PlGF (also known as sFlt-1/PlGF) or the amount of endoglin divided by the amount of PlGF (also known as endoglin/PlGF).

The term "comparing" as used herein encompasses comparing the ratio with a reference defined elsewhere. It is to be understood that a comparison as used herein refers to any type of comparison made between the ratio and the reference. It has been found in the studies of the present invention that a reduced or non-increased risk of developing pre-eclampsia correlates with a ratio determined for sFlt-1 or endoglin and PlGF that is the same or reduced compared to the reference value. More preferably, the reference value for this ratio is about 46, about 45, about 40 or about 35 or less, preferably it is about 33 or less. Even more preferably, the reference value for the ratio determined for sFlt-1 and PlGF is about 38. The aforementioned cut-off values differ significantly compared to other (non-specific) exclusion methods used for diagnosis on the day of the visit in the prior art (see e.g. Stepan supra) and achieve surprisingly high predictive negative predictive values when applied in the method of the invention.

In another preferred embodiment of the method of the invention, the method further comprises suggesting a management measure for the patient based on the diagnosis.

The term "advice" as used herein means to establish a proposal that can be applied to a subject or prohibit patient management measures or a combination thereof from being applied to a subject. However, in a particular embodiment, it should be understood that the term does not include applying actual management measures anyway. Patient management measures as used herein refer to all measures that can be applied to a subject suffering from preeclampsia to cure, avoid or manage the health condition. For example, patient management measures include the extent of monitoring (e.g., close, periodic, or weak monitoring), hospitalization or ambulatory (ambulant) maintenance, application or avoidance of medication, or lifestyle advice. Preferably, (i) if the subject is not diagnosed as not at risk of developing preeclampsia, then the patient management measures are selected from the following measures: close monitoring, hospitalization, administration of hypotensive agents and lifestyle advice; (ii) the patient management measure is flow monitoring if the subject is diagnosed as not being at risk of developing preeclampsia.

The present invention also relates to a method of managing a subject suspected of having preeclampsia, comprising the steps of the above-mentioned method for diagnosing whether a pregnant subject is not at risk for preeclampsia within a short time window, and a further step of managing the subject according to the established diagnosis. Preferably, the management comprises flow maintenance, regular or weak monitoring, avoidance of drug administration, especially administration of hypotensive agents or prenatal glucocorticoids (e.g. betamethasone), the latter for accelerating fetal lung development in women at risk of preterm birth.

The invention also relates to the use of at least one of the biomarkers sFlt-1, endoglin and PlGF, or at least one detection agent that specifically binds to said biomarker, in a sample of a pregnant subject for diagnosing whether said subject is not at risk of developing preeclampsia within a short period of time. The invention also includes the use of at least one of the biomarkers sFlt-1, endoglin and PlGF in a sample of a pregnant subject or at least one detection agent that specifically binds to said biomarker in the recommendation of patient management measures as set out elsewhere herein.

In addition, the present invention contemplates the use of at least one of the biomarkers sFlt-1, endoglin and PlGF, or at least one detection agent that specifically binds to said biomarker, in a sample of a pregnant subject for the preparation of a diagnostic entity or a pharmaceutical entity or composition for diagnosing whether said subject is not at risk of developing preeclampsia within a short period of time. The invention also comprises the use of at least one of the biomarkers sFlt-1, endoglin and PlGF, or at least one detection agent that specifically binds to said biomarker, in a sample of a pregnant subject for the preparation of a diagnostic entity or a pharmaceutical entity or composition for advising patient management measures as given elsewhere herein.

The present invention relates to a device suitable for diagnosing whether a pregnant subject is not at risk of developing preeclampsia within a short period of time by performing the method of the invention, comprising:

a) an assay unit comprising at least one detection agent that specifically binds to at least one angiogenic biomarker selected from the group consisting of sFlt-1, endoglin, and PlGF, said unit being suitable for determining the amount of sFlt-1, endoglin, and/or PlGF in a sample from a pregnant subject; and

b) an evaluation unit comprising a data processor executing an algorithm for comparing the amount to a reference, whereby the subject is diagnosed not to be at risk of developing preeclampsia within a short period of time if the amount is the same or decreased as compared to the reference in the case of sFlt-1 and endoglin and if the amount is the same or increased as compared to the reference in the case of P1GF, wherein the reference allows for a diagnosis with a negative predictive value of at least about 98%.

