CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/555,349, filed March 22, 2004 and U.S. Provisional Application No. 60/637,944, filed December 21, 2004, the teachings of both of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Preeclampsia (gestational proteinuric hypertension) is a multisystem maternal disorder that complicates 6-8% of all pregnancies. It is the second most common cause of maternal mortality in the United States, accounting for 12-18% of all pregnancy-related maternal deaths (around 70 maternal deaths per year in the United States and an estimated 50,000 maternal deaths per year worldwide). It is also associated with a high perinatal mortality and morbidity, due primarily to iatrogenic prematurity. The only definitive treatment for preeclampsia is delivery of the fetus and placenta. Neurologic manifestations are common in severe preeclampsia, and include seizures or coma (eclampsia), stroke, hypertensive encephalopathy, headaches, and visual aberrations. Despite intensive research efforts, the etiology of preeclampsia and its neurologic manifestations remains unknown.

The symptoms of preeclampsia typically appear after the 20th week of pregnancy and are usually detected by routine monitoring of the woman's blood pressure and urine. However, these monitoring methods are ineffective for diagnosis of the syndrome at an early stage, which could reduce the risk to the subject or developing fetus, if an effective treatment were available. Currently, there are no known cures for preeclampsia. Preeclampsia can vary in severity from mild to life threatening. A mild form of preeclampsia can be treated with bed rest and frequent monitoring. For moderate to severe cases, hospitalization is recommended and blood pressure medication or anticonvulsant medications to prevent seizures are prescribed. If the condition becomes life-threatening to the mother or the baby, the pregnancy is terminated and the baby is delivered pre-term. Clearly, there is a need for novel approaches for diagnosing and treating preeclampsia, which is a significant public health problem.

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery that proteomic analysis of cerebrospinal fluid (CSF) can accurately distinguish severe preeclampsia from both mild preeclampsia and normotensive controls. These data demonstrate that proteomic technology can be used to identify important protein biomarkers for the diagnosis of preeclampsia and candidate targets for treatment. In particular, Applicants found that women with severe preeclampsia have nanomolar amounts of free hemoglobin in their CSF and that increased levels of oxidized protein metabolites (as indicated by protein carbonylation) may have both pathophysiologic and therapeutic implications.

In certain embodiments, the present invention provides a method of identifying at least one marker for preeclampsia in CSF. This method comprises: (a) obtaining a CSF sample from at least one pregnant woman who suffers from preeclampsia; (b) separating proteins from the CSF sample; (c) obtaining a proteomic profile of the CSF sample obtained in (a); and (d) identifying at least one protein that has a different level in the CSF sample obtained in (a) as compared to a control CSF sample, wherein the identified protein is a CSF marker for preeclampsia. In certain cases, the identified protein is present at a higher level in the preeclampsia CSF sample than in the control CSF sample. In other cases, the identified protein is present at a lower level in the preeclampsia CSF sample than in the control CSF sample. In one specific embodiment, the proteomic profile in this method is represented in the form of mass spectra. The identified protein can be used as a CSF marker for the status of preeclampsia (e.g., severe preeclampsia). An example of the identified CSF markers for preeclampsia is free hemoglobin, including hemoglobin a chain, hemoglobin β chain, and glycated isoforms of hemoglobin a chain or hemoglobin β chain.

In certain embodiments, the present invention provides a method of diagnosing or aiding in the diagnosis of severe preeclampsia in a pregnant woman. Such method comprises: (a) measuring the level of free hemoglobin in a cerebrospinal fluid (CSF) sample obtained from pregnant woman; and (b) comparing the level of free hemoglobin in the CSF sample with a reference value, wherein a higher level of free hemoglobin in the CSF sample relative to the reference value indicates that the woman has severe preeclampsia or is at increased risk of developing severe preeclampsia. The free hemoglobin level in CSF can be measured using, for example, an immunological assay (e.g., an ELISA), a protein chip assay, or surface-enhanced laser desorption/ionization (SELDI). As used herein, the term "hemoglobin" includes hemoglobin a chain, hemoglobin β chain, and modified forms of hemoglobin such as glycated isoforms of hemoglobin α chain and glycated isoforms of hemoglobin β chain.

In certain embodiments, the present invention provides a method for monitoring the progression or regression of preeclampsia in a pregnant woman. Such method comprises: (a) measuring the level of free hemoglobin in a first cerebrospinal fluid (CSF) sample obtained from the woman; and (b) measuring the level of free hemoglobin in a second CSF sample obtained from the same pregnant woman later in her pregnancy, wherein a higher level of free hemoglobin in the second CSF sample relative to the free hemoglobin level in the first CSF sample indicates preeclampsia progression and a lower level of free hemoglobin level in the second CSF sample relative to the free hemoglobin level in the first CSF sample indicates preeclampsia regression. The free hemoglobin level in CSF can be measured using an immunological assay (e.g., an ELISA), a protein chip assay, or surface-enhanced laser desorption/ionization (SELDI). This method includes measurement of hemoglobin α chain, hemoglobin β chain, and/or modified forms of hemoglobin such as glycated isoforms of hemoglobin α chain and glycated isoforms of hemoglobin β chain.

