HK1194813B

HK1194813B - Dynamic of sflt-1 or endoglin/plgf ratio as an indicator for imminent preeclampsia and/or hellp syndrome

Info

Publication number: HK1194813B
Application number: HK14108088.0A
Authority: HK
Inventors: Barbara DENK; Martin Hund
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2011-11-09
Filing date: 2012-11-08
Publication date: 2018-02-02

Description

Dynamic ratio of SFlt-1 or Endoglin/PlGF as indicator of critical preeclampsia and/or HELLP syndrome

The present invention relates to diagnostic methods and tools. In particular, the present invention relates to a method of diagnosing whether a pregnant subject is at risk for developing preeclampsia within a short period of time, the method comprising: (a) determining the amount of the biomarker sFlt-1 or Endoglin and the amount of PlGF in a first and a second sample of said subject, wherein said first sample is obtained before said second sample, (b) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample and a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample, and (c) comparing the values of said first and second ratios, and if compared to the value of said first ratio, said second ratio is increased by at least about 3-fold, i.e. diagnosing the subject as being at risk of developing pre-eclampsia in the short period. The invention also relates to a method of distinguishing between pregnant women at risk of developing early-onset preeclampsia (early-onset-preeclampsia) and pregnant women without risk of developing early-onset preeclampsia. Furthermore, the invention also comprises a device and a kit for carrying out said method. And to a system for performing an optimized risk assessment of pre-eclampsia as described herein, as well as reagents and kits for use in carrying out the methods described herein.

Pregnancy may have different complications, which are related, on the one hand, to pregnancy-related mortality of the pregnant woman and, on the other hand, to increased morbidity and mortality of the newborn. Maternal mortality of 14.5 individuals per 100000 live births (live births) is more common in pregnant women over the age of 39, which may be due to bleeding, thrombotic pulmonary embolism (thrombotic pulmonary embolism), infection, cardiomyopathy, cardiovascular and non-cardiovascular diseases, and hypertensive disorders, with pre-eclampsia being the most common (Berg2010, obsterics and Gynecology:116: 1302-1309).

Preeclampsia is associated with approximately 2% to 8% of pregnant women and is the leading cause of maternal and fetal death worldwide (Duley2009, Semin Perinato: 33: 130-37.) preeclampsia is generally defined as pregnancy related or induced hypertension characterized by hypertension and proteinuria where hypertension is defined as a blood pressure of 140mmHg (systolic) to 90mmHg (diastolic) or more in two independent measurements with at least 6 hours between the two measurements proteinuria is shown as a protein at 300mg/dL or more in a24 hour urine sampleund Geburtshilfee.V.,August2008。

The pathogenesis of preeclampsia is largely unknown. However, it is believed to be caused by placental dysfunction associated with impaired remodeling of helical arteries (spiral arteries). Flow defects (flow defects) that occur during the development of pre-eclampsia are associated with ischemia, which ultimately leads to the release of anti-angiogenic factors (anti-angiogenic factors) such as sFlt-1 and Endoglin into the blood circulation.

To date, the only way to treat preeclampsia has been to deliver preterm infants by vaginal or caesarean delivery to terminate pregnancy. As discussed above, the development of preeclampsia before gestation week 34 will seriously threaten the viability of the mother and fetus. Therefore, efforts should be made to delay production and improve survival of the newborn.

For preeclampsia, especially early onset preeclampsia that occurs as early as gestation between 20 and 34 weeks, early, reliable diagnosis is critical for clinical control of this disease. It is clear that pregnant women with preeclampsia require special care, such as close observation, helpful treatment, and hospitalization in specialized hospitals with maternal and infant intensive care units (MFICUs) once severe preeclampsia develops. In particular, early onset preeclampsia is troublesome to clinicians due to its serious side effects and adverse consequences commonly associated therewith. Furthermore, reliable diagnosis and prediction for early stage preeclampsia are also critical for planning preventive and therapeutic intervention studies (Ohkuchi2011, Hypertension58: 859-.

Recently, it has been suggested that angiogenic factors and their antagonists are indicators of preeclampsia. Specifically, 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 the reports on individual factors and their changes in healthy individuals and patients with or at risk of pre-eclampsia (see, e.g., Rana2007, Hypertension50: 137-142; WO2004/008946), there are also reports of the ratio of sFlt-1 to PlGF or the ratio of Endoglin to PlGF as a diagnostic or prognostic parameter.

A single ratio of sFlt-1 and PlGF has been reported as a prognostic factor for pre-eclampsia during early pregnancy (Crispi2008, ultrasounds Obstet Gynecol31: 303-309). In addition, the relevance of the respective proportions of sFlt-1 and PlGF at different time points during pregnancy to the risk of pre-eclampsia was shown (DeVivo2008, actaObstetricia et Gynecology 87: 837-. The degree of change in the prognosis of preeclampsia has also been explored (Kusanovic2009, supra).

However, the proportions studied to date have not been able to distinguish between early-onset and late-onset preeclampsia (DeVivo2008, supra). Even though there are reports that the ratio of Endoglin and PlGF determined in the third trimester of pregnancy is usually associated with early-onset preeclampsia, there are no reports in the prior art for indicators of critical preeclampsia in pregnant women that appear healthy.

Thus, there is currently no, but urgent need for a reliable assay for identifying pregnant women who appear to be healthy but who are at risk for pre-eclampsia, and particularly at risk for early onset pre-eclampsia.

The technical problem addressed by the present invention can be seen as providing means and methods that serve the above needs. The technical problem is solved by the embodiments described in the claims and hereinafter.

Accordingly, the present invention relates to a method of diagnosing whether a pregnant subject is at risk for developing preeclampsia within a short period of time, the method comprising:

a) determining the amount of the biomarker sFlt-1 or Endoglin and the amount of PlGF in a first and a second sample of said subject, wherein said first sample is obtained before said second sample;

b) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample and a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample, and

c) comparing the values of the first and second ratios, and diagnosing the subject as being at risk for developing preeclampsia within the short term if the value of the second ratio is increased by at least 3-fold as compared to the value of the first ratio.

The method of the invention is preferably an ex vivo (ex vivo) method. Also, steps other than those explicitly set forth above may be included. For example, additional steps may be with respect to sample pre-treatment, or evaluation of the results obtained by the method. The method can be implemented manually or assisted by automation. Preferably, steps (a), (b) and/or (c) may be carried out fully or partially by automated assistance, for example using suitable robots and sensing equipment (sensory equipment) for the determination in step (a), computer-implemented algorithms in the data processing device for step (b), or comparison and/or diagnostic algorithms in the data processing device for step (c).

Thus, the present invention also preferably relates to a system for optimizing a risk assessment based on clinical prediction rules for classifying pregnant subjects, the system comprising:

a) an analyzer unit configured to contact in vitro a portion of a second sample from a pregnant subject comprising a ligand with specific binding affinity for sFlt-1 and/or Endoglin, and configured to contact in vitro a portion of a sample from a pregnant subject comprising a ligand with specific binding affinity for PlGF,

b) an analyzer unit configured to detect a signal from the portion of the sample from the subject contacted by the ligand,

c) a computing device having a processor and in operable communication with the analyzer unit,

d) a non-transitory machine-readable medium comprising a plurality of processor-executable instructions that, when executed, calculate an amount of sFlt-1 and/or Endoglin, calculate an amount of PlGF, calculate a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in the sample, and compare the calculated ratio to a first ratio obtained from a first sample, thereby optimizing risk assessment based on clinical prediction rules for classifying pregnant subjects.