The term "device" as used herein relates to a system comprising the aforementioned units, which are operatively connected to each other to allow a diagnosis according to the method of the invention. Preferred detection agents that can be used in the analytical unit are disclosed elsewhere herein. The analytical unit preferably comprises a detection agent immobilized on a solid support such that the solid support is contacted with a sample comprising the biomarker whose amount is to be determined. Furthermore, the analysis unit may further comprise a detector that determines the amount of the detection agent that specifically binds to the one or more biomarkers. The determined quantity can be transmitted to an evaluation unit. The evaluation unit comprises a data processing element, such as a computer, with an executing algorithm, which performs the calculation of the ratio, the comparison of the calculated ratio and the evaluation of the comparison result by executing a computer-based algorithm, which implements the steps of the method of the invention shown in detail elsewhere herein. The results may be provided as an output of the parametric diagnostic raw data. It will be appreciated that such data will typically need to be interpreted by a clinician. However, expert system devices are also contemplated in which the output results contain processed diagnostic raw data, the interpretation of which does not require a specialized clinician.

In a preferred embodiment of the device of the invention, the analysis unit comprises a detection agent for determining the amount of the biomarkers sFlt-1 or endoglin and PlGF in a sample of said subject, and wherein the algorithm executed in the evaluation unit compares the ratio of the amount of sFlt-1 or endoglin to PlGF with said reference value, and if said ratio is identical or reduced compared to said reference value, thereby diagnosing that the subject is not at risk of developing preeclampsia within a short period of time. Preferably, the algorithm executed in the evaluation unit further calculates the ratio of the amount of sFlt-1 or endoglin to PlGF.

In another preferred embodiment of the invention, the evaluation unit further comprises an executed algorithm that makes recommendations for patient management measures based on the diagnosis shown elsewhere herein.

As can be seen from the above, in accordance with some embodiments of the present disclosure, portions of some steps of the methods disclosed and described herein may be performed by a computing device. The computing device may be, for example, a general purpose computer or a portable computing device. It is also understood that multiple computing devices may be used together (e.g., via a network or other data transmission method) to perform one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, notebook computers, personal data assistants ("PDAs") (e.g., BLACKBERRY brand devices), cellular devices, tablet computers, servers, and the like. Typically, a computing device contains a processor capable of executing a plurality of instructions (e.g., a software program).

The computing device may access the memory. The memory is a computer-readable medium and may comprise, for example, a single storage device or multiple storage devices located within the computing device or accessible to the computing device over a network. Computer readable media can be any available media that can be accessed by a computing device and includes both unstable and stable media. Additionally, the computer readable media may be one or both of removable and non-removable media. By way of example, and not limitation, computer readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store a plurality of instructions that can be accessed by a computing device and executed by a processor of the computing device.

According to embodiments of the present disclosure, software may include instructions that, when executed by a processor of a computing device, may perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to generate signals that control the operation of other machines, such that the signals may be operated to alter materials remote from the computer. These descriptions and representations are the means used by those skilled in the data processing arts to most effectively convey the substance of their work to others skilled in the art, for example.

The plurality of instructions may also comprise an algorithm, which is generally considered to be a self-consistent sequence of steps leading to a desired result. The steps are those requiring physical manipulations of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of being stored, transferred, converted, combined, compared, and otherwise manipulated. It has proven convenient at times, principally for reasons of common usage, to refer to these signals as values, characters, display data, numbers, or the like, when referring to physical items or phenomena in which such signals are contained or represented. It should be borne in mind, however, that all of these and similar terms are to be associated with the appropriate physical quantities and are merely convenient labels applied to these quantities. According to some embodiments of the disclosure, the instructions comprise an algorithm, and the algorithm is performed by executing the instructions for performing a comparison between the measured amount of one or more markers disclosed herein and a suitable reference. The results may be output as parametric diagnostic raw data or as absolute or relative quantities. According to various embodiments of the systems disclosed herein, the computing device of the systems disclosed herein may provide a "diagnosis" based on a comparison of the calculated "amount" to a reference or threshold. For example, the computing device of the system may provide the indicator in the form of a word, symbol, or value that indicates a particular diagnosis.

The computing device may also access an output device. Exemplary output devices include, for example, facsimile machines, displays, printers, and documents. According to some embodiments of the invention, a computing device may perform one or more steps of the methods disclosed herein and then provide an output via an output device that relates to the results, indications, ratios, or other factors of the methods.

