Patent Publication Number: US-2007111326-A1

Title: Diagnostic method for proteinaceous binding pairs, cardiovascular conditions and preeclampsia

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
      In accordance with 35 U.S.C. § 119(e), this application claims the benefit of provisional application U.S. Ser. No. 60/736,659 filed Nov. 14, 2005, the disclosure of which is specifically incorporated by reference herein. 
    
    
     TECHNICAL FIELD OF THE INVENTION  
      The Field of the invention relates to the detection of placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-1), and related molecules in biological samples that are preferably obtained from patients.  
     BACKGROUND OF THE INVENTION  
      Immunodiagnostics enables the detection, diagnosis, prognosis of diseases, dysfunctions, and other conditions afflicting or affecting animals, including humans. It has become highly desirable to perform immunodiagnostics testing with the aid of automated testing equipment that minimizes the investigator&#39;s time handling samples and data. The rapid commercial growth of immunodiagnostics since 1980 has been made possible in part by technology permitting the rapid and efficient isolation of antibodies and/or antibody fragments that bind with sufficient specificity to markers found in biological samples, so that the marker can be recognized. Even more desirable for some immunodiagnostics testing has been the use of monoclonal antibodies, which in many instances allows the skilled artisan to carefully tailor the performance, specificity, and sensitivity of an assay to particular needs. Antibodies also tend to be predictable molecules that are somewhat amenable to improvement by genetic re-engineering. Hence, they have become essential elements of modern immunodiagnostics agents.  
      Other reagents are available for the detection of markers in biological samples, but the need to carefully characterize these agents and develop unique techniques for their use in immunoassays has somewhat discouraged their use in modern immunodiagnostics. This is particularly true when the non-antibody reagent is a polypeptide or protein.  
      VEGF and PlGF belong to a family of regulatory peptides that can control blood vessel formation and vascular permeability. These proteins are believed to interact with Flt-1 and KDR/FLK1 to achieve this function (Mattei et al., Genomics, 32, 168-169, (1996)). There are currently 3 putative isoforms of PlGF identified: PlGF1, PlGF2, and PlGF3. PlGF2 can bind with heparin. PlGF2 is believed to be capable of binding neuropilin-1 in human umbilical vein endothelial cells in a heparin-dependent fashion. Neuropilin-1 is also believed to be able to bind with PlGF1 with lower affinity (Migdal et al., J Biol Chem,    273 ,  22272   -22278 (1998)).  
      PlGF is believed to be capable of stimulating angiogenesis and collateral growth in ischemic heart and limb with good efficiency (Luttun et al., Nature Med 8, 831-840 (2002)). Activation of Flt-1 by PlGF can cause angiogenesis. Both VEGF and PlGF bind to Flt-1, however, PlGF binding with Flt-1 is believed to cause different biological effects than VEGF binding to Flt-1.  
      In pregnant women suffering from preeclampsia, increased soluble Flt-1 (sFlt-1) may cause decreased circulating levels of free VEGF and especially PlGF, resulting in endothelial cell dysfunction that could be relieved by exogenous VEGF and PlGF (Maynard et al., J Clin Invest, 111, 649-658 (2003)). Serum levels of PlGF were significantly lower in women who later had preeclampsia, than in women who did not later develop preeclampsia, in a study reported by Levine et al. (New Eng J Med, 350, 672-683 (2004)). The study suggested that the difference might be perceptible by about 13 to about 16 weeks of gestation, and the greatest difference in PlGF levels was apparent closer to the onset of preeclampsia. Levine et al. also suggested that an increase in the total sFlt-1 level in the blood was also more pronounced in the preeclamptic women. Levine et al. therefore suggested that increased levels of total sFlt-1 and lower levels of PlGF could predict the subsequent development of preeclampsia.  
      sFlt-1 is believed to be an alternately spliced form of Flt-1 resulting in a soluble variant of the Flt-1 protein and can bind both vascular endothelial growth factor (VEGF) with high affinity (Kendall et al., Biophys Res Commun, 226, 324-328 (1996)) and PlGF. Domain deletion studies of the sFlt-1 have shown that (s)Flt-1 domains 2 and 3 permit binding to VEGF with almost the same affinity as sFlt-1 and that domain 2 alone binds only 60-fold less tightly than the full-length sFlt-1.  
     SUMMARY OF THE INVENTION  
      The invention involves the use of a proteinaceous binding partner, other than a portion of an antibody, used to detect the quantity or concentration of a second binding partner, other than a portion of an antibody, in a biological sample. Only one antibody or portion thereof is preferably used in the inventive method. Preferred binding partners of the invention include, but are not limited to, placental growth factor (PlGF) and soluble fms-like tyrosine kinase (sFlt-1), which is a portion of Flt-1 generated by alternative splicing of the Flt-1 gene product and is capable of binding with PlGF.  
      In certain preferred embodiments, the invention also provides a method of determining the quantity of sFlt-1 that is not bound to PlGF (“free sFlt-1”) and a method of determining the quantity of PlGF that is not bound to sFlt-1 (“free PlGF”).  
      Moreover, the invention provides a method of determining the ratio of free sFlt-1 to free PlGF.  
      In another preferred embodiment, the ratio of free sFlt-1 to free PlGF is used to diagnose, predict, monitor, or monitor therapy of preeclampsia.  