The term "preeclampsia" as used in this application refers to a pathological condition characterized by hypertension and proteinuria. "preeclampsia" occurs in pregnant female subjects, where hypertension is also referred to as pregnancy induced hypertension. Preferably, the presence of pregnancy induced hypertension in the subject is identified by two blood pressure determinations of 140mmHg (systolic) to 90mmHg (diastolic) or more, wherein the two determinationsThe patient with pre-eclampsia related to liver may further develop HELLP syndrome within 24 hours, and therefore, patients at risk of pre-eclampsia according to the present invention, preferably also have a potential risk of HELLP syndrome, with adverse consequences of high risk, such as premature placental peeling, renal failure, subcapsular bleeding, recurrent eclampsia, premature labor, or even pre-maternal/or fetal death related to the syndrome of pre-eclampsia and the syndrome of follow-up diseases such as HELLP or eclampsia, and the detailed medical guidance of follow-up diseases such as that of Bupleuropy, Japanese eclampsia, and hepatitis, see the medical Guidelines for Clinical sciences, see the medical science ü, the medical Guidelines for Clinical sciences, and Clinical sciences, see the medical sciences for example, the Clinical sciences, and the scientific research institute of human, and the Clinical sciences, and medical sciences for exampleund Geburrstellfe.V., August 2008. Preeclampsia occurs in up to 10% of pregnancies, usually in the second or third trimester. However, there are also some women who develop preeclampsia as early as the 20 th week of pregnancy.

Within weeks 20 to 34 of gestation, preeclampsia is also referred to as early onset preeclampsia, and preeclampsia occurring later than week 34 of gestation is also referred to as late onset preeclampsia. It is known that common early onset preeclampsia is associated with more severe side effects and adverse consequences than the generally relatively mild late onset preeclampsia.

By "at risk of developing preeclampsia" is meant that the pregnant subject has a statistically significantly increased likelihood that preeclampsia will occur within a future prognostic window as compared to a pregnant subject that is not at risk of preeclampsia. Preferably, said probability is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99% or up to 100%. For more details on the statistics, see elsewhere in this application.

The term "subject" as used in the present application relates to an animal, preferably a mammal, more preferably a human. The subject of the present invention is desirably a pregnant subject, i.e. a pregnant female. Preferably, the subject of the invention should not exhibit preeclampsia symptoms. Such preeclampsia symptoms are preferably clinical symptoms as described in detail in the rest of the application. More preferably, the symptoms comprise at least one symptom selected from the group consisting of: pain in the upper abdomen, headache, visual disturbance, edema. However, the subject of the invention may also exhibit at least one of the symptoms described above, and thus has been suspected of suffering from preeclampsia.

More preferably, the pregnant subject of the invention is between about week 15 and about week 34 of gestation, preferably, between about week 15 and about week 30 of gestation.

The methods of the invention can be used in a routine screening format for pregnant subjects that appear healthy. However, the pregnant subjects contemplated by the present invention may also belong to risk groups with higher pre-eclampsia incidences. Generally, pregnant subjects with obesity, hypertension, autoimmune diseases, such as lupus erythematosus, thrombophilia or diabetes (diabetes mellitis) have a higher incidence of preeclampsia. It is also applicable to subjects suffering from preeclampsia, eclampsia and/or HELLP syndrome in a previous pregnancy. Furthermore, women of older age who are first pregnant also show a propensity to develop preeclampsia. However, the likelihood of developing preeclampsia decreases with the number of pregnancies.

The term "diagnosing" as used herein refers to determining whether a subject is at risk for developing preeclampsia within a short period of time. Preferably, the short term is a period of less than 4 weeks, preferably between about 2 and about 3 weeks, and more preferably, the period of time is about 16 days. Those skilled in the art will appreciate that such a determination is not generally intended to be 100% correct for the subject being diagnosed. However, this term requires that the determination be correct for a statistically significant portion of the subjects (e.g., a cohort in a cohort study). One skilled in the art can readily determine whether a portion is statistically significant by using a variety of known statistical evaluation tools, such as the determination of confidence intervals, the determination of p-values, Student's t-tests, Mann-Whitney tests, and the like. See downy and Wearden, Statistics for Research, John Wiley & Sons, New York1983 for details. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

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

The "first sample" in the method of the invention is obtained before said "second sample". It will be appreciated that the first and second samples are samples of the same type of sample material, i.e. both from the same type of bodily fluid, cell, tissue or organ. Furthermore, said first sample is prior to a second sample, preferably from two subsequent routine medical examinations during pregnancy, wherein said first sample is taken in a prior examination and said second sample is taken in a subsequent examination.

Preferably, the first sample has been obtained from about 1 week to about 15 weeks, preferably from about 2 weeks to about 6 weeks, more preferably from about 4 weeks to about 5 weeks prior to the second sample.

The term "sFlt-1" as used in this application refers to a polypeptide which is a soluble form of fms-like tyrosine kinase 1. The polypeptide is also known in the art as soluble VEGF receptor 1(sVEGF R1) (see, e.g., Sunderji2010, Am JObstet Gynecol202:40e 1-7). It was identified in human umbilical vein endothelial cell conditioned medium. The endogenous sFlt1 receptor is chromatographically and immunologically similar to human sFlt1 and binds [125I ] VEGF with similar high affinity. Human sFlt1 appeared to form a VEGF-stabilized complex with the extracellular domain of KDR/Flk-1 in vitro. Preferably, sFlt1 refers to human sFlt1 as described in Kendall1996, Biochem Biophs Res Commun226(2): 324-; the amino acid sequences are described, for example, in the human sFlt-1 sequence Genebank accession number P17948, GI:125361, the mouse sFlt-1 sequence BAA24499.1, GI:2809071(Genebank is available from NCBI, USA website: "http:// www.ncbi.nlm.nih.gov/entrez). The term also encompasses variants of the human sFlt1 polypeptide described above. Such variants have at least the same basic biological and immunological properties as the previously described sFlt1 polypeptide. In particular, they 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 an ELISA assay using polyclonal or monoclonal antibodies specifically recognizing the sFlt1 polypeptide. Furthermore, it is understood that the amino acid sequence of a variant referred to in the present invention may differ from a particular sFlt1 polypeptide sequence by at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of said variant still has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identity to a particular sFlt1 polypeptide sequence, preferably to the full-length human sFlt1 sequence, 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 the amino acid sequence segments in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) used for optimal alignment. The percentage of sequence identity is calculated by determining the number of positions at which the amino acid residues in the two amino acid sequences are identical, obtaining the number of matched positions, dividing the number of matched positions by the number of total positions in the window of comparison, and multiplying the result by 100. Optimal alignment of the sequences being compared can be performed by the local homology algorithm disclosed by Smith1981, Add.APL.Math.2:482, the homology alignment algorithm described by Needleman1970, J.mol.biol.48:443, Pearson1988, Proc.Natl.Acad Sci. (USA)85:2444, by computerized implementation of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis), or by visual inspection. After identifying the two sequences for comparison, optimal alignment and hence degree of identity is preferably determined using GAP and BESTFIT. Preferably, a default value of 5.00 for the gap weight and a default value of 0.30 for the length of the gap weight are used. Such variants may be allelic variants, or any other species-specific homologues, orthologs, or orthologs. Such variants may be allelic variants, or any other species-specific homologues, orthologs. Furthermore, variants described herein include fragments or subunits of a particular sFlt-1 polypeptide or variant types described above, provided that such fragments have the basic biological and immunological properties described above. Such fragments may be, for example, degradation products of sFlt-1 polypeptides. Such variants are considered to have the same basic biological and immunological properties if they can be detected by the same specific assay as mentioned in the present specification, for example an ELISA assay using a polyclonal or monoclonal antibody specifically recognizing the sFlt1 polypeptide. Preferred assays are described in the accompanying examples. Variants that differ by post-translational modifications, such as phosphorylation, myristylation, are also included in the variants described herein. sFlt-1 may be detected in bound or free form, or as the amount of total sFlt-1 in a sample.