The invention further relates to a system for aiding in the diagnosis of whether a pregnant subject is not at risk of developing preeclampsia for a short period of time by performing the method of the invention, comprising:

a) an analysis unit configured to contact the sample with a detection agent(s) that specifically binds at least one marker selected from the group consisting of sFlt-1, endoglin, and PlGF for a time sufficient to allow the detection agent and the at least one marker from the sample to form a complex;

b) an analyzer unit configured to measure an amount of complex formed, wherein the amount of complex formed is proportional to an amount of at least one marker present in the sample;

c) a computing device having a processor in operative communication with the analysis unit; and

d) a non-transitory machine-readable medium comprising a plurality of instructions executable by a processor, which when executed convert an amount of a complex formed to an amount of at least one biomarker to reflect an amount of the at least one marker present in a sample, compare the amount to a reference, facilitate establishing a risk assessment optimized according to clinical prediction rules for classifying a subject at risk of developing preeclampsia within a short period of time according to a comparison result with the reference.

Preferred embodiments of the present disclosure include a system for optimizing risk assessment to classify subjects at risk of developing preeclampsia according to clinical prediction rules. Examples of systems include clinical chemistry analyzers, coagulation chemistry analyzers (chemical or biological reactions), immunochemistry analyzers, urine analyzers, nucleic acid analyzers, which are used to detect the results of or processes of chemical or biological reactions. More specifically, exemplary systems of the present disclosure may include a Roche Elecsys (TM) system ande immunoassay analyzer, Abbott ArchitectM and AxsymTM analyzers, Siemens Centaur and ImmuliteTM analyzers, and Beckman Coulter UniCelTM and AcessTM analyzers, and the like.

Embodiments of the system may include one or more analyzer units for implementing the present disclosure. The analysis unit of the system disclosed herein is in operative communication with the computing device disclosed herein via any known wired connection, bluetooth, LANS, or wireless signal. Furthermore, in accordance with the present disclosure, an analysis unit may comprise a module within a separate device or larger instrument that performs one or both assays (e.g., for qualitative and/or quantitative evaluation of a sample for diagnostic purposes). For example, the analysis unit may perform or assist in the aspiration, metering, mixing of the sample and/or reagents. The analysis unit may comprise a reagent holding unit for holding reagents for performing the assay. The reagents may be arranged, for example, in the form of containers or cassettes containing individual reagents or groups of reagents, placed in appropriate containers or locations within a holding compartment or conveyor. The detection reagent may also be in a form immobilized on a solid support in contact with the sample. Furthermore, the analysis unit may comprise methods and/or detection components that may be optimized for a specific analysis.

According to some embodiments, the analysis unit may be configured for optical detection of an analyte (e.g. a marker in a sample). An exemplary analysis unit configured for optical detection includes a device configured to convert electromagnetic energy into an electrical signal, including single element and multi-element or array optical detectors. In accordance with the present disclosure, an optical detector is capable of monitoring an optical electromagnetic signal and providing an electrical output signal or response signal relative to a baseline signal that is indicative of the presence and/or concentration of an analyte in a sample located in an optical path. Such devices may also include, for example, photodiodes (including avalanche photodiodes), phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS detectors (including CMOS array detectors), photomultiplier tubes, and arrays of photomultiplier tubes. According to certain embodiments, the optical detector (e.g., photodiode or photomultiplier) may contain additional signal conditioning or processing electronics. For example, the optical detector may comprise at least one preamplifier, electronic filter or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance and current gain (current mirror) preamplifiers.

Furthermore, one or more analysis units of the present disclosure may comprise a light source for emitting light. For example, the light source of the analysis unit may comprise at least one light emitting element (e.g. a light emitting diode, an electrokinetic radiation source, such as an incandescent lamp, an electroluminescent lamp, a gas discharge lamp, a high intensity discharge lamp, a laser) for measuring the analyte concentration of the sample tested or to enable energy transfer (e.g. by fluorescence resonance energy transfer or catalytic enzymes).

In addition, the analysis unit of the system may include one or more incubation units (e.g., for maintaining the sample or reagent at a specified temperature or temperature range). In some embodiments, the analyzer unit may comprise a thermal cycler, including a real-time thermal cycler, for subjecting the sample to repeated temperature cycles and monitoring changes in the amount of amplification product in the sample.

Furthermore, the analysis unit of the system disclosed herein may comprise or be operatively connected to a reaction vessel or vessel feeding unit. Exemplary feed units include liquid handling units, such as pipetting units, that deliver samples and/or reagents to reaction vessels. The suction unit may comprise a reusable washable needle, such as a steel needle, or a disposable pipette tip. The analysis unit may further comprise one or more mixing units, such as a shaker to shake the container containing the liquid, or an agitator to mix the liquid in the container or the reagent container.

The invention also relates to a kit suitable for carrying out the method of the invention, comprising at least one detection agent for determining the amount of an angiogenic biomarker selected from the group consisting of sFlt-1, endoglin and PlGF, and instructions for carrying out said method.