In certain embodiments, the present invention provides a method of assessing the efficacy of a treatment for preeclampsia in a pregnant woman. Such method comprises: (a) measuring the level of free hemoglobin in a first CSF sample obtained from the pregnant woman before treatment; (b) measuring the level of free hemoglobin in a second CSF sample from the same pregnant woman after treatment; and (c) comparing the level determined in (a) with the level determined in (b), wherein a decrease in the free hemoglobin level in the second CSF sample relative to the free hemoglobin level in the first CSF sample indicates that the treatment is efficacious for treating preeclampsia.

In certain embodiments, the present invention provides a kit that comprises: (a) a capture reagent that binds to hemoglobin; and (b) instructions to detect hemoglobin by contacting a CSF sample with the capture reagent and detecting the hemoglobin retained by the capture reagent. An example of the capture reagent includes an antibody that specifically binds to hemoglobin (e.g., hemoglobin a chain, hemoglobin β chain, and modified forms of hemoglobin). Optionally, the capture reagent is immobilized onto a solid matrix. In certain cases, the kit further comprises a wash solution that selectively allows retention of the bound hemoglobin to the capture reagent as compared with other proteins after washing. Optionally, the kit further comprises a second capture reagent that binds to a second CSF marker for preeclampsia.

In certain embodiments, the present invention provides a method of identifying a compound that ameliorates or treats preeclampsia. Such method comprises: (a) administering a candidate compound to a female; (b) comparing the level of free hemoglobin in a test CSF sample obtained from the subject with the level of free hemoglobin in a control CSF sample, wherein if the free hemoglobin level is lower in the test CSF sample than in the control CSF sample, the candidate compound is a compound that ameliorates or treats preeclampsia. The free hemoglobin level in CSF can be measured using an immunological assay (e.g., an ELISA), a protein chip assay, or surface-enhanced laser desorption/ionization (SELDI). This method includes measurement ofhemoglobin a chain, hemoglobin β chain, and modified forms of hemoglobin such as glycated isoforms of hemoglobin a chain and glycated isoforms of hemoglobin β chain. In certain specific embodiments, the subject is a female human such as a pregnant woman. Examples of the candidate compound include a polypeptide, a nucleic acid, an antibody, a small molecule, and a peptidomimetics.

In certain embodiments, the present invention provides a method of diagnosing or aiding in the diagnosis of preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a cerebrospinal fluid (CSF) sample obtained from the pregnant woman; and (b) comparing the level of the marker associated with preeclampsia in the CSF sample with a reference value, wherein a higher level of the marker associated with preeclampsia in the CSF sample relative to the reference value indicates that the pregnant woman has preeclampsia or is at increased risk of developing preeclampsia, the preeclampsia is severe preeclampsia.

In certain embodiments, the present invention provides a method of diagnosing or aiding in the diagnosis of preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a cerebrospinal fluid (CSF) sample obtained from the pregnant woman; and (b) comparing the level of the marker associated with preeclampsia in the CSF sample with a reference value, wherein a lower level of the marker associated with preeclampsia in the CSF sample relative to the reference value indicates that the pregnant woman has preeclampsia or is at increased risk of developing preeclampsia.

In certain embodiments, the present invention provides a method for monitoring the progression or regression of preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a first cerebrospinal fluid (CSF) sample isolated from the pregnant woman; and (b) measuring the level of the marker in a second CSF sample from the same pregnant woman at a later time, wherein an increase in the level of the marker associated with preeclampsia level in the second CSF sample relative to the level of the marker in the first CSF sample indicates preeclampsia progression and a decrease in the level of the marker in the second CSF sample relative to the level of the marker in the first CSF sample indicates preeclampsia regression.

In certain embodiments, the present invention provides a method for monitoring the progression or regression of preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a first CSF sample obtained from the pregnant woman; and (b) measuring the level of the marker in a second CSF sample from the same pregnant woman at a later time, wherein an decrease in the level of the marker associated with preeclampsia level in the second CSF sample relative to the level of the marker in the first CSF sample indicates preeclampsia progression and a increase in the level of the marker in the second CSF sample relative to the level of the marker in the first CSF sample indicates preeclampsia regression.

In certain embodiments, the present invention provides a method of assessing the efficacy of a treatment for preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a first CSF sample obtained from the pregnant woman before treatment; (b) measuring the level of the marker associated with preeclampsia in a second CSF sample from the same pregnant woman after treatment; and (c) comparing the level determined in (a) with the level determined in (b), wherein a decrease in the level of the marker in the second CSF sample relative to the level of the marker in the first CSF sample indicates that the treatment is efficacious for treating preeclampsia.

In certain embodiments, the present invention provides a method of assessing the efficacy of a treatment for preeclampsia (e.g., severe preeclampsia) in a pregnant woman, comprising: (a) measuring the level of a marker associated with preeclampsia in a first CSF sample obtained from the pregnant woman before treatment; (b) measuring the level of the marker associated with preeclampsia in a second CSF sample from the same pregnant woman after treatment; and (c) comparing the level determined in (a) with the level determined in (b), wherein a increase in the level of the marker in the second CSF sample relative to the level of the marker in the first CSF sample indicates that the treatment is efficacious for treating preeclampsia.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows optimization of Surface-Enhanced Laser Desorption and Ionization (SELDI) analysis of CSF. 5-μL aliquots of undiluted CSF pooled from patients with severe preeclampsia (n=7) and normal controls (n=7) were air dried onto WCX2 protein chip arrays (Ciphergen), overlaid with 50 percent SPA, and read in the SELDI reader (Ciphergen). Matrix and buffer alone was used as a negative control. Representative proteomic profiles are shown. Systematic manual analysis of SELDI tracings identified a region at 14 to 17 kDa on the proteomic array with several visually conspicuous-peaks that appeared to differentiate between the 'diseased' (severe preeclampsia) and 'normal' sample pool and were not present in the negative control samples (highlighted).