The term "Endoglin" in the present application refers to a polypeptide having a molecular weight of 180kDa when unreduced, a molecular weight of 95kDa when reduced, and a molecular weight of 66kDa in the reduced and N-deglycosylated form. The polypeptides are capable of forming dimers and binding to TGF-beta and TGF-beta receptors. 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 Endoglin, S-Endoglin and L-Endoglin, have been described. L-Endoglin consists of 633 amino acids in total and contains a cytoplasmic tail of 47 amino acids, while S-Endoglin consists of 600 amino acids in total and contains a cytoplasmic tail of 14 amino acids. Preferably, the Endoglin used in the present application is a soluble Endoglin. The soluble Endoglin described herein is preferably as described in EP1804836B 1. Furthermore, it is understood that the amino acid sequence of a variant referred to in the present invention may differ by at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of said variant still has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identity to the amino acid sequence of the specific Endoglin. Such variants may be allelic variants, splice variants, or any other species-specific homologues, orthologs, or orthologs. Furthermore, the variants described in this application include fragments or subunits of the particular Endoglin or variant types described above, provided that the fragments have the basic immunological and biological properties described above. Such fragments may be, for example, degradation products of endoslin. Said variants are considered to have the same basic biological and immunological properties if they can be detected by the same specific assay as mentioned in the present specification, for example an ELISA assay using a polyclonal or monoclonal antibody specifically recognizing said Endoglin polypeptide. Preferred assays are described in the accompanying examples. Variants that differ by post-translational modifications, such as phosphorylation, myristylation, are also included in the variants described herein. Endoglin can be detected in bound or free form, or as the total amount of Endoglin in a sample.

The term "PlGF (placental growth factor)" as used in the present application refers to a growth factor from placenta, which is a polypeptide having a length of 149 amino acids and being highly homologous to the platelet-derived growth factor-like region of human Vascular Endothelial Growth Factor (VEGF). Like VEGF, PlGF has angiogenic activity in vitro and in vivo. For example, the biochemical and functional characteristics of PlGF from transfected COS-1 cells revealed to be a glycosylated dimeric secretory protein capable of stimulating endothelial cell growth in vitro (Maqlione1993, Oncogene8(4): 925-31). Preferably, PlGF refers to human PlGF, more preferably human PlGF having an amino acid sequence as set forth in Genebank accession number P49763, GI: 17380553. The term encompasses variants of the specific human PlGF. Said variants having at least the same basic biological and immunological properties as the aforementioned specific PlGF. Variants are considered to have the same basic biological and immunological properties if they can be detected by the same specific assay as mentioned in the present specification, e.g. an ELISA assay using a polyclonal or monoclonal antibody specifically recognizing said specific PlGF polypeptide. Preferred assays are described in the accompanying examples. Furthermore, it is understood that the amino acid sequence of a variant of the invention may differ by at least one amino acid substitution, deletion and/or addition, wherein the amino acid sequence of said variant still has at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identity to the specified pigf polypeptide sequence. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and described elsewhere in this application. Such variants may be allelic variants, or any other species-specific homologues, orthologs, or orthologs. Furthermore, the variants described in the present application include fragments of the particular PLGF polypeptide or of the above-mentioned variant types, provided that these fragments have the basic biological and immunological properties described above. Such fragments may be, for example, splice variants or degradation products of the PLGF polypeptide. Furthermore, variants that differ by post-translational modifications, such as phosphorylation, myristylation, are also included in the variants described herein. PlGF can 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 referred to in this specification relates to measurement of quantity or concentration, preferably semi-quantitative or quantitative. The measurement may be made directly or indirectly. Direct measurement involves measuring the amount or concentration of a peptide or polypeptide based on the signal obtained from the peptide or polypeptide itself, as well as the signal intensity that is directly related to the number of molecules of the peptide 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 secondary components (i.e., components other than the peptide or polypeptide itself) or biological readout systems (biological read out systems), such as measurable cellular responses, ligands, tags or enzymatic reaction products.

According to the invention, the determination of the amount of the peptide or polypeptide can be achieved by all known means for determining the amount of peptide in a sample. Such means include immunoassay devices and methods employing labeled molecules, in a variety of different sandwich, competition, or other assay formats. The assay will produce a signal indicating the presence or absence of the peptide or polypeptide. Moreover, the intensity of the signal is preferably directly or indirectly related to (e.g., inversely proportional to) the amount of polypeptide in the sample. Other suitable methods include measuring physical or chemical properties specific to the peptide or polypeptide, such as its precise molecular weight or NMR spectrum. The method comprises, preferably, 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. Additional methods include methods based on microplate ELISA, fully automated or robotic immunoassays (e.g.by Elecsys)^TMAnalyzer performed), CBA (enzymatic cobalt binding assay, e.g., by Roche-Hitachi)^TMAnalyzer) and latex agglutination test assays (e.g., as may be performed by Roche-Hitachi)^TMAnalyzer run).

Preferably, determining the amount of the peptide or polypeptide comprises the steps of: (a) contacting said peptide or polypeptide with a cell capable of eliciting a cellular response, the intensity of said cellular response being indicative of the amount of said peptide or polypeptide, for a sufficient period of time, (b) measuring said cellular response. To measure cellular responses, preferably, the sample or treated sample is added to a cell culture and the cellular response is measured internally or externally. The cellular response may include measurable reporter gene expression or secretion of substances, such as peptides, polypeptides and small molecules. The expression or substance will produce an intensity signal that correlates with the amount of the peptide or polypeptide. In a preferred embodiment, the contacting, removing, and measuring steps can be performed by an analyzer unit of the system disclosed herein. According to some implementations, the steps may be performed by a single analyzer unit of the system, or by more than one analyzer unit in operable communication with each other. For example, according to one particular embodiment, the system disclosed herein may include a first analyzer unit for performing the contacting and removing steps, and a second analyzer unit operatively connected to the first analyzer unit by a transport unit (e.g., a robot), the second analyzer unit performing the measuring steps.