The term "kit" as used herein refers to a collection of the aforementioned components, preferably separated or provided in a single container. The container also contains instructions for carrying out the method of the invention. These instructions may be in the form of a manual or may be provided by computer program code which, when executed on a computer or data processing device, is capable of performing the calculations and comparisons referred to in the methods of the invention and determining a diagnosis accordingly. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g. an optical disc), or directly on a computer or data processing device. Furthermore, the kit may preferably contain standard amounts of the biomarkers described elsewhere herein for calibration purposes.

In a preferred embodiment of the kit of the invention, the kit comprises a detection agent for determining the amount of sFlt1 and/or endoglin in a sample of a pregnant subject and a detection agent for determining the amount of PlGF.

In some embodiments, the kits disclosed herein comprise at least one ingredient or packaged combination of ingredients for practicing the disclosed methods. By "packaged combination" is meant that the kit provides a single package containing a combination of one or more of the components disclosed herein, such as probes (e.g., antibodies), controls, buffers, instructions for reagents (e.g., conjugates and/or substrates), and the like. Kits comprising a single container are also included within the definition of "packaged combination". In some embodiments, the kit comprises at least one probe, such as an antibody having specific affinity for an epitope of a biomarker disclosed herein. For example, the kit may include an antibody labeled with a fluorophore or an antibody that is a member of a fusion protein. In this kit, the probe may be immobilized and may be immobilized in a specific conformation. For example, immobilized probes can be provided in a kit to specifically bind to a target protein, detect a target protein in a sample, and/or remove a target protein from a sample.

According to some embodiments, the kit comprises at least one probe, which may be immobilized, in at least one container. The kit may also include multiple probes, optionally immobilized, in one or more containers, e.g., multiple probes may be present in a single container or in separate containers, e.g., where each container contains a single probe.

In some embodiments, a kit can include one or more non-immobilized probes and one or more solid supports that include or do not include immobilized probes. Some embodiments may comprise some or all of the reagents and materials required to immobilize one or more probes to a solid support, or to bind immobilized probes to a particular protein within a sample.

In certain embodiments, a single probe (comprising multiple copies of the same probe) can be immobilized on a single solid support and provided in a single container. In other embodiments, two or more probes (e.g., specific probes) each specific for a different target protein or different formats of a single target protein are provided in a single container. In some such embodiments, the immobilized probe may be provided in a plurality of different containers (e.g., in a single use format), or a plurality of immobilized probes may be provided in a plurality of different containers. In other embodiments, the probe can be immobilized on a variety of different types of solid supports. The kits disclosed herein contemplate any combination of one or more immobilized probes and one or more containers, any combination of which may be selected to result in a kit suitable for the intended use.

The container of the kit can be any container suitable for packaging and/or containing one or more of the components disclosed herein, including, for example, probes (e.g., antibodies), controls, buffers, and reagents (e.g., conjugates and/or substrates). Suitable materials include, but are not limited to, glass, plastic, cardboard boxes or other paper products, wood, metal, and any alloys thereof. In some embodiments, the container may completely encase one or more immobilized probes or may simply cover the probes to minimize contamination by dust, oil, etc., and exposure to light. In some other embodiments, the kit may comprise a single container or a plurality of containers, and where a plurality of containers is present, each container may be the same as all other containers, different from the others, or different from some but not all of the other containers.

In one aspect of the invention, a method is contemplated that assists in the elimination of preeclampsia within a short time window, comprising:

a) determining the amount of at least one angiogenic biomarker mentioned herein in a sample of a pregnant subject, the determining comprising: (i) contacting the sample with a detection agent that specifically binds to the at least one angiogenic biomarker for a time sufficient to allow the detection agent and the biomarker from the sample to form a complex; (ii) measuring the amount of complex formed, wherein the amount of complex formed is proportional to the amount of at least one biomarker present in the sample; and (iii) converting the amount of complex formed into an amount of at least one biomarker that reflects the amount of the at least one biomarker present in the sample;

b) comparing the amount to a reference; and

c) aiding the elimination of preeclampsia within a short time window based on the results of the comparison in step b).

In one aspect, a suitable detection agent may be an antibody that specifically binds to at least one angiogenic biomarker in a sample of a subject to be studied by the method of the invention. In one aspect, another detection agent that may be used may be an aptamer that specifically binds to at least one angiogenic biomarker in a sample. In yet another aspect, the sample is removed from complexes formed between the detection agent and the at least one angiogenic biomarker prior to measuring the amount of complex formed. Thus, in one aspect, the detection agent can be immobilized on a solid support. In yet another aspect, the sample can be removed from the complex formed on the solid support by application of a wash solution. The complex formed should be proportional to the amount of at least one angiogenic biomarker present in the sample. It will be appreciated that the specificity and/or sensitivity of the detection agent to be applied defines the extent to which the proportion of the at least one angiogenesis biomarker capable of being specifically bound is contained in the sample. Additional details on how the assay is performed are also found elsewhere herein. The amount of complex formed should be converted to an amount of at least one angiogenic biomarker, which reflects the amount that is actually present in the sample. Such an amount may on the one hand be substantially the amount present in the sample or on the other hand be a certain proportion thereof, due to the relationship between the complex formed and the amount present in the original sample.