      Other proteinaceous binding pairs amenable for detection or quantitation in accordance with the invention include but are not limited to atrial natriuretic peptide (ANP), brain natriuretic peptide (aka, b-type natriuretic peptide) (BNP) with natriuretic peptide receptor a/guanylate cyclase a (NPR1) (also known as atrial natriuretic peptide receptor, type a (ANPRA or NPRA), as atrionatriuretic peptide receptor, Type A and as GUC2A, which is believed to map to gene locus 1q21-q22; and insulin-like growth factor receptor (IGF-1) and its receptor (IGFR1). 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       FIG. 1  illustrates the ability of sFlt-1 to interact with PlGF. Although the binding of two molecules of sFlt-1 with a homodimer of PlGF has been suggested by observations of experimental systems, the inventors recognize that this state may not exist in vivo or may exist at insignificant levels, but is presented in  FIG. 1  because the inventive method is useful for the detection of such complexes, if they exist.  
       FIG. 2  depicts a histogram of PlGF levels observed by an immunoassay employing a monoclonal antibody used to capture free PlGF and a polyclonal antibody used to detect PlGF in a small number of human samples collected and investigated under ethically appropriate conditions.  FIG. 2  demonstrates that low levels of PlGF are associated with preeclamptic pregnancies (PE), and also that the ability to separate non-preeclamptic (Normal) from preeclamptic pregnancies using two antibodies to PLGF is in need of improvement.  
       FIG. 3  depicts a histogram of data collected using a monoclonal antibody as a first sbp for sFlt-1 and a polyclonal antibody as a second sbp for sFlt-1. These data show that there is a significant overlap in the range of total sFlt-1 values for non-preeclamptic pregnant women (Normal) with the range of total sFlt-1 values for preeclamptic women (PE). According to these data, there would be a need to improve the ability to discriminate between normal and preeclamptic pregnancies based on inspection of sFlt-1 levels observed by an immunoassay using two antibodies to sFlt-1 in diagnostic samples obtained from pregnant women.  
       FIG. 4  depicts data obtained in a manner similar to that of the data depicted in  FIG. 3 , except that two monoclonal antibodies to sFlt-1 were used instead of a combination of a polyclonal and a monoclonal antibody. The data presented in  FIGS. 3 and 4  indicate that an immunoassay for sFlt-1 comprising a polyclonal and monoclonal antibody for sFlt-1 outperforms a similar immunoassay comprising two monoclonal antibodies. Even though based on general principles one would expect that this assay would provide better quantitation than the assay of  FIG. 3 , it surprisingly provide less ability to discriminate non-preeclamptic specimens from preeclamptic specimens.  
       FIG. 5  depicts a histogram of data collected using one preferred embodiment of the present invention. These data were collected with an immunoassay comprising a microparticle-labeled monoclonal antibody to sFlt-1 so that total sFlt-1 in the sample would be bound to the microparticle. The bound sFlt-1 was detected by sFlt-1-binding portion of PlGF labeled with acridinium. This assay determines the amount of free sFlt-1 in the specimen. These data show that there is a significant reduction in the overlap of free sFlt-1 values for non-preeclamptic pregnant women (Normal) with the range of values of free sFlt-1 for preeclamptic women (PE). According to these data, use of a portion of PlGF as a sbp for free sFlt-1 significantly improves the ability to discriminate between normal and preeclamptic pregnancies based on inspection of sFlt-1 levels in diagnostic samples obtained from pregnant women.  
       FIG. 6  collects the data described above and presents it in a single graphic representation.  
       FIG. 7  normalizes the data presented in  FIG. 6  to a single non-preeclamptic sample.  
       FIG. 8  compares data collected from the “most normal” preeclamptic woman in the study (“mP-20”) to data collected from the samples of non-preeclamptic women. Measurements of sFlt-1 are of total sFlt-1 for the monoclonal+polyclonal antibody format of this assay, and for the monoclonal+monoclonal format of this assay, whereas for the “Free Recpt” data, an sFlt-1-binding portion of PlGF was used as one sbp in a sandwich immunoassay, and therefore, bound only to free sFlt-1. These data show that, in accordance with aspects of the present invention, the ratio of free sFlt-1 to free PlGF (ranging from about 0 to about 1.0) is a better predictor of lower risk (i.e., normal pregnancies) than the ratio of PlGF to total sFlt-1 in the sample.  
       FIG. 9  depicts in tabular form the increased ability of the present invention to discriminate non-preeclamptic from preeclamptic samples. These data show that for non-preeclamptic specimens determined with a two-antibody based immunoassay the ratio of free sFlt-1 to free PlGF is observed to be higher than when using embodiments of the invention. Accordingly, these data show that the invention provides superior discrimination of non-preeclamptic specimens from preeclamptic samples. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
      In describing the invention, the following terms and abbreviations will be used.  
      Abbreviations used to Describe the Invention  
      The abbreviation “sbp” refers to a “specific binding partner”. All biological materials have some affinity for each other, however, specific binding partners are those that bind together in a specific way to perform a biological function. For example, sFlt-1 binds to PlGF with a biologically significant affinity, and by so doing, is able to modulate the biological activity of PlGF. Accordingly, sFlt-1 and PlGF are specific binding partners. Similarly, the membrane bound counterpart to sFlt-1, i.e., Flt-1, is also a sbp of PlGF. Moreover, VEGF is also a sbp with Flt-1. Another type of sbp that is useful in the context of the invention is an antibody and the molecule comprising the epitope to which it binds. While this type of sbp is essential to the function of some of the embodiments of the present invention, most embodiments of the invention are directed to determining the presence or quantity of one specific binding partner by detecting under assay conditions the binding to the other sbp, wherein neither the first or second sbp is an antibody or portion of an antibody.  