Figure 2 shows use of SELDI to identify discriminatory protein peaks in the CSF of women with severe preeclampsia. Individual samples of CSF from women with severe preeclampsia, mild preeclampsia, and normotensive controls were analyzed in a blinded fashion by SELDI using the dry on-chip protocol as described. Representative proteomic profiles in the 14 to 17 kDa region of the proteomic spectrum are shown. As evidenced by the profiles, five distinct protein peaks (designated #1 through #5) were noted. Peak #1 is present in all CSF samples but not in the negative control (binding buffer), and was therefore used as a reference peak. Peaks #2 though #5, on the other hand, were present only in the CSF of patients with severe preeclampsia and not in the CSF of patients with mild preeclampsia or normotensive controls. These are the four discriminatory peaks. The peaks and their estimated mass (kDa; 95 percent CI) are shown.

Figure 3 shows utility of preeclamptic proteomic biomarker (PPB) score to discriminate severe preeclampsia from mild preeclampsia and normal controls. Having identified four discriminatory protein peaks and developed the PPB score, Applicants were able to measure the utility of this scoring system to discriminate patients with severe preeclampsia from all other study patients (mild preeclampsia and normotensive controls). (A) The distribution of patients with and without severe preeclampsia and their respective PPB score is shown. The single outlier (designated by an arrow) was a patient with severe preeclampsia by pulmonary edema only. Review of the medical record suggested that, according to the obstetric care provider, the pulmonary edema likely resulted from iatrogenic fluid overload. (B) A receiver operating curve (ROC) analysis of the PPB score is shown, which demonstrates that the PPB score is able to separate these groups using a cut-off of >1 with 85.7 percent sensitivity and 100 percent specificity (area under the ROC curve = 0.920; 95% CI = 0.728-0.989).

Figure 4 shows in-gel tryptic digests. To identify the discriminatory protein biomarkers in CSF that make up the PPB score, one-dimensional SDS-PAGE electrophoresis followed by in-gel tryptic digestion of excised bands was performed as described in the Materials and Methods section above. (A) A representative SDS- PAGE gel is shown. The negative control included water only. The positive control lane contained mass spectrometry molecular weight standards (equine myoglobin and cytochrome C [Ciphergen Biosystems]). Protein bands of interest were designated A through F. Band E appears to be specific for severe preeclampsia. (B) When comparing the SELDI analysis (spectral and pseudo-gel views) with SDS-PAGE gels, band E corresponds with the location of the discriminatory proteomic peaks of interest (highlighted). (C) A representative SELDI analysis of the in-gel tryptic digests for the positive control lane and bands E and F identified in Figure 4A is shown. The small protein peaks in the positive control (water) lane represent autolytic products of trypsin. (D) The identity of each protein band was confirmed by comparing the corresponding peptide map with a database of tryptic digest peptide maps of known proteins as reported by Profound (http://prowl.rockefeller.edu). The accession numbers indicated are those designated by SwissProt (http://www.expasy.ch).

Figure 5 shows on-chip antibody capture assay. To confirm the identity of the discriminatory PPB biomarkers, on-chip antibody capture assays were performed as described in the Materials and Methods section above. The SELDI spectra from a patient with severe preeclampsia and a control patient using anti-hemoglobin antibody (middle lane) and mouse IgG as negative control (lower lane) are shown. The SELDI spectrum of CSF from patients with severe preeclampsia obtained with the dry on- chip protocol is also shown as a reference (upper lane). A series of peaks are present in the SELDI spectrum obtained using anti-hemoglobin antibody (but not negative control) that effectively recapitulates the PPB score as seen with the WCX2 dry on- chip protocol. From these data, Applicants conclude that peaks #2 and #4 from Figure 2 represent the a- (15.126 kDa [SwissProt]) and /3-chains (15.867 kDa) of hemoglobin, respectively. It is likely that peaks #3 and #5 from Figure 2 represent post-translational (glycolated) isoforms of modifications of the a- and /3-chains of hemoglobin, respectively.

Figure 6 shows hemoglobin and cystatin C ELISA. ELISA for hemoglobin (A) and for cystatin C (B), a reference biomarker also identified in CSF, were performed as described in the Materials and Methods section above. Hemoglobin levels in the CSF of women with severe preeclampsia were significantly higher than that in mild preeclampsia or normal controls. The patient without hemoglobin in her CSF (arrow in Figure 6A) was patient #2 (Table 1) who had severe preeclampsia by pulmonary edema only that was attributed in large part to iatrogenic fluid overload. Levels of cystatin C in the CSF were not significantly different between the three groups. Individual results and mean are shown. * p<0.001.