Also in an aspect of the aforementioned method, step a) may be performed by an analysis unit (in an aspect, an analysis unit as defined elsewhere herein). In other aspects, any or all of steps a) to c) may be performed by an analysis unit as defined elsewhere herein.

In one aspect of the method of the invention, the amount determined in step a) is compared to a reference. In one aspect, the reference is a reference as defined elsewhere herein. In yet another aspect, the reference takes into account a proportional relationship between the amount of complex measured and the amount present in the original sample. Thus, in one aspect of the method of the invention, the reference applied is an artificial reference that reflects the limitations of the detection agent used. On the other hand, this relationship may also be taken into account when making the comparison, for example comprising a step of normalization and/or correction calculation of the measured quantity before actually comparing the value of the measured quantity with the reference. Also, the step of normalizing and/or correcting the measured quantity employs such a comparison step that the limitations of the detection agent used are correctly reflected. In one aspect, the comparison is performed automatically, for example, with the assistance of a computer system or the like.

Making a secondary diagnosis based on a comparison by explicitly assigning the subject in step b) to a group of subjects not at risk of developing preeclampsia within a short time window or implicitly excluding it from the group. As already discussed elsewhere herein, the distribution of the subjects studied is unlikely to be 100% correct in the cases studied. The subject groups to which study subjects are assigned are human groups in that they are established based on statistical considerations (i.e., based on some pre-selected degree of likelihood that the methods of the invention should be run). Thus, the method may establish a secondary diagnosis that may in one aspect require further enhancement of the diagnosis by other techniques. In another aspect of the invention, the auxiliary diagnosis is automatically established, for example, with the aid of a computer system or the like.

In one aspect of the method of the invention, the method further comprises the step of treating/prescribing/suggesting or managing the subject based on the results of the aided diagnosis established in step c) as shown elsewhere herein.

In one aspect of the aforementioned method, steps b) and/or c) are performed by an evaluation unit as shown elsewhere herein.

All references cited in this specification are incorporated herein by reference in their entirety and the disclosures explicitly mentioned in this specification.

Brief Description of Drawings

FIG. 1 shows the sFlt-1/PlGF ratio at different gestational weeks. Open circles represent cases in which Preeclampsia (PE) was not detected within 1 week after sampling (visit), and gray circles are cases with Preeclampsia (PE). (A) n-94, no PE cases were detected below the cutoff value of 46; (B) n is 269; below the cutoff value of 38, only a few PE cases were detected.

FIG. 2 shows the sFlt-1/PlGF ratio at different weeks of pregnancy. Open circles represent cases in which no Preeclampsia (PE) was detected within 2 weeks after sampling (visit). Grey circles represent cases with Preeclampsia (PE). Below the cutoff value of 46, no PE cases were detected. (A) n-94, no PE cases were detected below the cutoff value of 46; (B) n is 269; below the cutoff value of 38, only a few PE cases were detected.

FIG. 3(A) shows a bivariate distribution analysis of sFlt-1/PlGF ratio and days until onset of pre-eclampsia (PE); (B) same as in (a), but with larger groups.

FIG. 4(A) shows the ROC curve and its statistical analysis for the absence of Preeclampsia (PE) onset within 1 week after sampling (visit). An AUC (area under the curve) of 1.0 achieves a perfect diagnostic test, an AUC of 0.5 is a useless diagnostic test. The confidence interval of the AUC reflects the accuracy of the estimate based on the existing data; n is 94; (B) same as in (a), except that n is 269.

FIG. 5(A) shows the ROC curve and its statistical analysis for the absence of Preeclampsia (PE) onset within 2 weeks after sampling (visit). An AUC (area under the curve) of 1.0 achieves a perfect diagnostic test, an AUC of 0.5 is a useless diagnostic test. The confidence interval of the AUC reflects the accuracy of the estimate based on the existing data; n is 94; (B) same as in (a), except that n is 269.