      The term “proteinaceous” is used herein to describe polypeptides, protein fragments, and proteins which are large enough to form at least one helix, sheet, or other significant (polypeptidyl) secondary structure. The term proteinaceous includes both unmodified and modified polypeptides, such as glycosylated, proteolytically cleaved, prenylated, and dimerized polypeptides and proteins. Each proteinaceous binding partner preferably comprises at least one element of secondary structure, such as a helical or sheet structure (e.g., by way of example, an α-helix or β-sheet). Accordingly, each proteinaceous binding partner preferably comprises at least 8 amino acid residues, more preferably at least 50 amino acid residues, and yet more preferably at least 100 amino acid residues. A proteinaceous binding partner can also comprise multiple polypeptide strands folded together so as to form a complex protein.  
      The terms “biological specimen” or “biological sample” are used interchangeably (unless expressly indicated to the contrary), and refer to any material originating from a human or other animal that contains proteinaceous molecules. The inventive method can usefully be performed on any suitable sample, including without limitation sewage, clothing, carpeting, respiratory condensates, and tissue biopsies. Preferred “biological samples” of the present invention include blood, blood plasma, blood serum, urine, feces, lymph, and saliva. When substantially solid biological samples are used, it will most commonly be preferable to extract or solubilize a portion of the sample prior to performing or continuing the inventive method.  
      The term “PlGF” refers to placental growth factor, and is sometimes referred to as PGF. The gene encoding PlGF is currently believed to map to gene locus 14q24-q31. PlGF encompasses all three isoforms currently known in the art and any others that are currently not well characterized to the extent that they bind with the PlGF sbp used in any particular embodiment of the invention.  
      The term “label” or “detectable label” means any suitable molecule allowing the direct or indirect quantitative or relative measurement of the molecule to which it is attached. Suitable labels useful in the context of the invention include solids, enzymes, enzyme substrates, enzyme inhibitors, coenzymes, enzyme precursors, apoenzymes, fluorescent substances, pigments, chemiluminescent compounds, luminescent substances, coloring substances, magnetic substances, metal particles such as gold colloids, radioactive substances, and the like. Useful enzymatic markers include without limitation dehydrogenases, oxidoreductases such as reductases and oxidases; transferases that catalyze the transfer of functional groups, such as amino, carboxyl, methyl, acyl, and phosphate groups; hydrolases that hydrolyze bonds such as ester, glycoside, ether, and peptide bonds; lyases; isomerases; ligases; and the like. Multiple enzymes can also be used in a conjugated form for detection.  
      Useful solid labels include but are not limited to microtiter plates, particles, microparticles and microscope slides.  
      When the detectable marker is an enzyme, detection of the labeled molecule also can be facilitated by enzymatic cycling. For example, when the detectable label is alkaline phosphatase, measurements can be made by observing the fluorescence or luminescence generated from a suitable substrate, such as an umbelliferone derivative. Useful umbelliferone derivatives include without limitation 4-methyl-umbellipheryl phosphate.  
      Other useful labels include phosphorylated phenol derivatives such as nitrophenyl phosphate, luciferin derivatives, dioxetane derivatives.  
      Preferred fluorescent and chemiluminescent labels useful in the context of the invention include fluorescein isothiocyanate; rhodamine derivatives such as rhodamine B isothiocyanate and tetramethyl rhodamine isothiocyanate; dancyl chloride (5-(dimethylamino)-1-naphtalenesulfonyl chloride), dancyl fluoride, fluorescamine (4-phenylspiro[furan-2(3H), 1′-(3′H)-isobenzofuran]-3,3′-dione); phycobiliproteins such as phycocyanine and physoerythrin; acridinium salts; luminol compounds such as lumiferin, luciferase and aequorin; imidazoles; oxalic acid esters; chelate compounds of rare earth elements such as europium (Eu), terbium (Th) and samarium (Sm); and coumarin derivatives such as 7-amino-4-methylcoumarin.  
      Accordingly, it will be appreciated that a wide variety of detectable markers useful in the context of the present invention are available. It will also be appreciated that any suitable detection means can be used to quantify the amount of a molecule attached to a detectable label, such as but not limited to the use of electrodes, spectrophotometric measurement of color, light, or absorbance, and visual inspection.  
     Specific Embodiments of the Invention  
      The present invention provides a method of determining the concentration or amount of a proteinaceous specific binding-pair present in a biological specimen. The binding-pair comprises two proteinaceous moieties (i.e., a first proteinaceous moiety and a second proteinaceous moiety) that preferably bind directly to each other and neither proteinaceous binding partner is an antibody (or fragment of an antibody). Any suitable proteinaceous binding partners, including polypeptides and proteins, can be used in the inventive method. Antibodies and portions of antibodies can be used in the inventive method, but preferably less than two antibodies or portions thereof are used.  
      Alternatively, when more than two antibodies are used, one of the two proteinaceous specific binding partners of the method is labeled with the detectable marker that is observed in the method. For example, when absorbance at a particular wavelength is used, the chromophore is linked to the either the first or second sbp other than through use of an antibody or portion thereof. Similarly, if a scintillation or Geiger counter is used to detect a radioactive label (e.g.,  125 I), the sbp is indirectly, or more preferably directly, attached directly to the second sbp. When the first or second sbp is labeled indirectly through a means other than a first or second antibody, or portion of an antibody, then the directly labeled moiety is preferably either one having biospecific affinity for the first or second sbp (i.e., a third sbp) or is one having stringent affinity for a component of the first or second sbp. One example of an indirect label system having stringent affinity includes biotin and avidin. In this non-limiting example, the second sbp can be labeled with biotin and an avidin-like moiety (e.g., avidin, streptavidin, extravidin) can be directly labeled (with, e.g., an acridinium ester or other chemiluminescent acridinium derivative).  