Figure 7 shows direct spectrophotometry. To investigate further the presence of free hemoglobin in CSF, direct spectrophotometry of individual CSF samples was performed as described in the Materials and Methods section above. (A) Absorbance spectra of CSF from a representative control patient and two patients with severe preeclampsia are shown. The presence of hemoglobin is evident as a peak optical density at 414 nm. Oxyhemoglobin shows an absorbance peak at 414 nm, while bilirubin displays a shoulder at 455 nm. (B) A sample of CSF from a representative control patient (PPB=O) demonstrates high absorbance at these wavelengths due to an unidentified interfering substance(s).

Figure 8 shows estimation of protein carbonylation. To investigate the functional importance of nanomolar amounts of free hemoglobin in the CSF of women with severe preeclampsia, protein carbonylation in individual CSF samples was estimated as a measure of oxidative modification as described in the Materials and Methods section above. (A) A representative western blot with five individual CSF samples (PPB score 0 or 4) stained with rabbit anti-dinitrophenyl (DNP) antibody to identify proteins with incorporated DNP residues (arrow) is shown. Images are compared with quantitative standards in which known amounts of DNP residues are incorporated in a standard mixture of proteins on a dot blot, and the level of protein oxidation is reported in fhiol of DNP per μg protein. (B) Individual results and mean for all 22 patients are shown along with their PPB score. * p=0.0173.

DETAILED DESCRIPTION OF THE INVENTION

In certain aspects, the present invention provides markers for preeclampsia and methods that are useful for determining preeclampsia status of a pregnant woman by measuring one or more of these markers. The measurement of these markers in patient samples, such as cerebrospinal fluid (CSF) samples, provides information useful to diagnose or aid in the diagnosis of preeclampsia (e.g., severe preeclampsia). Specifically, biomarkers of the present invention were identified by comparing mass spectra of CSF samples obtained from two groups of pregnant subjects: subjects with preeclampsia and normal subjects. The subjects were diagnosed according to standard clinical criteria.

One or more markers shown to be associated with preeclampsia can be used in the present method. In certain embodiments, a marker or markers (e.g. proteins) in CSF that are present at higher levels in preeclampsia than in pregnant woman without preeclampsia are assessed. An increased level of such a marker(s) in CSF indicates the pregnant woman has preeclampsia or is at risk of developing preeclampsia. In other embodiments, a marker or markers in CSF that are present at lower levels in preeclampsia than in pregnant women without preeclampsia are addressed. A lower level of such a marker(s) in CSF indicates the pregnant woman has preeclampsia or is at risk of developing preeclampsia.

In one embodiment, the marker is free hemoglobin in CSF. Thus, in certain aspects, the present invention relates to methods of measuring free hemoglobin in CSF for determining preeclampsia status. Applicants have demonstrated, using proteomic technology (SELDI-TOF mass spectroscopy) coupled with standard molecular and biochemical identification assays, that women with severe preeclampsia have nanomolar amounts of free hemoglobin in their CSF, which is not present in women with mild preeclampsia or normotensive controls.

Adult hemoglobin consists of two a- and two /3-polypeptide chains, each containing a non-peptide haem group that reversibly binds a single oxygen molecule. Hemoglobin circulates within erythrocytes. Although critical to oxygenation, free hemoglobin in the circulation is toxic to tissues by altering the vascular redox balance during the auto-oxidation of haem from its ferrous to ferric state (Motterlini et al., Am J Physiol 1995, 269:648-55) and possibly through the induction of globin-centered free radicals (Svistunenko et al., J Biol Chem 1997, 272:7114-21). As such, an elaborate scavenging system exists to clear free hemoglobin from the circulation, involving binding of free hemoglobin to haptoglobin and clearance of the hemoglobin-haptoglobin complex by macrophages through CD163 -mediated endocytosis (Graversen et al., Int J Biochem Cell Biol 2002, 34:309-14). However, scavenging of free hemoglobin in CSF is far more limited. Applicants propose that increased levels of free hemoglobin in the CSF of women with severe preeclampsia not only serves as a marker for the disease, but is also directly responsible for the cerebrovascular manifestations commonly seen in such patients.

As used herein, the terms "marker" and "biomarker" refer to an organic biomolecule, preferably, a polypeptide or protein, which is differentially present in a sample taken from a woman having preeclampsia as compared to a comparable sample taken from a woman, referred to as a "normal" woman/subject who does not have preeclampsia. A marker is differentially present in samples from women having preeclampsia, if it is present at an elevated level in the women with preeclampsia, as compared to samples from normal subjects. Alternatively, a marker can be differentially present in samples from normal subjects, if it is present at an elevated level in the normal subjects as compared to samples of women having preeclampsia. An example of the subject markers is free hemoglobin present in a CSF sample.

1. Test Sample Preparation

In certain aspects, samples of the subject methods can be collected from pregnant women, e.g., pregnant women in whom preeclampsia status is to be assessed. The pregnant women may be pregnant women who have been determined to have a high risk of preeclampsia based on their personal or family history. Other patients include pregnant women who are known to have preeclampsia and for whom the test is being used to determine the effectiveness of therapy or treatment they are receiving. Also, patients could include healthy pregnant women who are having a test as part of a routine examination, or to establish baseline levels (e.g., control or reference level) of the biomarkers. In other aspects, samples may be collected from pregnant non-human mammals, or non-pregnant women, for example, in methods of identifying a compound for preeclampsia.