FIG. 6 shows boxplots obtained using the sFlt-1/PlGF ratio (A) and the endoglin/PlGF ratio (B); n is 94 for 1 week PE and 88 for 4 weeks PE. The left side of each figure shows measurements from patients with abnormal doppler sonography results (mPI-UtA >95 percentile) and the right side shows measurements from patients with normal results. In addition, patients who developed (red boxes) or did not develop (green boxes) PE/HELLP within one week (left panel) and four weeks (right panel) were measured separately. The graph shows that, especially in patients with abnormal Doppler ultrasonography outcomes, both the sFlt-1/PlGF ratio and the endoglin/PlGF ratio have the potential to predict whether a patient will or will not develop PE within one and four weeks. Especially for the prediction of PE/HELLP within four weeks, the sFlt-1/PlGF ratio appears to differentiate more accurately than the endoglin/PlGF ratio.

Fig. 7 shows a box plot obtained using the sFlt-1/PlGF ratio (box plot n is 269). The left side of each figure shows measurements from patients with abnormal doppler sonography results (mPI-UtA >95 percentile) and the right side shows measurements from patients with normal results. In addition, patients who developed or did not develop PE/HELLP within one week (a), two weeks (B) and four weeks (C) were measured separately. The graph shows that, particularly in patients with abnormal doppler sonography outcomes, sFlt-1/PlGF has the potential to predict whether a patient will or will not develop PE within one week, two weeks, and four weeks.

FIG. 8: for the Prediction (PROGNOSIS) study (with 500 patients), a cutoff value for the sFlt1-PlGF ratio for pre-eclampsia over four weeks was determined, as well as the performance of the cutoff value. To avoid overfitting, a cross-validation method, Monte Carlo cross-validation, was applied. To this end, all data is divided into two disjoint subsets (training and test sets) on a2 to 1 scale. After this segmentation, the model is determined on the training set and the prediction is evaluated on the test set. This method was repeated 1999 times on randomly selected training set-test set-partitions. The result is 1999 predictive models with 1999 cut-offs, reflected by the box plot, with a median of, for example, about 38. This is the same cut-off value as found in the large population to rule out preeclampsia within one week (example 4).

Examples

The following examples are merely illustrative of the present invention. In no event, they should be construed as limiting the scope of the invention.

Example 1: determination of blood levels of sFlt-1, Endoglin and PlGF

Blood levels of sFlt-1, PlGF, and endoglin were determined using commercially available immunoassays. Specifically, the following measurements were used.

Using a probe from Roche Elecsys^TM-or cobas e^TMA series of analyzers tested for sFlt-1 using a sandwich immunoassay. The assay comprises two monoclonal antibodies specific for each polypeptide. The first of these antibodies was a biotinylated antibody and the second was an antibody labeled with a Tris (2, 2' -bipyridyl) ruthenium (II) complex. In a first incubation step, both antibodies are incubated with the sample. A sandwich complex is formed comprising the peptide to be assayed and two different antibodies. In the next incubation step, streptavidin-coated beads are added to this complex. The beads are bound to the sandwich complex. The reaction mixture is then aspirated into the measurement chamber, where the beads are measuredThe measuring chamber is magnetically trapped on the surface of the electrode. Application of a voltage then induces chemiluminescent emission from the ruthenium complex as measured by a photomultiplier tube. The amount of luminescence depends on the amount of the sandwich complex on the electrode. The sFlt-1 test is commercially available from Roche Diagnostics GmbH, Mannheim, Germany. Additional details regarding this assay are found in the package insert. The measurement range for sFlt-1 includes amounts between 10 and 85,000 pg/ml.

With a catalyst selected from the group consisting of&Available from D Systems, Inc, Minneapolis, USHuman endothelin/CD 105 immunoassay measures endoglin. This assay utilizes a quantitative sandwich enzyme immunoassay technique. Monoclonal antibodies specific for endoglycoproteins were coated onto microplates. Standards and samples are pipetted into the wells and any endoglin present is bound by the immobilized antibody. After washing away any unbound material, an enzyme-linked monoclonal antibody specific for endoglin is added to the wells. After washing to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells, showing a color proportional to the amount of endoglin bound in the initial step. The color development was terminated and the color intensity was measured. Additional details regarding this assay are found in the package insert. The range of measurements for endoglin includes amounts between 0.001ng/L and 10 ng/ml.

PlGF is tested in a sandwich immunoassay in ELECSYS using two antibodies specific for PlGF^TM-or cobas e^TMSerial analyzers (detailed above). The PlGF test is commercially available from Roche diagnostics GmbH, Mannheim, Germany. Additional details regarding this assay are found in the package insert. The range of PlGF measurements includes amounts of 3 to 10,000 pg/ml.

Example 2: analysis of preeclampsia as a result within 1 week

In the table used to generate the ROC curve, a cutoff value with the target NPV can be selected and the sensitivity, specificity and PPV at this cutoff value estimated. For all of these ratios, confidence intervals may be obtained based on the current sample size.