Preferably, determining the amount of the peptide or polypeptide comprises the step of measuring a specific intensity signal obtainable from the peptide or polypeptide in the sample. As mentioned above, the signal may be the intensity of the signal observed in the mass spectrum or NMR spectrum specific for the peptide or polypeptide at the m/z variable specific for 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) measuring the amount of bound ligand. The bound ligand will generate an intensity signal. Binding in the context of the present invention includes both covalent and non-covalent binding. The ligand in the present invention may be any compound, such as a peptide, polypeptide, nucleic acid or small molecule, that binds to a peptide or polypeptide in the present application. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, e.g., receptors or binding partners for the peptides or polypeptides, and fragments thereof comprising the binding domain of the peptide, and aptamers, e.g., nucleic acid or peptide aptamers. Methods for preparing such ligands are well known in the art. For example, the identification and production of suitable antibodies and aptamers may be provided by a supplier. One of ordinary skill in the art is familiar with methods for developing derivatives of the above ligands with higher affinity and specificity. For example, random mutations may be introduced into the nucleic acid, peptide or polypeptide. The resulting derivatives are then tested for binding by screening procedures known in the art, such as phage display. Antibodies referred to herein include polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F (ab)2 fragments that bind antigen or hapten. The invention also includes single chain antibodies, as well as humanized hybrid antibodies, in which the amino acid sequence of a non-human donor antibody exhibiting the desired antigen specificity is combined with the amino acid 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 relevant amino acid residues of the donor antibody. The hybrids can be prepared by several methods known in the art. Preferably, the ligand or agent binds specifically to the peptide or polypeptide. Specific binding according to the present invention means that the ligand or reagent does not substantially bind to other peptides, polypeptides or substances present in the sample to be analyzed, i.e.: cross-reaction occurs. Preferably, the specific binding peptide or polypeptide has a binding affinity that is at least 3-fold stronger, more preferably at least 10-fold stronger, and even more preferably at least 50-fold stronger than any other related peptide or polypeptide. Such non-specific binding may be tolerable if it can still be clearly distinguished and measured, for example, 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. Further suitable methods for determining the polypeptide or peptide are described in the subsequent section.

First, the binding of the ligand can be measured directly by, for example, NMR or surface plasmon resonance. The measurement of ligand binding is, according to a preferred embodiment, performed by an analyzer unit of the system described herein. Thereafter, the measured binding amount may be entered by a computing device of the system described hereinAnd (5) line calculation. Second, if the ligand also serves as an enzymatically active substrate for the peptide or polypeptide of interest, the enzymatic reaction product can also be measured (e.g., by measuring, e.g., the amount of protease by measuring the amount of cleaved substrate on a Western Blot). Alternatively, the ligand itself may exhibit enzymatic properties, and the "ligand/peptide or polypeptide" complex or the ligand to which the peptide or polypeptide binds, respectively, may be contacted with a suitable substrate, the intensity signal generated thereby enabling detection. 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 period of time. A sufficiently long period of time refers to the time necessary to produce a detectable, preferably measurable amount of product. The time necessary for a given (e.g., detectable) amount of product to occur can be measured without having to measure the amount of product. Third, the ligand may be covalently or non-covalently coupled to a label that allows the ligand to be detected and measured. Labeling can be performed by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) a second ligand to the first ligand. The second ligand should specifically bind to the first ligand. The second ligand may be coupled to a suitable label and/or act as a target (receptor) for a third ligand which binds to the second ligand. The role of a second, third or even higher order ligand is generally to enhance the signal. Suitable secondary or higher order ligands may include antibodies, secondary antibodies, and the well known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate may also be "tagged" with one or more tags known in the art. Such tags can thus serve as 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. For peptides or polypeptides, the tag is preferably located at the N-terminus and/or C-terminus. Suitable labels are any labels which can be detected by a suitable detection methodGeneral labels include gold particles, latex beads, 9, 10-acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels (e.g., "magnetic beads", including paramagnetic or superparamagnetic labels), and fluorescent labels, enzymatically active labels include, e.g., horseradish peroxidase, alkaline phosphatase, β -galactosidase, luciferase, and derivatives thereof suitable substrates for detection include Diaminobenzidine (DAB), 3'-5,5' -tetramethylbenzidine, NBT-BCIP (4-nitrotetrazolium chloride and 5-bromo-4-chloro-3-indole-phosphate, available from stock Roche Diagnostics), CDP-Star^TM(Amersham Biosciences)，ECF^TM(Amersham biosciences). Suitable enzyme-substrate combinations can produce colored reaction products, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using a light-sensitive film or a suitable photographic system). For the measurement of the enzymatic reaction, the above criteria are similarly applied. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, texas red, fluorescein, and Alexa dyes (e.g., Alexa 568). Further fluorescent labels are available from e.g. molecular probes (Oregon). Quantum dots are also contemplated as fluorescent labels. Common radioactive labels include 35S, 125I, 32P, 33P, and the like. The radiolabel may be detected by any known suitable method, for example light-sensitive film or a phosphoimager (phosphorimager). Suitable measurement methods according to the invention also include precipitation (especially immunoprecipitation), electrochemiluminescence (electro-chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), sandwich enzyme immunoassays (sandwich enzyme immuno assays), electrochemiluminescence sandwich immunoassays (ECLIA), dissociation-enhanced lanthanide fluorescence immunoassays (DELFIA), Scintillation Proximity Assay (SPA), turbidimetry, nephelometry, turbidimetry-enhanced turbidimetry or nephelometry, or solid phase immunoassays. Other methods known in the art (e.g., gel electrophoresis, 2D gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)) Western blot and mass spectrometry) can be used alone or in combination with labels or other detection methods described above.

The amount of the peptide or polypeptide may also, preferably, be determined according to the following steps: (a) contacting a sample comprising said peptide or polypeptide with a solid support comprising a ligand for said peptide or polypeptide, (b) preferably, removing unbound peptide or polypeptide and remaining sample material, and (c) measuring the amount of peptide or polypeptide bound to said support. Preferably, the ligand is selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, and is preferably present in an immobilized form on the solid support. Materials for preparing solid supports are well known in the art and include, among other known materials, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and/or silicon chips or surfaces, nitrocellulose strips, membranes, sheets, duracytes, wells and walls of reaction trays, 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 or modified cellulose, polyacrylamide, agarose and magnetite. The nature of the support according to the object of the invention may be soluble or insoluble. Suitable methods of immobilizing (immobilizing)/immobilizing (immobilizing) the carrier are well known in the art and include, but are not limited to, ionic, hydrophobic, covalent interactions and the like. It is also possible to consider the use of "suspended arrays" as arrays according to the invention (Nolan2002, Trends Biotechnol.20(1): 9-12). In such a suspension array, the carrier, such as a microbead or microsphere, is present in a suspended state. The array consists of different microbeads or microspheres, which may be labeled, carrying different ligands. Methods for preparing such arrays, e.g. based on solid phase chemistry or photolabile protecting groups, are well known in the art (US5,744,305).

The term "amount" as used herein includes the absolute amount of the peptide or polypeptide, the relative amount or concentration of the peptide or polypeptide, and values or parameters related thereto or derived therefrom. These values or parameters include intensity signal values from all characteristic physical or chemical properties obtained by direct measurement of the peptide, such as intensity values in a mass spectrum or NMR spectrum. Furthermore, all values or parameters obtained by indirect measurements as described elsewhere in the specification, such as the level of response in the response of the peptide as determined by a biological readout system, or the intensity signal obtained by specific binding to a ligand, are also included. It will be understood that values related to the aforementioned quantities or parameters may also be obtained by all standardized mathematical operations. According to a preferred embodiment of the invention, the determination of said "amount" is performed by a system as disclosed herein, wherein the computing device determines the above-mentioned "amount" based on the contacting and measuring steps performed by one or more analyzer units of the system.