The markers can be measured in different types of biological samples, preferably biological fluid samples such as CSF. Other biological fluid samples may include blood, blood serum, plasma, vaginal secretions, urine, tears, and saliva. If desired, the sample can be prepared to enhance detectability of the markers. For example, a blood serum sample from the subject can be fractionated. Any method that enriches for the protein of interest can be used. Sample preparations, such as prefractionation protocols, are optional and may not be necessary to enhance detectability of markers depending on the methods of detection used. For example, sample preparation may be unnecessary if antibodies that specifically bind markers are used to detect the presence of markers in a sample.

Typically, sample preparation involves fractionation of the sample and collection of fractions determined to contain the biomarkers. Methods of prefractionation include, for example, size exclusion chromatography, ion exchange chromatography, heparin chromatography, affinity chromatography, sequential extraction, gel electrophoresis and liquid chromatography. Examples of methods of fractionation are described in PCT/US03/00531 (incorporated herein in its entirety).

As an example, a sample is pre-fractionated by anion exchange chromatography. Anion exchange chromatography allows pre-fractionation of the proteins in a sample roughly according to their charge characteristics. For example, a Q anion-exchange resin can be used, and a sample can be sequentially eluted with eluants having different pH's. Anion exchange chromatography allows separation of biomolecules in a sample that are more negatively charged from other types of biomolecules. Proteins that are eluted with an eluant having a high pH is likely to be weakly negatively charged, and a fraction that is eluted with an eluant having a low pH is likely to be strongly negatively charged. Thus, in addition to reducing complexity of a sample, anion exchange chromatography separates proteins according to their binding characteristics.

As another example, biomolecules in a sample can be separated by high- resolution electrophoresis, e.g., one or two-dimensional gel electrophoresis. A fraction containing a marker can be isolated and further analyzed by gas phase ion spectrometry. Preferably, two-dimensional gel electrophoresis is used to generate two-dimensional array of spots of biomolecules, including one or more markers. See, e.g., Jungblut and Thiede, Mass Spectr. Rev. 16:145-162 (1997). The two- dimensional gel electrophoresis can be performed using methods known in the art. See, e.g., Deutscher ed., Methods In Enzyniology vol. 182. In certain cases, biomolecules in a sample are separated by, e.g., isoelectric focusing, during which bioniolecules in a sample are separated in a pH gradient until they reach a spot where their net charge is zero (isoelectric point). This first separation step results in one- dimensional array of biomolecules. The biomolecules in one-dimensional array is further separated using a technique generally distinct from that used in the first separation step. Typically, two-dimensional gel electrophoresis can separate chemically different biomolecules in the molecular mass range from 1000-200,000 Da within complex mixtures. The pi range of these gels is about 3-10 (wide range gels).

As another example, high performance liquid chromatography (HPLC) can also be used to separate a mixture of biomolecules in a sample based on their different physical properties, such as polarity, charge and size. HPLC instruments typically consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Biomolecules in a sample are separated by injecting an aliquot of the sample onto the column. Different biomolecules in the mixture pass through the column at different rates due to differences in their partitioning behavior between the mobile liquid phase and the stationary phase. A fraction that corresponds to the molecular weight and/or physical properties of one or more markers can be collected. The fraction can then be analyzed by gas phase ion spectrometry to detect markers. For example, the spots can be analyzed using either MALDI or SELDI as described below.

Optionally, a marker can be modified before analysis to improve its resolution or to determine its identity. For example, the markers may be subject to proteolytic digestion before analysis. Any protease can be used. Proteases, such as trypsin, that are likely to cleave the markers into a discrete number of fragments are particularly useful. The fragments that result from digestion function as a fingerprint for the markers, thereby enabling their detection indirectly. This is particularly useful where there are markers with similar molecular masses that might be confused for the marker in question. Also, proteolytic fragmentation is useful for high molecular weight markers because smaller markers are more easily resolved by mass spectrometry. Optionally, the identity of the markers can be further determined by matching the physical and chemical characteristics of the markers in a protein database (e.g., SwissProt). 2. Detection and Measurement of Markers

Biomarkers such as hemoglobin are preferably captured with capture reagents immobilized to a solid support, such as any biochip described herein, a multiwell microtiter plate or a resin. A preferred mass spectrometric technique for use in the invention is Surface Enhanced Laser Desorption and Ionization (SELDI), as described, for example, in U.S. Patent No. 5,719,060 and No. 6,225,047, in which the surface of a probe that presents the analyte to the energy source plays an active role in desorption/ionization of analyte molecules. In this context, the term "probe" refers to a device adapted to engage a probe interface and to present an analyte to ionizing energy for ionization and introduction into a gas phase ion spectrometer, such as a mass spectrometer. A probe typically includes a solid substrate, either flexible or rigid, that has a sample-presenting surface, on which an analyte is presented to the source of ionizing energy.

One version of SELDI, called "Surface-Enhanced Affinity Capture" or "SEAC," involves the use of probes comprised of a chemically selective surface ("SELDI probe"). A "chemically selective surface" is one to which is bound either the adsorbent, also called a "binding moiety,'" or "capture reagent," or a reactive moiety that is capable of binding a capture reagent, e.g., through a reaction forming a covalent or coordinate covalent bond.