      In a first embodiment of the inventive method, the first proteinaceous specific binding partner is labeled with a detectable label, which therefore forms a labeled moiety. The labeled moiety is contacted with the biological specimen and the degree of binding between the labeled moiety and the component of the biological specimen that is the second binding partner is determined. The degree of binding indicates either the concentration or amount of the first binding partner or the second binding partner present in the specimen. The determination of the degree of binding can be a relative value, but is preferably quantitative. This degree of binding can be determined by any suitable means, such as by causing the bound pair to agglutinate, bind to a solid support, or migrate at a differential rate through a liquid medium. Preferably, the degree of binding is determined by causing the bound pair to adhere to a solid support, separating the unbound labeled moiety from the bound label moiety and determining either the bound or unbound (or both) fraction of the labeled moiety.  
      The binding pair used in the invention is preferably not a pair of proteins that interact in the mammalian immune system such as the T cell receptor and major histocompatibility complex, CD40 and CD40L, or an antibody and its target.  
      The method optionally can be performed with a cartridge, test strip, or in a unitary package adapted to be used by a semi-automated or fully-automated immunoanalyzer. Automated diagnostic assays in the context of the invention are preferably performed in a system that delivers samples and reagents to a reaction vessel, performs incubations, and optionally washes unbound labeled moiety from the bound labeled moiety, without user intervention, once the sample and reagents are inserted into the system. Such a system optionally can be distinguished from manual or less-automated systems by the ability of the system to perform at least eight assays, preferably at least 16 assays, more preferably at least 64 assays, and most preferably at least 128 assays in a 48-hour period without user intervention after inserting the sample and the reagents into the system. The system is preferably also able to calculate the concentration or quantity of the target protein of the binding pair automatically, i.e., without the need for human calculation or input once the samples are loaded into the system.  
      The method also can be performed in a cartridge format or in a test strip assay. In such an assay, the assay reagents are preferably provided as a unit-dose loadable into disposable instrument and the unit-dose contains all the reagents necessary to assay to perform the method. Such a unit dose instrument for example can comprise a plastic housing comprising a disposable membrane-like structure of nylon, nitrocellulose, or other suitable material. The sample can be preprocessed or loaded directly onto a loading zone. The sample can then optionally flow across the membrane-like structure through a plurality of zones contained on the membrane. The membrane-like structure optionally further contains a detergent or lateral flow-aid and also optionally contains an absorbant to collect excess fluid and/or encourage the lateral flow across the membrane. Additionally, the inventive method can be performed with multi-pack systems in which each pack comprises sufficient reagents to perform 2, 4, 8, 10, or 12 assays, or preferably one assay.  
      The method can also be performed in a microfluidic device designed to analyze samples in the microliter range (e.g., less than 50 μL, preferably less than 12 μL, and optionally less than 4 μL of fluid). Such microfluidic devices can optionally contain flow aids, propulsion devices (including but not limited to expansion gels, waxes, and gases), nanovalving and the like to assist the transportation of the biological fluid or assay reagents or both through the microfluidic device.  
      The method optionally can be configured as a sandwich assay. Sandwich assays comprise binding the labeled moiety to the other specific binding partner of the binding pair, and another specific labeling reagent. Multiple sandwich assays are within the scope of the invention. Mainly for the sake of illustration and ease of comprehension, but not by limitation, the following sandwich assays are illustrated by the use of PlGF as the first binding partner and sFlt-1 as the second binding partner. However, any suitable pair of proteinaceous specific binding partners can be substituted for the PlGF and/or sFlt-1 in the following illustrations.  
      In a first sandwich assay within the scope of the invention an antibody to PlGF (α-PlGF) is bound to a microtiter plate which antibody is previously or subsequently bound to PlGF or an sFlt-1-binding fragment thereof. In the context of the invention, the PlGF is thus labeled with a solid substrate and is a labeled moiety. The microtiter plate can be optionally washed to ensure that substantially no free PlGF is on the microtiter plate. A sample is contacted to the plate, which sample is known to contain, or suspected of containing sFlt-1. Thus, the use of PlGF:sFlt-1 binding is used in the assay. After washing, the quantity of sFlt-1 in the sample can be detected by contacting the plate with a labeled antibody. The antibody can be labeled with any suitable detectable label.  
      In a second sandwich assay within the scope of the invention the microtiter plate of the first sandwich assay is replaced a microparticle. Preferred microparticles include but are not limited magnetic microparticles, particularly those averaging between 0.2 and 7.0 microns in size, haptenated microparticles, microparticles impregnated by one or preferably at least two fluorescent dyes (particularly those that can be identified after individual isolation in a flow cell and excitation by a laser), ferrofluids (i.e., magnetic particles less than about 0.1 μm in size), and other microparticles removable by collectable or removable by filtration.  
      In a third and fourth sandwich assay within the scope of the invention, the PlGF is conjugated directly to the microtiter plate or to the microparticle, respectively.  
      In a fifth and sixth sandwich assay, the PlGF is biotinylated or labeled with a suitable hapten, such as for example, adamantine, fluorescein isothiocyanate, or carbazole. This allows the formation of aggregates when contacted with a multi-valent antibody or (strept)avidin containing moiety, or alternatively allows easy attachment of the PlGF to a solid substrate such as a microtiter plate, microscope slide, or microparticle. In embodiments employing aggregation, any suitable separation or detection means can be used, such as precipitation or filtration of the aggregates or liquid chromatography of the aggregates.  