The term "calculating a first ratio" or "calculating a second ratio" in this application refers to calculating the ratio of the amount of sFlt-1 or Endoglin to the amount of PlGF by dividing the amount of sFlt-1 or Endoglin by the amount of PlGF, or any other similar mathematical operation that relates the amount of sFlt-1 or Endoglin to the amount of PlGF. Preferably, the ratio is calculated by dividing the amount of sFlt-1 or Endoglin by the amount of PlGF. Said calculation is performed on the respective values determined in said first and second samples to obtain said first and second ratios, respectively.

The term "comparing" in this application comprises comparing the first ratio and the second ratio as defined above. It is to be understood that comparison as used in this application refers to any manner of comparison between the calculated first ratio value and the calculated second ratio value. In studies throughout the present invention it has been found that an increased risk of developing preeclampsia is associated with an increase in the value of the first ratio by a factor of about 3 or more than the value of the second ratio. The comparison referred to in the method of the invention may be performed manually or by a computing device (system disclosed in the present application).

The comparison referred to in step (c) of the method of the invention may be done manually or computer-assisted. The values of the ratios may, for example, be compared with each other and the comparison is made automatically by a computer program executing an algorithm for the comparison. As a result of comparing said values, the obtained slope value indicates a multiple of the difference of said second proportional value from said first proportional value. In a further comparison step, it is determined whether the slope value is equal to, greater than or less than 3 times. If the slope value is about 3 or greater (i.e., when an increase of about 200% or more is observed), a diagnosis of risk of developing preeclampsia in the short term is made ("rule-in"). Similarly, a slope value below a factor of 3 (i.e., an observed increase of less than 200%, said value being substantially unchanged or decreased) will make a diagnosis that there is no risk of developing preeclampsia within a short period of time ("rule-out"). The evaluation of the first and second ratio value comparison results may also be performed automatically. The computer program performing the evaluation will provide the required evaluation in a suitable output format. For example, the comparison results are given in the form of raw data (absolute or relative quantities), and in some cases in the form of words, phrases, symbols or numerical indications representing specific diagnostic results. The "likelihood of confirmation" and/or "likelihood of exclusion" diagnosis may be provided by the computing device of the system disclosed herein based on a comparison between the calculated first and second ratios as described herein. For example, a computing device of the system may provide an indication in the form of a word, phrase, symbol, or value representing one of a "confirmed likelihood" diagnosis or a "excluded likelihood" diagnosis.

The term "about" as used herein refers to +/-20%, +/-10%, +/-5%, +/-2% or +/-1% of the parameter or value indicated. This also takes into account customary deviations due to measurement techniques etc.

Advantageously, it has been found throughout the studies of the present invention that a strongly increased ratio between the amount of sFlt-1 or Endoglin and the amount of PlGF (the ratio of sFlt1/PlGF or Endoglin/PlGF) in pregnant subjects who do not exhibit, or exhibit only limited clinically significant symptoms of pre-eclampsia at the time the study sample was taken is an indication of critical pre-eclampsia, i.e. the development of pre-eclampsia and/or critical HELLP syndrome within a short period of several weeks. Furthermore, it has also been found that the preeclampsia suffered by the subject at risk is often of the more severe early onset preeclampsia type. In particular, a 3-fold or greater increase was found to be a reliable predictor of the aforementioned critical preeclampsia, with better sensitivity and specificity above 98% in practice. Whereas a weaker increase does not show a close correlation with the development of critical preeclampsia. Clearly, the above-described dramatic increase in either the absolute amount or the ratio of the biomarkers is predictive.

Thanks to the present invention, a more reliable diagnosis of critical preeclampsia, in particular the risk of critical early onset preeclampsia, can be made on the basis of reliable indications which appear to be independent of the absolute amount of the above-mentioned biomarkers found in the subject itself. Furthermore, the method of the present invention as an aid to diagnosis can avoid time-consuming, expensive and cumbersome diagnostic procedures such as the current scoring systems. Because the close observation and specific care required of pregnant women with preeclampsia can be better assessed and taken into account in the management of medical services, medical services management would greatly benefit from the methods of the present invention.

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

In a preferred embodiment of the method of the invention, said preeclampsia is early onset preeclampsia.

Thus, the methods of the invention can be used to diagnose a subject at risk of developing early onset preeclampsia in the short term, particularly for such pregnant subjects between weeks 15 and 30 of gestation. As discussed previously, early onset preeclampsia generally has more severe consequences than late onset preeclampsia, and thus patients require helpful measures to ameliorate the consequences of such preeclampsia. For example, a patient at risk may be admitted to a hospital with a maternal and infant intensive care unit at an early stage.

In another preferred embodiment of the method of the invention, the method further comprises recommending at least one helpful measure for preeclampsia if the subject is diagnosed with an increased risk of preeclampsia in the short term.

The term "recommendation" as used herein refers to a suggestion regarding a helpful measure or combination of measures that may be applied to a subject. It is to be understood that the term does not necessarily encompass the administration of the treatment per se.

As discussed previously, subjects with preeclampsia require special medical care. Thus, if a subject is diagnosed as being at risk for preeclampsia, such diagnosis may help establish appropriate helpful measures for the subject in advance, i.e., before the onset of such preeclampsia becomes clinically apparent. Preferably, the at least one assisting measure is selected from the group consisting of: close monitoring, hospitalization, administration of hypotensive agents, and/or lifestyle recommendations. With respect to the fetus, it may be advisable to administer betamethasone in preparation for later improvement of the respiratory function of the neonate in preterm delivery.

The invention also relates to a method of distinguishing between a pregnant subject at risk of developing early onset preeclampsia and a pregnant subject not at risk of developing early onset preeclampsia, the method comprising:

b) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample and a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample;

c) comparing the values of the first and second ratios, wherein the value of the second ratio is at least 3-fold increased if compared to the value of the first ratio, i.e., diagnosing the subject as being at risk for developing early onset preeclampsia.

Preferably, the early onset preeclampsia is initiated in the short term as set forth in detail elsewhere in this application.

It is also preferred that the second sample has been taken no later than at week 30 of pregnancy, i.e. prior to week 30 of pregnancy.

As already discussed above, reliable identification of subjects at risk of developing early onset preeclampsia is a crucial task in health management. In particular, pregnant women with preeclampsia require special care, and the present invention allows for a better assessment of the care needs and allows for medical services management considerations. In particular, the above method may even detect early-onset preeclampsia if said second sample is obtained around week 30 of pregnancy. It will be appreciated that if a subject still has no preeclampsia symptoms or exhibits only limited preeclampsia symptoms by the 30 th week of gestation, i.e., after a second sample has been obtained, it will be almost diagnosable as non-early onset preeclampsia because clinically significant preeclampsia typically occurs after the 34 th week of gestation. Since the above-described method already takes into account the dynamic changes of the biomarkers, it is more reliable to diagnose the correct type of preeclampsia.