The phrase "reactive moiety" here denotes a chemical moiety that is capable of binding a capture reagent. Epoxide and carbodiimidizole are useful reactive moieties to covalently bind polypeptide capture reagents such as antibodies or cellular receptors. Nitriloacetic acid and iminodiacetic acid are useful reactive moieties that function as chelating agents to bind metal ions that interact noncovalently with histidine containing peptides. A "reactive surface" is a surface to which a reactive moiety is bound. An "adsorbent" or "capture reagent" can be any material capable of binding a biomarker of the invention. Suitable adsorbents for use in SELDI, according to the invention, are described in U.S. Patent No. 6,225,047.

One type of adsorbent is a "chromatographic adsorbent," which is a material typically used in chromatography. Chromatographic adsorbents include, for example, ion exchange materials, metal chelators, immobilized metal chelates, hydrophobic interaction adsorbents, hydrophilic interaction adsorbents, dyes, mixed mode adsorbents (e.g., hydrophobic attraction/electrostatic repulsion adsorbents). "Biospeciflc adsorbent" is another category, for adsorbents that contain a biomolecule, e.g., a nucleotide, a nucleic acid molecule, an amino acid, a polypeptide, a simple sugar, a polysaccharide, a fatty acid, a lipid, a steroid or a conjugate of these (e.g., a glycoprotein, a lipoprotein, a glycolipid). In certain instances, the biospecific adsorbent can be a macromolecular structure such as a multiprotein complex, a biological membrane or a virus. Illustrative biospecific adsorbents are antibodies, receptor proteins, and nucleic acids. A biospecific adsorbent typically has higher specificity for a target analyte than a chromatographic adsorbent.

Another version of SELDI is Surface-Enhanced Neat Desorption (SEND), which involves the use of probes comprising energy absorbing molecules that are chemically bound to the probe surface ("SEND probe"). The phrase "Energy absorbing molecules" (EAM) denotes molecules that are capable of absorbing energy from a laser desorption ionization source and, thereafter, contributing to desorption and ionization of analyte molecules in contact therewith. The EAM category includes molecules used in MALDI, frequently referred to as and is exemplified by cinnamic acid derivatives, sinapinic acid (SPA), cyano-hydroxy-cinnamic acid (CHCA) and dihydroxybenzoic acid, ferulic acid, and hydroxyaceto-phenone derivatives. The category also includes EAMs used in SELDI, as enumerated, for example, by U.S Patent No. 5,719,060.

Another version of SELDI, called Surface-Enhanced Photolabile Attachment and Release (SEPAR), involves the use of probes having moieties attached to the surface that can covalently bind an analyte, and then release the analyte through breaking a photolabile bond in the moiety after exposure to light, e.g., to laser light. For instance, see U.S. Patent No. 5,719,060. SEPAR and other forms of SELDI are readily adapted to detecting a biomarker or biomarker profile, pursuant to the present invention.

The detection of the biomarkers according to the invention can be enhanced by using certain selectivity conditions, e.g., adsorbents or washing solutions. The phrase "wash solution" refers to an agent, typically a solution, which is used to affect or modify adsorption of an analyte to an adsorbent surface and/or to remove unbound materials from the surface. The elution characteristics of a wash solution can depend, for example, on pH, ionic strength, hydrophobicity, degree of chaotropism, detergent strength, and temperature.

Pursuant to one aspect of the present invention, a sample is analyzed by means of a "biochip," a term that denotes a solid substrate having a generally planar surface, to which a capture reagent (adsorbent) is attached. Frequently, the surface of a biochip comprises a plurality of addressable locations, each of which has the capture reagent bound there. A biochip can be adapted to engage a probe interface and, hence, function as a probe, which can be inserted into a gas phase ion spectrometer, preferably a mass spectrometer. Alternatively, a biochip of the invention can be mounted onto another substrate to form a probe that can be inserted into the spectrometer.

A variety of biochips is available for the capture of biomarkers, in accordance with the present invention, from commercial sources such as Ciphergen Biosystems (Fremont, CA), Packard BioScience Company (Meriden, CT), Zyomyx (Hayward, CA), and Phylos (Lexington, MA). Exemplary of these biochips are those described in U.S. Patents Nos. 6,225,047, 6,329,209, and in PCT Publication Nos. WO 99/51773 and WO 00/56934.

More specifically, biochips produced by Ciphergen Biosystems have, surfaces, presented on an aluminum substrate in strip form, to which are attached, at addressable locations, chromatographic or biospecific adsorbents. The surface of the strip is coated with silicon dioxide.

Illustrative of Ciphergen ProteinChip® arrays are biochips H4, SAX-2, WCX- 2, and IMAC-3, which include a functionalized, crosslinked polymer in the form of a hydrogel, physically attached, to the surface of the biochip or covalently attached through a silane to the surface of the biochip. The H4 biochip has isopropyl functionalities for hydrophobic binding. The SAX-2 biochip has quaternary ammonium functionalities for anion exchange. The WCX-2 biochip has carboxylate functionalities for cation exchange. The IMAC-3 biochip has nitriloacetic acid functionalities that adsorb transition metal ions, such as Cu4+ and Ni+*, by chelation. These immobilized metal ions, in turn, allow for adsorption of biomarkers by coordinate bonding.