      Similarly, the inventive method comprises competitive inhibition assays. A competitive inhibition assay can be configured with a single specific binding partner or also as a sandwich assay. Useful competitive inhibition assays include those in which a labeled second specific binding partner (or fragment thereof) (“labeled 2 nd  sbp”) is synthesized or isolated from a source other than the biological sample to be assayed, and labeled with a direct or indirect label. The amount of the 2 nd  sbp in the tested biological sample is then determined by measuring the extent to which the labeled 2 nd  sbp is prevented from binding to the first sbp. By way of illustration, and not limitation, and using the illustrative convention used above, sFlt-1 or a PlGF-binding fragment thereof can be labeled with any suitable detectable label, including without limitation those discussed above. When immobilized PlGF is contacted with a biological sample, the sFlt-1 in the biological sample will compete with the labeled sFlt-1 for binding to the PlGF. The reduction in label binding to the immobilized PlGF then indicates the amount of sFlt-1 in the biological sample which is known to contain, or is suspected of containing, s-Flt-1.  
      The skilled artisan will appreciate, therefore, that the invention provides many embodiments in which the binding interaction of a first polypeptide or protein with a second polypeptide or protein is used to measure the amount or concentration of the second polypeptide or protein. The specific binding partners used to illustrate embodiments of the invention above, i.e., PlGF and sFlt-1, are of course among the preferred specific binding partners that are suitable for use with the invention. Additional preferred embodiments include other angiogenic growth factors and their receptors, such as without limitation, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VPF and the like. Similarly, the biologically significant variants of the growth factors, such as without limitation, VEGF-A 206 , VEGF-A 189 , VEGF-A 165 , VEGF-A 145 , and VEGF-A 121  are preferred specific binding partners of the invention. Similarly, receptors for these molecules, such as KDR, or Flk-1 (fms tyrosine-like kinase-1) (also VEGFR or VEGFR2) are preferred first or second binging partners of the present invention.  
      Other preferred specific binding partners of the invention include natriuretic factors, a natriuretic factor receptor, an insulin-like growth factor (IGF), or an IGF-like receptor. Examples of natriuretic factors include atrial natriuretic peptide (ANP), B-type or brain natriuretic peptide (BNP), and c-type natriuretic peptide (CNP). Any suitable member of the IGF, IGFR, and IGFBP family can be used as the first or second specific binding member or both.  
      It will be readily appreciated that the first specific binding partner or the second specific binding partner can be a chimera or fusion comprising amino acid residues from other polypeptides. Similarly, the specific binding partners can be full-length or truncated. Particularly preferred truncations useful in the context of the invention are those that cleave a transmembrane region from a soluble extracellular domain of the protein, although the method can also be performed using membrane-bound or bindable binding partners. When a specific binding partner is membrane bound the membrane optionally can be part of a cellular structure, synthetic, or removed from a cellular context (e.g., in a vesicle, liposome, or emulsion). This extracellular domain itself can be “full length”, truncated at the N-terminus or C-terminus or both, and can be fused to exogenous polypeptides.  
      The invention also provides a method of determining the concentration of sFlt-1 that does not have a specific binding partner bound to the PlGF binding site of sFlt-1. The method includes contacting a sample that is known or suspected of containing sFlt-1 that does not have a specific binding partner bound to the PlGF binding site of sFlt-1 with a first specific binding partner (sbp) of sFlt-1 capable of forming a sbp:sFlt-1 complex and with a second sbp, wherein the second sbp is specifically labeled with a detectable label or a solid structure. The first or second sbp is PlGF or an sFlt-1-binding fragment of PlGF. The first sbp, the second sbp, and the sFlt-1, if present, then form a ternary complex which is detected as an indication of the amount of sFlt-1 in the sample.  
      In one preferred embodiment the total sFlt-1 in the biological sample is captured with an Flt-1 binding antibody and the portion of sFlt-1 in the sample that is not bound to PlGF or a similar binding partner that binds to the PlGF-binding site of sFlt-1 is detected with a labeled fragment of the epidermal growth factor (EGF) superfamily. Suitable members of the EGF superfamily include, but are not limited to, any suitable portion of PlGF or VEGF. In yet more preferred embodiments that EGF superfamily member is labeled with a luminophore or an enzyme capable of producing a detectable product, such as without limitation, horse radish peroxidase, fluorescein, or acridinium.  
      Another preferred embodiment comprises affixing PlGF on a solid surface, such as without limitation a microparticle. This reagent will capture only free sFlt-1. Without desiring to be bound by any particular theory it is believed that the sFlt-1 in the tested biological sample does not bind to the solid-bound PlGF because the binding site between PlGF and sFlt-1 is already bound in non-free sFlt-1. The complex can then be detected in any suitable manner. Suitable direct and indirect detection reagents include an antibody, antibody-fragment, or aptamer to the sFlt-1.  
      Any of the reagents in the foregoing embodiments can be readily biotinylated through prior art methods. Accordingly, the PlGF can be biotinylated which will facilitate its fixture to a solid phase or another detectable molecule, and the detection reagent can be biotinylated so that it is detectable with a specific binding partner for biotin. Preferred specific binding partners for biotin in the context of the invention include antibodies and aptamers to biotin, avidin and strepavidin.  