In general, the invention includes the use of the biomarkers sFlt-1 or Endoglin and PlGF, or detection reagents that specifically bind to the biomarkers, in first and second samples from a pregnant subject to diagnose whether the subject is at risk for developing pre-eclampsia in the short term.

Thus, preferably as shown in the above methods, the biomarker or detection reagent thereof may be used to diagnose in a pregnant subject whether the subject is at risk of developing preeclampsia in the short term. More preferably, the ratio of sFlt-1 or Endoglin to PlGF is calculated in the first sample and the second sample, and the resulting ratios are then compared to determine a fold increase or change between the two resulting ratios, wherein an increase of up to about 3 fold or more is used as an indication that the subject is at risk of developing preeclampsia in the short term.

In addition, in general, the invention relates to the use of the biomarkers sFlt-1 or Endoglin and PlGF, or detection reagents that specifically bind to said biomarkers, in a first and second sample of a pregnant subject to distinguish between a pregnant subject at risk of developing early onset preeclampsia and a pregnant subject not at risk of developing early onset preeclampsia.

Preferably as shown in the above methods, the biomarkers or detection reagents thereof can be used to distinguish between pregnant subjects at risk of developing early onset preeclampsia and pregnant subjects not at risk of developing early onset preeclampsia. More preferably, the ratio of sFlt-1 or Endoglin to PlGF is calculated in the first sample and the second sample, and the resulting ratios are then compared to determine a fold increase or change between the two resulting ratios, wherein an increase of up to about 3 fold or more is used as an indication that the subject is at risk of developing early onset preeclampsia in the short term.

One aspect of the invention relates to a method for establishing an aid (aid) for optimizing a risk assessment based on clinical prediction rules classifying pregnant subjects, the method comprising:

a) by passing

(i) Contacting the first sample with a detection reagent that specifically binds sFlt-1, Endoglin, and/or PlGF for a time sufficient for the detection reagent to form a complex with the marker in the sample,

(ii) measuring the amount of complex formed, wherein said amount of complex formed is proportional to the amount of marker present in said sample,

(iii) converting the amount of complex formed to an amount of marker that reflects the amount of marker present in the sample, and

(iv) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample,

thereby obtaining a first ratio;

b) by passing

(i) Contacting the second sample with a detection reagent that specifically binds sFlt-1, Endoglin, and/or PlGF for a time sufficient for the detection reagent to form a complex with the marker in the sample,

(iv) calculating a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample,

thereby obtaining a second ratio;

c) comparing said first ratio with said second ratio; and

d) establishing an aid for optimizing the risk assessment based on clinical prediction rules classifying the pregnant subject according to the comparison result obtained in step c).

Yet another aspect of the invention relates to a system for establishing an aid for optimizing risk assessment based on clinical prediction rules classifying pregnant subjects, the system comprising:

b) an analyzer unit configured to detect a signal from the portion of the sample from the subject contacted with the ligand,

c) a computing device having a processor and in operable communication with the analyzer unit, an

Suitable detection reagents may, on the one hand, be antibodies which specifically bind to at least one marker in a sample of a subject to be investigated by the method of the invention, i.e. detection reagents which bind to sFlt-1, Endoglin or PlGF. In one aspect, another applicable detection reagent may be an aptamer (aptamer) that specifically binds to at least one marker in the sample. In another aspect, the sample is separated from the complex formed between the detection reagent and the at least one marker prior to measuring the amount of the complex. Thus, in one aspect, the detection reagent may be immobilized on a solid support. Yet another aspect is that the sample can be separated from the complex formed on the solid support by applying a wash solution. The complex formed is proportional to the amount of at least one marker present in the sample. It will be appreciated that the specificity and/or sensitivity of the detection reagent to be used determines the degree of proportion of the at least one marker contained in the sample that can be specifically bound. There is also a more detailed description of how the determination is carried out in other parts of the application. The amount of complex formed will be converted to the amount of at least one marker, which reflects the amount of marker actually present in the sample. This amount may on the one hand be substantially the amount present in the sample and on the other hand be a certain proportion assumed by the relationship of the complex formed and the amount in the original sample.

In a further aspect of the above method, step a) may be carried out by an analyser unit, in one aspect as defined elsewhere in this application.

Establishing an aid for optimizing the risk assessment based on the comparison in step d) made by assigning the subjects to a group of subjects with increased risk or a group of subjects with decreased risk as described elsewhere in the application. As already discussed above, in the case studied, the classification for the subject to be studied must not be 100% correct. Furthermore, the groups of subjects to which the subjects to be studied are classified are artificial groups, since these groups are established on the basis of statistical considerations, i.e. the method of the invention is operated on the basis of a pre-selected degree of probability. In one aspect of the invention, the assistance means for optimizing the risk assessment is established automatically, for example, by a computing device as disclosed and described herein.

In one aspect of the method of the invention, the method further comprises the step of suggesting or managing the subject, and/or adjusting the concentration of disease monitoring, according to the results established in step d) as set forth in detail elsewhere in this application.

Yet another aspect of the invention comprises a system for establishing an aid for optimizing risk assessment based on clinical prediction rules for classifying subjects with pneumonia, the system comprising:

A preferred embodiment of the present application includes a system for optimizing risk assessment based on clinical prediction rules classifying pregnancy. Examples of such systems include those used to detect the results of or monitor the progress of a chemical or biological reaction, clinical chemistry analyzers, coacervation chemistry analyzers (coagulochemistry analyzers), immunochemical analyzers, urine analyzers, nucleic acid analyzers. More specifically, an exemplary system of the present application includes Roche Elecsys^TMSystems ande Immunolassay Analyzer, Abbott Architect^TMAnd Axsym^TMAnalyzer, Siemens Centaur^TMAnd Immulite^TMAnalyzer, and Beckman Coulter UniCel^TMAnd AcessT^TMAn analyzer, and the like.

Embodiments of the system can include one or more analyzer units for practicing the subject matter of the present application. The analyzer unit of the system disclosed herein may be in operable communication with the computing device disclosed herein via any known wired connection, bluetooth, LANS, or wireless signal. Furthermore, according to the present application, the analyzer unit may comprise a separate device or element for sample detection, e.g. one or both of qualitative and/or quantitative evaluation, in a larger apparatus for diagnostic purposes. For example, the analyzer unit may perform or assist in pipetting, metering (dosing), mixing of samples and/or reagents. The analyzer unit may comprise a reagent holding unit for holding a reagent for performing the assay. The arrangement of reagents may be, for example, in containers or cassettes containing individual reagents or a group of reagents, placed in suitable holders or positions in a storage chamber or conveyor. The detection reagent may also be immobilized on a solid support that is in contact with the sample. The analyzer unit may also include processing and/or detection components optimized for a particular analysis.

According to some embodiments, the analyzer unit may be configured to optically detect an analyte, e.g. a marker, in the sample. Examples of analyzer units for optical detection include devices configured to convert electromagnetic energy into electrical signals, including single and multi-element or array optical detectors. In accordance with the present disclosure, an optical detector can monitor a photo-electromagnetic signal and provide an electrical output signal representative of the presence and/or concentration of an analyte within a sample disposed in an optical path, or a response signal relative to a baseline signal. The device may also include, for example, photodiodes, including avalanche photodiodes (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 tube, may include additional signal conditioning or processing electrical components. For example, the optical detector may include at least one preamplifier, electronic filter, or integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance, and current gain (current mirror) preamplifiers.