A substrate with an adsorbent is contacted with the CSF sample for a period of time sufficient to allow biomarker that may be present to bind to the adsorbent. After the incubation period, the substrate is washed to remove unbound material. Any suitable washing solutions can be used; preferably, aqueous solutions are employed. An energy absorbing molecule then is applied to the substrate with the bound biomarkers, As noted, an energy absorbing molecule is a molecule that absorbs energy from an energy source in a gas phase ion spectrometer, thereby assisting in desorption of biomarkers from the substrate. Exemplary energy absorbing molecules include, as noted above, cinnamic acid derivatives, sinapinic acid and dihydroxybenzoic acid. Preferably sinapinic acid is used.

Once captured on a substrate, e.g., biochip or antibody, any suitable method can be used to measure one or more markers in a sample. For example, markers can be detected and/or measured by a variety of detection methods including for example, gas phase ion spectrometry methods, optical methods, electrochemical methods, atomic force microscopy and radio frequency methods. Using these methods, one or more markers can be detected.

In one embodiment, methods of detection and/or measurement of the biomarkers use mass spectrometry and, in particular, SELDI. SELDI refers to a method of desorption/ ionization gas phase ion spectrometry (e.g., mass spectrometry) in which the analyte is captured on the surface of a SELDI probe that engages the probe interface. In "SELDI MS," the gas phase ion spectrometer is a mass spectrometer. SELDI technology is described in more detail above.

In another embodiment, an immunoassay can be used to detect and analyze markers in a sample. An immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a marker). The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a marker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically reactive with that marker and not with other proteins, except for polymorphic variants and alleles of the marker. This selection may be achieved by subtracting out antibodies that cross-react with the marker molecules from other species.

Using the purified markers or their nucleic acid sequences, antibodies that specifically bind to a marker (e.g., hemoglobin) can be prepared using any suitable methods known in the art. See, e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane, Antibodies: A Laboratory Manual (1988); Goding, Monoclonal antibodies: Principles and Practice (2d ed. 1986); Kohler & Milstein, Nature 256:495-497 (1975); Huse et al, Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546 (1989).

Generally, a sample obtained from a subject can be contacted with the antibody that specifically binds the marker. Optionally, the antibody can be fixed to a solid support to facilitate washing and subsequent isolation of the complex, prior to contacting the antibody with a sample. Examples of solid supports include glass or plastic in the form of, e.g., a microtiter plate, a stick, a bead, or a microbead. Antibodies can also be attached to a probe substrate or a protein chip.

After incubating the sample with antibodies, the mixture is washed and the antibody-marker complex formed can be detected. This can be accomplished by incubating the washed mixture with a detection reagent. This detection reagent may be, e.g., a second antibody which is labeled with a detectable label. Exemplary detectable labels include magnetic beads, fluorescent dyes, radiolabels, en∑ymes (e.g., horse radish peroxide, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads. Alternatively, the marker in the sample can be detected using an indirect assay, wherein, for example, a second, labeled antibody is used to detect bound marker- specific antibody, and/or in a competition or inhibition assay wherein, for example, a monoclonal antibody which binds to a distinct epitope of the marker is incubated simultaneously with the mixture.

Methods for measuring the amount or presence of an antibody-marker complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index (e.g., surface plasmon resonance, ellipsometry, a resonant mirror method, a gating coupler waveguide method or interferometry). Optical methods include microscopy (both confocal and non-confocal), imaging methods and non¬ imaging methods. Electrochemical methods include voltametry and amperometry methods. Radio frequency methods include multipolar resonance spectroscopy. Useful assays are well known in the art, including, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay.

Immunoassays can be used to determine presence or absence of a marker in a sample as well as the quantity of a marker in a sample. The amount of an antibody- marker complex can be determined by comparing to a standard. A standard can be, e.g., a known compound or another protein known to be present in a sample. It is understood that the test amount of marker need not be measured in absolute units, as long as the unit of measurement can be compared to a control.

When the sample is measured and data is generated, e.g., by mass spectrometry, the data is then analyzed by a computer software program. In certain cases, the biomarkers bound to the substrates can be detected in a gas phase ion spectrometer. The biomarkers are ionized by an ionization source such as a laser, the generated ions are collected by an ion optic assembly, and then a mass analyzer disperses and analyzes the passing ions, The detector then translates information of the detected ions into mass-to-charge ratios. Detection of a biomarker typically will involve detection of signal intensity. Thus, both the quantity and mass of the biomarker can be determined.

Generally, data generated by desorption and detection of markers can be analyzed with the use of a programmable digital computer. The computer program analyzes the data to indicate the number of markers detected, and optionally the strength of the signal and the determined molecular mass for each hiomarker detected. Data analysis can include steps of determining signal strength of a biomarker and removing data deviating from a predetermined statistical distribution. For example, the observed peaks can be normalized, by calculating the height of each peak relative to some reference. The reference can be background noise generated by the instrument and chemicals such as the energy absorbing molecule which is set as zero in the scale.

The computer can transform the resulting data into various formats for display. The standard spectrum can be displayed, but in one useful format only the peak height and mass information are retained from the spectrum view, yielding a cleaner image and enabling biomarkers with nearly identical molecular weights to be more easily seen, in another useful format, two or more spectra are compared, conveniently highlighting unique biomarkers and biomarkers that are up- or downregulated between samples. Using any of these formats, one can readily determine whether a particular biomarker is present in a sample.

Software used to analyze the data can include code that applies an algorithm to the analysis of the signal to determine whether the signal represents a peak in a signal that corresponds to a biomarker according to the present invention. The software also can subject the data regarding observed biomarker peaks to classification tree or ANN analysis, to determine whether a biomarker peak or combination of biomarker peaks is present that indicates a diagnosis of intra-amniotic inflammation.