      The structure of sFlt-1 is well-known in the art (See, Wiesmann et al., Cell, 91, 695-704 (1997); Davis-Smyth et al., EMBO J, 15, 4919-4927 (1996); Barleon et al., J Biol Chem, 272, 10382-10388 (1997); Cunningham et al., Biochem Biophys Res Commun, 231, 596-599 (1997); Fuh et al. (cited within Wiesmann et al.)). A preferred sFlt-1 specific binding partner is any suitable sFlt-1-binding fragment to PlGF. Preferred sFlt-1-binding fragments of polypeptides comprising at least about 90% of the second and third domains of sFlt-1. Truncated polypeptides of PlGF are also preferred, such as the 21 st  amino acid of PlGF through domain 3 of PlGF. Either or both of the PlGF and sFlt-1-binding fragment of PlGF can be labeled as disclosed elsewhere herein.  
      To facilitate detection of the interaction of the PlGF capture or detection reagent and the sFlt-1 that was free in the tested biological sample, an additional reagent can be added which is labeled by binding to a solid surface or a detectable label. Labeled antibodies are among the preferred additional reagents.  
      The invention also provides an immunoassay based on the competitive inhibition of a labeled sFlt-1 moiety by the quantity of sFlt-1 in the tested biological sample that does not have PlGF bound to the sFlt-1 (“free sFlt-1”). The method comprises contacting a sample that contains free sFlt-1 with a first sbp, in which the first sbp contains an sFlt-1-binding fragment of PlGF and a second sbp, in which the second sbp contains a fragment of sFlt-1 that is capable of binding to the sFlt-1 binding fragment of PlGF where at least the first sbp or the second sbp is labeled. The concentration of sFlt-1 present in the sample is then determined by measuring the decrease in binding between the first sbp and the second sbp caused by the sample.  
      The invention also provides a method of determining the ratio of free sFlt-1 to total sFlt-1 in a sample. The method comprises (i) determining the amount of sFlt-1 according to any of the foregoing embodiments, (ii) determining the total amount of sFlt-1 in the sample, and comparing the result of part (i) to part (ii). Any suitable method can be used to determine the total amount of sFlt-1 in the sample. Suitable methods for carrying out this step include, but are not limited to, sandwich immunoassays and competitive inhibition assays. If at least one antibody used in an immunoassay to determine the total sFlt-1 present in the assay binds to the binding site of PlGF or another factor present or possibly present in the biological sample (e.g., an anti-idiotypic antibody specific for the active site of a first or second sbp), then a portion of the sample optionally can be denatured to disrupt the binding of the sFlt-1 to other proteins in the biological sample. In this instance, any suitable technique can be used to denature the sFlt-1 such that proteins that would block the antibody binding to the active site of sFlt-1 are released. Suitable techniques include adding acid, base, salt, detergents or surfactants, organic bases or a combination of the foregoing and are within the skill of those in the art. To facilitate the binding of an antibody or another sbp used as a diagnostic reagent, the denaturant used to disrupt the binding of sFlt-1 to the sbp in the sample is preferably readily neutralized or removed from the sample. Preferably, however, the one or more antibodies used in an immunoassay to determine total sFlt-1 does not bind to the PlGF binding site of sFlt-1. The skilled artisan will appreciate that still other methods of measuring total sFlt-1 in the sample are readily available and within the scope of the present invention. Accordingly, the invention enables both the direct and indirect determination of each of (i) free sFlt-1, (ii) bound sFlt-1, and (iii) total sFlt-1. Any of these sFlt-1 values optionally can be further compared to the concentration of an EGF superfamily member, including without limitation VEGF, and preferably PlGF.  
      In a particularly preferred embodiment, an anti-sFlt immunoreagent is attached to a magnetic microparticle, and the biological specimen is contacted to the anti-sFlt-1 bound microparticle such that the sFlt-1 in the sample is bound to the magnetic microparticle. The complex can then be optionally washed in a suitable solution or buffer one or more times to remove unbound molecules that could interfere with the assay. Then labeled PlGF is contacted to microparticle containing complex and unbound labeled PlGF is removed or washed away from the magnetic microparticle. The amount of labeled PlGF bound to the magnetic microparticle then serves as an indication of the quantity of free sFlt-1 in the biological specimen because sFlt-1 bound by a sbp (which spb binds to the PlGF binding-site of sFlt-1) cannot efficiently bind the labeled PlGF.  
      In another embodiment of the claimed invention, the total sFlt-1 and the portion of the sFlt-1 bound to PlGF is measured. Any suitable method can be used to determine the quantity of total and PlGF-bound sFlt-1 (“bound sFlt-1”) present in the sample. One suitable method to determine the quantity of bound sFlt-1 in the sample is to detect the formation of a complex having at least three components including an anti-PlGF antibody, the bound sFlt-1 (which itself comprises at least sFlt-1 and PlGF), and an anti-sFlt-1 antibody. That is, to employ a two-antibody sandwich assay in which one antibody is specific for PlGF and at least one antibody is specific for sFlt-1. In accordance with other preferred embodiments of the invention, the antibodies are each preferably labeled with detectable labels. In an even more preferred embodiment of this embodiment, one antibody is labeled by attachment to a solid substrate and at least one antibody is labeled by conjugation to another label referred to herein.  
      In another embodiment, the detection of free sFlt-1 is performed with an antibody that binds to an epitope that is not accessible (i.e., hidden) when PlGF is bound to the sFlt-1. In this way respect, the assay of the invention is any traditional sandwich, competitive inhibition, or other conventional immunoassay (for sFlt-1), except that it only measures free sFlt-1. This allows comparison of the quantity of free sFlt-1 to the quantity of total PlGF, or more preferably, to the quantity of free PlGF. In further aspects of this embodiment, an antibody to the sFlt-1 binding site of PlGF can be substituted for the portion of the sFlt-1 used in other embodiments of this invention.  