Further, one or more analyzer units of the present application may include a light source for emitting light. For example, the light source of the analyzer unit may consist of at least one light emitting element (e.g., a light emitting diode, a power emitting source such as an incandescent lamp, an electroluminescent lamp, a gas discharge lamp, a high intensity discharge lamp, a laser) for measuring the concentration of the analyte in the sample to be measured, or to enable energy conversion (e.g., by fluorescence resonance energy transfer or catalytic enzymes).

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

The analyzer unit of the systems disclosed herein may also include or be operatively connected to a reaction vessel or cuvette (cuvette) delivery unit. Examples of transport units include liquid handling units, such as pipetting units, for delivering samples and/or reagents to reaction vessels. The pipetting unit may comprise a reusable wash-resistant needle, such as a steel needle, or a disposable pipetting head. The analyzer unit may also include one or more mixing units, such as a shaker for shaking the liquid-containing cuvette, or a paddle for mixing the liquid in the cuvette or reagent container.

The present invention also relates to a device suitable for diagnosing whether a pregnant subject is at risk of developing preeclampsia within a short period by carrying out the method described above, comprising:

a) an analyzer unit comprising a detection reagent that specifically binds sFlt-1 and/or Endoglin and a detection reagent that specifically binds PlGF, said unit being adapted to determine the amount of sFlt-1 and/or Endoglin and the amount of PlGF in a first sample and a second sample of a pregnant subject; and

b) an evaluation unit comprising a data processor having an execution algorithm for performing the steps of:

i) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample and a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample; and

ii) comparing the values of said first and second ratios, wherein a subject is diagnosed as being at risk of developing preeclampsia within the short term if the value of said second ratio is increased by at least a factor of 3 if compared to the value of said first ratio.

The term "device" as used in this application relates to a system comprising the above-mentioned units operatively connected to each other, which enables a diagnosis according to the method of the invention. Preferred detection reagents that can be used in the analytical unit are disclosed elsewhere in this application. The analysis unit (or analyzer unit) preferably comprises a detection reagent in immobilized form on a solid support, which is to be contacted with a sample comprising the biomarker whose amount is to be determined. Furthermore, the analysis unit may further comprise a detector for determining the amount of detection reagent specifically binding to the biomarker. The determined amount may be transferred to the evaluation unit. The evaluation unit comprises a data processing element, for example a computer, with an execution algorithm, which carries out the steps of the inventive method, which have been elaborated elsewhere in the application, by executing a computer-based algorithm, thereby carrying out a ratio calculation, comparing the calculated ratios, and evaluating the comparison result. The results can be presented as parameterized diagnostic raw data output. It will be appreciated that such data typically requires interpretation by a physician. But an expert system device is also contemplated wherein the output contains processed diagnostic raw data that does not require interpretation by a specialized physician.

According to some embodiments of the present application, the algorithm for comparing the first ratio and the second ratio disclosed in the present application is implemented and executed by executing instructions. The results can be output as parameterized diagnostic raw data, or given as absolute or relative quantities. According to various embodiments of the system disclosed herein, a "diagnosis" is provided by the computing device of the system disclosed herein based on a comparison of the calculated ratios. For example, a computing device of the system may provide an indication in the form of text, symbols, or numerical values representative of a particular diagnosis.

The present invention also relates to a device suitable for distinguishing between a pregnant subject at risk of developing early onset preeclampsia and a pregnant subject not suffering from early onset preeclampsia by carrying out the method described above, comprising:

a) an analysis unit comprising a detection reagent specifically binding to sFlt-1 and/or Endoglin and a detection reagent specifically binding to PlGF, said unit being suitable for determining the amount of sFlt-1 and/or Endoglin and the amount of PlGF in a first sample and a second sample of a pregnant subject; and

b) an evaluation unit comprising a data processor, said data processor executing an algorithm for performing the steps of:

ii) comparing the values of said first and second ratios, wherein a value of said second ratio is at least 3-fold increased if compared to the value of said first ratio, i.e. the subject is diagnosed as being at risk of developing early onset preeclampsia.

In addition, the invention also includes kits suitable for carrying out the above-described methods for diagnosing whether a pregnant subject is at risk for developing pre-eclampsia in the short term, comprising detection reagents for determining the amount of the biomarker sFlt-1 or Endoglin and the amount of PlGF, and instructions for carrying out the methods.

The term "kit" as used in this application refers to a collection of the above-mentioned components, which are preferably provided separately or in a single container. The container also comprises an operation instruction for implementing the method. These instructions may be in the form of an instruction manual or may be provided by computer program code which, when run on a computer or data processing device, is capable of performing the calculations and comparisons in the method of the invention and establishing 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 comprise a standard amount of a biomarker for calibration purposes, which biomarker is described elsewhere in the application.

The invention also relates to a kit suitable for distinguishing between a pregnant subject at risk of developing early onset preeclampsia and a pregnant subject not suffering from early onset preeclampsia by carrying out the method described above, comprising detection reagents for determining the amount of the biomarker sFlt-1 or Endoglin and the amount of pigf, and instructions for carrying out the method.

All references cited in this specification are incorporated herein by reference in their entirety for all disclosures and for all purposes to which this specification is specifically entitled.

Drawings

FIG. 1: the week number distribution of individual subjects in the patient group and healthy control group that resulted in Preeclampsia (PE) in this study is shown. The first visit, the second visit and the time difference in days (tine difference) are shown in the box plot.

FIG. 2: the sFlt-1/PlGF ratios for the PE group and healthy control groups were shown on a normal and logarithmic scale at different visits.

FIG. 3: different ratios of sFlt-1/PlGF are shown relative to gestational age and measurement time points.

FIG. 4: a graph showing both the time period to diagnosis of PE/HELLP versus the value of the sFlt-1/PlGF ratio (left), and the slope of the sFlt-1/PlGF ratio between the first and second visits for patients in the PE/HELLP group.

FIG. 5: shows the sFlt-1/PlGF ratio (A) at different gestational weeks and the Endoglin (sEng)/PlGF ratio (B) at different gestational weeks.

FIG. 6: sEng/PlGF ratios for PE and healthy controls are shown on a normal and logarithmic scale at different visits.

FIG. 7: showing different sEng/PlGF ratios with respect to gestational age and measurement time points

FIG. 8: a graph showing both the time period to diagnosis of PE/HELLP versus the value of sEng/PlGF ratio (left), and the slope of the sEng/PlGF ratio between the first and second visits for patients in the PE/HELLP group.

Examples

The following examples are intended only to illustrate the invention. They do not limit the scope of the invention in any way.

Example 1: measurement of PlGF, sFLT1 and Endoglin blood levels

Commercially available immunoassays are used to determine blood levels of PlGF, sFLT1 and Endoglin. Specifically, the following assay methods were applied.