For patients between weeks 24+/-0 and 40+/-0 after pregnancy, the cut-off value of 45 yielded 100% (95% lower confidence limit 96.58%) NPV to predict preeclampsia in terms of the sFlt-1/PlGF ratio determined in samples obtained at the first visit, with a sensitivity estimate of 100% (LCL 9566.37%) and a specificity estimate of 80.92% (LCL 9573.13%). The PPV was estimated to be 26.47% (LCL 9512.88%). 34: 106 subjects tested positive/negative at this cutoff value, 9 of the 34 subjects were expected to develop PE, but none of the 106 subjects developed PE.

For patients between weeks 24+/-0 and 40+/-0 after pregnancy, the cutoff value of 45 was estimated to yield an npv of 99.43% (95% lower confidence limit 97.96%) with a sensitivity estimate of 86.67% (LCL 9563.66%) and a specificity estimate of 80.60% (LCL 9576.56%) in terms of the sFlt-1/PlGF ratio determined in samples obtained at multiple visits. The PPV was estimated to be 13.40% (LCL 957.33%). 97: 351 subjects tested positive/negative at this cutoff predicted 13 of the 94 visits to develop PE, but only 2 of the 351 visits developed PE.

The results are also summarized in the following table:

table 1: AUC, LCL95 and UCL95 values

Reaction of	Biomarkers	auc	LCL95	ucl95
					Development to PE within 1 week	sFlt1 PlGF ratio	0.9211	0.8703	0.9719
Development to PE within 1 week	Endoglin PlGF ratio	0.9118	0.8448	0.9788
					Development to PE within 1 week	tst_res_sFlt_1_pg_ml	0.933	0.8889	0.9771
Development to PE within 1 week	tst_res_PlGF_pg_ml	0.8482	0.7608	0.9356
					Development to PE within 1 week	Endoglin _ ng _ ml	0.9254	0.8615	0.9892
Development to PE within 1 week-all visits	sFlt1 PlGF ratio	0.9108	0.8689	0.9528
					Development to PE within 1 week-all visits	Endoglin PlGF ratio	0.8949	0.8493	0.9405
Development to PE within 1 week-all visits	tst_res_sFlt_1_pg_ml	0.9087	0.849	0.9684
					Development to PE within 1 week-all visits	tst_res_PlGF_pg_ml	0.8618	0.8112	0.9124
Development to PE within 1 week-all visits	Endoglin _ ng _ ml	0.8928	0.8419	0.9437
					Development to PE within 2 weeks	sFlt1 PlGF ratio	0.9262	0.8788	0.9735
Development to PE within 2 weeks	Endoglin PlGF ratio	0.9108	0.8487	0.9729
					Development to PE within 2 weeks	tst_res_sFlt_1_pg_ml	0.9438	0.9041	0.9836
Development to PE within 2 weeks	tst_res_PlGF_pg_ml	0.8485	0.7681	0.9288
					Development to PE within 2 weeks	Endoglin _ ng _ ml	0.9238	0.865	0.9827
Development to PE within 2 weeks-all visits	sFlt1 PlGF ratio	0.8978	0.8566	0.939
					Development to PE within 2 weeks-all visits	Endoglin PlGF ratio	0.873	0.8305	0.9155
Development to PE within 2 weeks-all visits	tst_res_sFlt_1_pg_ml	0.8897	0.8356	0.9437
					Development to PE within 2 weeks-all visits	tst_res_PlGF_pg_ml	0.8568	0.8116	0.9021
Development to PE within 2 weeks-all visits	Endoglin _ ng _ ml	0.8615	0.8161	0.9069
					PE evolution within 4 weeks	sFlt1 PlGF ratio	0.89	0.8355	0.9445
PE evolution within 4 weeks	Endoglin PlGF ratio	0.8608	0.7977	0.9239
					PE evolution within 4 weeks	tst_res_sFlt_1_pg_ml	0.9034	0.8447	0.9621
PE evolution within 4 weeks	tst_res_PlGF_pg_ml	0.8331	0.7663	0.8999

PE evolution within 4 weeks	Endoglin _ ng _ ml	0.8651	0.801	0.9293
					PE development within 4 weeks-all visits	sFlt1 PlGF ratio	0.8655	0.8261	0.9049
PE development within 4 weeks-all visits	Endoglin PlGF ratio	0.8369	0.797	0.8768
					PE development within 4 weeks-all visits	tst_res_sFlt_1_pg_ml	0.861	0.8168	0.9053
PE development within 4 weeks-all visits	tst_res_PlGF_pg_ml	0.8262	0.7806	0.8717
					PE development within 4 weeks-all visits	Endoglin _ ng _ ml	0.8233	0.7801	0.8666