      The measurements of PlGF, and sFlt-1, including without limitation the measurements bound and free states of these molecules can be used for any suitable purpose. For example, the measurement of these markers can be used to predict the course of angina following a major cardiovascular event such as a non-lethal myocardial infarction. Similarly, the ability to measure these markers can be used to better understand the mode of action of heart medicines. Moreover, the accurate measurement of these markers permits more detailed investigations into the mechanisms of restenosis and neovascularization. The measurement of PlGF and sFlt-1 could find the greatest significance in demonstrating a lower risk of preeclampsia in pregnant women.  
      Preeclampsia affects about 5% of all pregnant women, and in some ethnic groups affects as many as about 10% of all pregnant women. The effects of preeclampsia can be severe and sometimes include death. Accordingly, there is a need to better separate normal pregnancies from pregnancies at high risk for preeclampsia.  
      The present inventors have discovered that the ratio of free sFlt-1 to free PlGF is a better predictor of risk of preeclampsia than is the ratio of total sFlt-1 to free PlGF. Because the quantity of free sFlt-1 is mathematically related to the quantity of total sFlt-1 and bound sFlt-1, these values can be used as a surrogate for the quantity of free sFlt-1, and can be compared to the quantity of PlGF in a biological sample within the scope of the present invention.  
      Many proteins of interest for medical diagnostics are present in low concentrations, e.g., at from less than 1 pg/mL to 0.1 mg/mL. Some of these proteins will bind to a protein receptor with affinities similar to that observed for antibody-antigen interactions. In an analogous fashion to enzymatic activity, it is possible to measure the amount of a free protein or the amount that is bound its native receptor.  
      Preeclampsia is a disease of late pregnancy that is currently diagnosed based on clinical symptoms of high blood pressure and protein in the urine. Recent literature has proposed that the precipitating event of the disease is a decrease in circulating levels of the angiogenic proteins Vascular Endothelial Growth Factor (VEGF) and Placental Growth Factor (PlGF). The resulting lack of vascularization in the placenta is then suggested to be responsible for the increase in blood pressure and proteinuria, clinically known as preeclampsia. The decrease in these two proteins is apparently due to the increased concentration of the soluble form of the receptor soluble fms-like tyrosine kinase 1(sFlt-1). The present invention covers an approach to measuring sFlt-1, which is free or bound to PlGF and its use as an assay component for measuring free and bound forms of PlGF. For detection of preeclampsia, the most relevant information is the levels of PlGF in relationship to that of the sFlt-1 which is not bound to PlGF. High concentrations of free sFlt-1 indicate that the PlGF concentrations are likely to be low due to the presence of a large excess of free receptor.  
      In the preferred embodiment, an antibody is used to bind all the circulating sFlt-1 (either bound or unbound to PlGF). A conjugate of a signal generating moiety and PlGF is then allowed to interact with the sFlt-1 bound to the solid phase. In this example, only the sFlt-1 free of PlGF would bind the conjugated PlGF. The unbound PlGF is then washed away and the necessary steps are taken to reveal the concentration of PlGF-conjugate.  
      The above format could also be constructed using VEGF, in the same manner as the PlGF as conjugate. Furthermore it would be possible to also use the heterodimer of VEGF and PlGF.  
      Another form of the assay would be to use PlGF bound to a solid phase and then capture any free PlGF which can then be detected with an conjugated antibody that binds sFlt-1. The same format can use VEGF on the solid phase.  
      Another form of the assay would measure free PlGF or VEGF by attaching the sFlt-1 to a solid phase and then capturing any free growth factor that is not bound to a soluble receptor. Detection again can be performed with a conjugated antibody that binds to the growth factor. This form of the assay would be specific for the biologically active form of the growth factors. In this format any degraded growth factor would not be detected improving the specificity of the assay to the biological event that causes preeclampsia.  
      When the relevant biological question is the amount of PlGF bound to receptor, it is also possible to use a solid phase that would capture sFlt-1 as in the first example and then use a conjugated antibody against PlGF or VEGF to measure the amount of bound growth factor. The utility of the approach would depend upon the successful correlation of disease state with the species measured.  
      Another form of the assay is to use immobilized receptor in a competitive format where the free ligand in the sample competes with labeled ligand. For example sFlt-1 on a solid phase be used to capture either the PlGF in the sample in a competitive format with labeled PlGF. This form of the assay would eliminate the requirement of an antibody in the assay.  
      The converse of the above example would be to immobilize with PlGF or VEGF and add sample and conjugated sFlt-1. In this format, free sFlt-1 would compete for sites on the solid phase.  
      The sources of the protein used in the assay could be derived from patient samples however the use of recombinant proteins expressed in either cell culture or in bacteria would be more practical approach.  
      The approach described here could be used to interrogate samples with regards to either receptor or associated ligand activity so long as the affinity between the ligand and the receptor is sufficiently high to permit the use of wash steps without such loss of the bound material to such an extent that it could not be detected in the signal generation of the assay protocol.  
      Assays that depend upon the inherent biological binding activity of the targeted proteins may provide superior information to assess the clinical situation of a patient. When a disease or medical condition involves a protein receptor, assays that measure the relative amount of that biological activity can be expected to lead to a more accurate clinical picture as compared to only knowing the mass of the protein.  
      In accordance with the foregoing methods, the present invention also provides an immunoassay comprising two proteinaceous specific binding partners, wherein at least one sbp is detectably labeled.  