Elecsys by Roche^TM-or cobas e^TMSerial analyzers sFlt1 was determined by sandwich immunoassay. The assay includes two monoclonal antibodies specific for the respective polypeptides. The first antibody was biotinylated and the second antibody was labeled with tris (2, 2' -bipyridine) ruthenium (II) -complex. In the first incubation step, both antibodies are incubated with the sample. A sandwich complex is formed containing the peptide to be determined and two different antibodies. In the next incubation step, streptavidin-coated beads are added to the complex. The beads are bound to the sandwich complex. The reaction mixture is then pumped into a measurement cell where the beads are magnetically captured to the electrode surface. Application of a voltage then induces chemiluminescent emission from the ruthenium complex, which is measured by a photomultiplier tube. The quantity of light emitted is dependent onAmount of intercalation compound on the electrode. The sFlt-1 assay is commercially available from Roche diagnostics GmbH, Mannheim, Germany. For more details on the assay, reference is made to the product description. The measurement range for sFlt1 includes amounts between 10 and 85000 pg/ml.

Endoglin measurement was performed using a commercially available from R&Quantikine from D Systems, Inc, Minneapolis, USA^TMHuman Endoglin/CD105 immunoassay. The assay employs a quantitative sandwich enzyme-linked immunoassay technique. Monoclonal antibodies specific for Endoglin have been pre-coated to microplates. Standards and samples are pipetted into the wells, where any Endoglin present binds to the immobilized antibody. After washing away all unbound material, an enzyme-linked monoclonal antibody specific for Endoglin was added to the wells. After washing away all unbound antibody-enzyme reagent, the substrate solution is added to the wells and color development is performed in proportion to the amount of Endoglin bound in the initial step. The development was terminated and the intensity of the color was measured. For more details on the assay, reference is made to the product description. The measured range of Endoglin includes amounts of 0.001ng/L to 10 ng/ml.

In Elecsys^TM-or cobas e^TMSerial analyzer PlGF was tested by sandwich immunoassay (see above for details) using two PlGF-specific antibodies. The PlGF test is commercially available from Roche Diagnostics GmbH, Mannheim, Germany. For more details on the assay, reference is made to the product description. The range of PlGF measurements includes amounts ranging from 3 to 10000 pg/ml.

Example 2: analysis of biomarkers sFlt-1 and PlGF in patients and healthy controls who resultantly suffered from Pre-eclampsia

A total of 286 patients enrolled at different sites in europe were studied. Pregnant women with gestational ages of at least 15+0 weeks up to 30+0 weeks were included in the study. The reference value for the sFlt-1/PlGF ratio generally decreases slightly until about week 28, and thus no physiological increase in said value is expected during this period. The diagnosis at each patient visit included in the study was "no preeclampsia or HELLP (PE/HELLP)" or "suspected PE/HELLP". The diagnosis of these patients may be PE/HELLP and the study analyzed whether elevated values are indicative of a critical diagnosis of PE/HELLP. Two visits were made to each woman: the last visit (visit 2) before week 30+0, and one previous visit (visit 1). If there is more than one selection for visit 1, the selection is closest to being made 3 weeks before visit 2.

Blood levels of PlGF and sFlt-1 were determined and assessed as described in example 1 above. The results are summarized in tables 1-11 below:

table 1: diagnosis of the end result

Table 2: gestational age at first visit

Table 3: gestational age at second visit

Table 4: number of days between visits

Table 5: sFlt-1/PlGF ratio at first visit

Table 6: sFlt-1/PlGF ratio at second visit

Table 7: absolute Change in sFlt-1/PlGF ratio between visits

Table 8: percent gain of sFlt-1/PlGF ratio

Table 9: list of patients with gain of sFlt-1/PlGF ratio of 100% or more

Table 10: final results for the gain grouping of the sFlt-1/PlGF ratio

Table 11: Sens/Spec dependent on gain as a cutoff value

As can be seen from this European PE study, a dramatic increase in the ratio of sFlt-1/PlGF (3-fold or more as proposed in the present application) appears to be a clear indication of critical PE/HELLP.

Example 3: analysis of biomarkers Endoglin and PlGF in patients with consequent preeclampsia and healthy controls

Patient samples similar to those in example 2 were studied and evaluated for PlGF and Endoglin (s-Eng) blood levels. The results are as follows:

table 12: sEng/PlGF ratio at first visit

Table 13: sEng/PlGF ratio at first visit

Table 14: absolute change of sEng/PlGF ratio between accesses

Table 15: percent gain of sEng/PlGF ratio

Table 16: list of patients with sEng/PlGF proportional gain of 100% or more

Table 17: sEng/PlGF proportional gain of packets versus end result

The gain of 100%/200% was taken as the cutoff value, which can be converted to clinical sensitivity/specificity:

table 18: Sens/Spec dependent on gain as a boundary

The different ratios determined for sFlt-1 and PlGF at different time points in pregnancy for healthy controls and patients with pre-eclampsia are also graphically shown in FIG. 5A. The same graph for the Endoglin/PlGF ratio is shown in FIG. 5B. It is clear that the ratios of sFlt-1/PlGF and Endoglin/PlGF exhibit similar distributions and, therefore, are similar predictors of pre-eclampsia.

Claims

1. Use of the biomarkers sFlt-1 or Endoglin and PlGF, or of detection reagents that specifically bind to said biomarkers, in the manufacture of a kit or device for diagnosing whether a pregnant subject is at risk of developing preeclampsia in the short term, said diagnosis comprising:

b) calculating a first ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said first sample and a second ratio from the amount of sFlt-1 or Endoglin and the amount of PlGF determined in said second sample,

c) comparing the values of the first and second ratios, wherein if the value of the second ratio is increased by at least 3-fold compared to the value of the first ratio, the subject is diagnosed as being at risk of developing preeclampsia within the short term,

wherein the short term is a period of less than 4 weeks.

2. The use of claim 1, wherein said first sample is obtained 4 to 5 weeks prior to said second sample.

3. The use of claim 1 or 2, wherein said pregnant subject is between the 15 th and 34 th week of gestation.

4. The use of claim 3, wherein said pregnant subject is between the 15 th and 30 th week of gestation.

5. Use according to any one of claims 1 to 4, wherein said short term is a period of between 2 and 3 weeks.

6. Use according to any one of claims 1 to 5, wherein said preeclampsia is early onset preeclampsia.

7. The use of any one of claims 1 to 6, wherein the diagnosis further comprises: if the subject is diagnosed with an increased risk of developing preeclampsia within the short term, at least one helper measure for preeclampsia is advised on the subject.

8. The use of claim 7, wherein the at least one assisting measure is selected from the group consisting of: close monitoring, hospitalization, administration of hypotensive agents, and/or lifestyle recommendations.

9. Use of the biomarkers sFlt-1 or Endoglin and PlGF, or of detection reagents that specifically bind to said biomarkers, in the preparation of a kit or device for distinguishing between a pregnant subject at risk of developing early onset preeclampsia and a pregnant subject not at risk of developing early onset preeclampsia, said distinguishing comprising:

c) comparing the values of the first and second ratios, wherein the value of the second ratio is increased by at least a factor of 3 if compared to the value of the first ratio, i.e. the subject is diagnosed as being at risk of developing early onset preeclampsia.

10. The use of claim 9, wherein said first sample is obtained 4 to 5 weeks prior to said second sample.

11. The use of claim 9 or 10, wherein the pregnant subject is between the 15 th and 34 th week of gestation.

12. The use of claim 11, wherein said pregnant subject is between the 15 th and 30 th week of gestation.

13. The use of any one of claims 1 to 12, wherein the first and second samples are blood, serum, or plasma samples.