Example 3: ratio of biomarkers in pregnant females: comparison of sFlt-1/PlG and Endoglin/PlGF with Doppler ultrasonography results to predict diagnostic Performance of PE/HELLP within a short time window

If the patient develops PE/HELLP syndrome within one/four weeks after the visit, mPI-UtA, the sFlt-1/PlGF ratio, and the endoglin/PlGF ratio are compared as categorical criteria (using cutoff values). The population here is pregnant females with available doppler ultrasonography results:

table 2: mPI-UtA, comparison of sFlt-1/PlGF ratios and Endoglin/PlGF ratios as sorting criteria

Both ratios appear to be superior to doppler ultrasonography for both prediction tasks. The sFlt-1/PlGF ratio appears to perform better than the endoglin/PlGF ratio (with the chosen cutoff).

If a patient develops PE within one/four weeks after a visit, the sFlt-1/PlGF ratio and the endoglin/PlGF ratio serve as the classification criteria (cut-off values are used). The population here is all pregnant females with abnormal doppler ultrasonography results:

table 3: sFlt-1/PlGF ratio and endoglin/PlGF ratio as classification criteria

In particular, the sFlt-1/PlGF ratio appears to be superior to the endoglin/PlGF ratio on a four week predictive task.

Table 4: ROC-related statistics of the values shown in FIG. 4A

Table 5: ROC-related statistics of the values shown in FIG. 5A

Example 4: results within 1 week were analysis of preeclampsia, large cohort (n ═ 269)

In the table used to generate the ROC curve, a cutoff value with the target NPV can be selected and the sensitivity, specificity and PPV at this cutoff value estimated. For all of these ratios, confidence intervals may be obtained based on the current sample size.

For patients between weeks 24+/-0 and 40+/-0 after pregnancy, the cutoff value of 38 resulted in an NPV of 98.9% (lower limit of bilateral 95% confidence interval (LCL 95): 97.3%) to predict preeclampsia in terms of the sFlt-1/PlGF ratio determined in the samples obtained at the first visit, with a sensitivity estimate of 88.2% (LCL 95: 72.6%) and a specificity estimate of 79.8% (LCL 95: 75.9%). PPV was estimated to be 24.4% (LCL 95: 17.1%). 123: 372 subjects tested positive/negative at this cutoff value, 30 of the 123 test positives were expected to develop PE and 4 of the 372 test negatives to develop PE. The results are also summarized in the following table:

table 5: AUC, LCL95 and UCL95 values, Large cohort

Claims (10)

1. Use of at least one of the biomarkers sFlt-1, endoglin and PlGF or at least one detection agent that specifically binds to said biomarker in a sample of a pregnant subject for the preparation of a diagnostic entity or a pharmaceutical entity or composition for diagnosing whether said subject is not at risk of developing preeclampsia within a short period of time, wherein said diagnosis comprises the method steps of:

a) determining the amount of the following biomarkers in a sample of the subject: (i) sFlt-1 and PlGF, or (ii) endoglin and PlGF;

b) calculating the ratio of the amounts of (i) sFlt-1 and PlGF, or (ii) endoglin and PlGF, in the sample determined in step a); and

c) comparing the ratio of the amounts to a reference value, whereby the subject is diagnosed not at risk of developing preeclampsia within a short period if the ratio of the amounts is the same or decreased compared to the reference value, wherein the reference value allows for a diagnosis with a negative predictive value of at least 98%, wherein the short period is less than 4 weeks.

2. The use of claim 1, wherein the reference value is 46 or less.

3. The use of claim 1, wherein the reference value is 38 or less.

4. Use according to claim 2, wherein the reference value is 33.

5. The use of claim 1, wherein the short period is a period of 1 to 2 weeks.

6. The use of claim 1, wherein the pregnant subject is between weeks 20 and 40 of pregnancy.

7. The use of claim 1, wherein the sample is a blood, plasma, serum or urine sample.

8. The use of claim 1, wherein the method further comprises suggesting a patient management measure based on the diagnosis.

9. The use of claim 8, wherein (i) if the subject is not diagnosed as not at risk of developing preeclampsia, then the patient management measures are selected from the following measures: close monitoring, hospitalization, administration of hypotensive agents and lifestyle advice; (ii) the patient management measure is flow monitoring if the subject is diagnosed as not being at risk of developing preeclampsia.

10. The use of claim 1, wherein the subject has been identified as being at risk of developing preeclampsia, eclampsia, and/or HELLP syndrome based on abnormal doppler sonography results.