      Additionally, in accordance with the foregoing the invention provides a composition of matter for determining the ratio of free sFlt-1 to free PlGF, as well as compositions of matter for determining the total (i) sFlt-1 and bound sFlt-1 or (ii) the total PlGF and bound PlGF, or both (i) and (ii).  
     EXAMPLES  
      The invention is illustrated with data obtained from various immunoassays for total PlGF, free PlGF, total sFlt-1, and free sFlt-1. A selection of these data deemed to be most illustrative of the invention and inventive concepts are presented in the attached drawings and discussed briefly in the Brief Description of the Drawings. As is clear from the entirety of this description of the invention, the examples are meant to illustrate the claimed invention rather than to limit its scope.  
     Further Examples  
      The following additional examples provide more detail regarding two preferred embodiments of the present invention  
     Example 2  
      This example illustrates the inventive method in an assay used to detect free sFlt-1 in a biological sample using a portion of PlGF as a sbp for sFlt-1.  
      A monoclonal antibody against sFlt-1 was coated on magnetic carboxyl-latex microparticles (4.7 microns) at a protein concentration of 0.1 mg/nL of microparticles at a concentration of 1% by weight in 50 mM sodium MES (2-morpholinoethanesulfonic acid) at pH 6.0. After 10 minutes, EDAC, (ethyl-3-(-3-dimethylaminopropyl)carbodiimide) was added and allowed to react for one hour before washing the particles with phosphate buffered saline. The particles were then diluted to 0.1% in a buffer for use in an automated immunochemical analyzer.  
      Acridinylated PlGF was prepared by dissolving PlGF in phosphate buffered saline and incubation with an acridinium-ester at a mass ratio of PlGF to acridinium of 150,00/1. The conjugate was then purified by HPLC chromatography and diluted to a concentration of approximately 75 ng/mL.  
      The following series of steps are then performed. A 0.05 mL aliquot of sample is added to a reaction vessel to which 0.05 mL of the 0.1% labeled microparticles is added. The reaction mixture is incubated for 18 minutes at 37 degrees centigrade. A magnet holds the particles while the reaction solution is removed. After the particles are washed, a 0.05 mL aliquot of conjugate solution is added. After incubation for 4 minutes, the particles are once again held to a magnet and the pellet of microparticles the conjugate solution is removed followed by washing of the particles once again. The remaining acridinium label is caused to emit light after the addition of sodium hydroxide and hydrogen peroxide. The photons released are measured and is linearly related to the calibrators run in the identical way.  
      Data were collected on test samples and compared to the results obtained with conventional immunoassays. The data suggested that the inventive method better discriminated non-preeclamptic samples from preeclamptic samples, particularly when observing the ratio of free sFlt-1 to free PLGF concentrations.  
     Example 3  
      This example illustrates the inventive method in an assay used to detect free PlGF in a biological sample using a portion of sFlt-1 as a sbp for PlGF.  
      Paramagnetic latex microparticles (4.7 microns), derivatized with carboxyl functional groups, was coated with anti-sFlt-1 antibody (containing domains 1-3 of the fms-like tyrosine kinase 1) at a protein concentration demonstrated to be sufficient to maximize the amount of protein absorbed to the surface area of the microparticles (2% solids by weight) in 50 mM MES, pH 5.5. In another embodiment, the sFlt-1 could be bound directly to a solid substrate. After 10 minutes, the non-absorbed sFlt-1 was removed by washing the particles multiple times with MES buffer. Following washing the particles, EDAC was added and allowed to react forming a covalent coupling of the sFlt-1 molecules to the particles. The particles were then washed with phosphate buffered saline to stop the reaction and remove unreacted EDAC. The particles are then diluted to 0.1% in buffer for use in an automated immunochemical analyzer.  
      Acridinium-labeled anti-PlGF antibody was prepared by incubating an polyclonal antibody (alternatively a monoclonal antibody could be used) with an acridinium-ester at a molar ratio of acridinium to antibody ranging from 1 to 100. Unconjugated acridinium was then separated from the acridinium-labeled antibody conjugate by size chromatography. The purified conjugate was then diluted in buffer to a concentration yielding the maximum signal to noise ratio in the assay.  
      The following series of steps were then performed. A 0.1 mL aliquot of sample was added to a reaction vessel to which 0.05 mL of the 0.1% labeled microparticles was added. The reaction mixture was incubated for 18 minutes at 37 degrees centigrade. Utilizing the paramagnetic property of the particles, a magnet attracts and holds the particles against the side of the reaction vessel while the reaction solution is removed. After the particles are washed, buffer is dispensed; a 0.05 mL aliquot of conjugate solution is added; the magnet removed and the mixture vortexed. After a 4-minute incubation, excess conjugate was removed by particle attraction to a magnet, washing and resuspension. The particles, now containing the sFlt-1/PlGF/anti-PlGF antibody (acridinium-labeled) sandwich, was then exposed to reactants causing the acridinium to emit light. The chemiluminescence, measured by the instrument, is directly proportional to the amount of PlGF (free) in the sample.  
      Data were collected on test samples and compared to the results obtained with conventional immunoassays. The data suggested that the inventive method better distinguished the preeclamptic state from the non-preeclamptic state by measuring the biological activity of the prophylactic or causative biological agent rather than using antibodies against the agent that would not necessarily distinguish between active and inactive forms of the protein.  
      All patents, patent applications, and publications mentioned herein are hereby incorporated by reference.  
      The present invention is amenable to many variations and includes modifications that can be derived from the description herein by a person skilled in the art. All such variations and modifications are considered to be within the scope and spirit of the present invention as defined by the following claims.