As a specific example, Applicants used SELDI-TOF mass spectroscopy to identify the biomarkers in CSF. Briefly, a single sample of the biologic specimen of interest was prepared by pooling small aliquots from all patients with "severe disease" (in this case, severe preeclampsia). A similar, pooled sample of all normotensive controls was prepared. These two samples were then used to optimize the conditions for analysis by SELDI-TOF mass spectroscopy, including the optimal dilution of the biologic samples, ProteinChip® array surface, metal affinities, binding buffer pH, washing conditions, and overlay matrix (energy absorbing molecules). The proteomic spectra are then systematically examined to identify regions of interest, and a scoring system is developed using Boolean indicators in which a score of 0 or 1 is assigned if the peak is absent or present, respectively (Figures 1 and 2). The resultant PPB score was then tested on individual samples to validate its ability to distinguish between the various clinical disease entities. In this instance, the PPB score was able to distinguish severe preeclampsia from both mild preeclampsia and normotensive controls with a sensitivity of 85.7 percent and a specificity of 100 percent (Figure 3).

3. Diagnosis Methods and Kits

In certain embodiments, the present invention relates to the proteomic analysis of CSF to obtain information that correlates with the severity of preeclampsia and the clinical outcome. Proteomic analysis of CSF, in accordance with the invention, provides a rapid, simple and reliable means of detecting in a patient who has or is at risk of developing preeclampsia.

In one embodiment, one or more of the markers (e.g., free hemoglobin in CSF) of the invention can be employed for determining preeclampsia status (e.g., severe preeclampsia) in a pregnant subject. For example, the concentration of a marker correlates with the severity of preeclampsia (e.g., mild or severe preeclampsia). It is known that neurologic manifestations, such as seizures or coma (eclampsia), stroke, hypertensive encephalopathy, headaches, and visual aberrations (scotomata, diplopia, amaurosis, homonymous hemianopsia, are common in severe preeclampsia (Douglas and Redman, Br Med J 1994, 309:1395-1400).

In another embodiment, the present invention contemplates use of a biomarker such as free hemoglobin level in CSF for detecting or predicting subarachnoid hemorrhage in a subject. Applicants found a correlation between CSF hemoglobin concentrations and erythrocyte counts. Although not wishing to be bound by theory, it is likely that the increase in free hemoglobin in the CSF of women with severe preeclampsia results from increased and selective trafficking of intact erythrocytes across the blood-brain barrier where they subsequently lyse releasing their hemoglobin content. It is known, for example, from data in patients with subarachnoid hemorrhage that erythrocytes lyse far more rapidly in CSF than in the circulation, with a decrease in average survival from 120 days in the circulation to 2-4 hours in CSF (Morgenstern et al., Ann Emerg Med 1998, 32: 297-304; Shah and Edlow, J Emerg Med 2002, 23: 67-74). In this method, the subject may include a non-pregnant subject (e.g., a female or a male).

In another embodiment, the present invention contemplates use of a biomarker such as free hemoglobin level in CSF for predicting a cerebrovascular disorder unrelated to hypertension in a subject. Applicants found an increase in free hemoglobin in the CSF of women with severe preeclampsia, and hypothesize that such women may represent a high-risk population for subsequent cerebrovascular disorders (e.g., stroke) unrelated to hypertension. Review of the literature suggests that this may indeed be true. See, e.g., Wilson et al., BMJ 2003, 326:845; Irgens et al., BMJ 2001, 323:1213-7; Kestenbaum et al., Am J Kidney Dis 2003, 42:982-9. In this method, the subject may include a non-pregnant subject (e.g., a female or a male).

In specific embodiments, the diagnostic/detection methods of the invention entails contacting a CSF sample from a patient with a substrate, having an adsorbent thereon, under conditions that allow binding between the biomarker and the adsorbent, and then detecting the biomarker bound to the adsorbent by gas phase ion spectrometry, for example, mass spectrometry. As described above, other detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Immunoassays in various foπnats, such as ELISA, likewise can be adapted for detection of biomarkers captured on a solid phase.

In certain embodiments, the present invention provides kits for aiding in the diagnosis of preeclampsia. The kits are used to detect or screen for the presence of biomarkers and combinations of biomarkers that are differentially present in samples from subjects with preeclampsia.

In one embodiment, the kit comprises a substrate having an adsorbent thereon, wherein the adsorbent is suitable for binding a biomarker of the invention, and a washing solution or instructions for making a washing solution, in which the combination of the adsorbent and the washing solution allows detection of the biomarker using gas phase ion spectrometry. In preferred embodiments, the kit comprises a immobilized metal affinity capture chip, such as the H4 chip. In another embodiment, a kit of the invention may include a first substrate, comprising an adsorbent thereon, and a second substrate onto which the first substrate is positioned to form a probe, which can be inserted into a gas phase ion spectrometer. In another embodiment, an inventive kit may comprise a single substrate that can be inserted into the spectrometer.

In a further embodiment, such a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert. For example, the instructions may inform a consumer how to collect the sample or how to wash the probe.

The biomarkers according to the invention also are useful in the production of other diagnostic assays for detecting the presence of the biomarker in a sample. For example, such assays may comprise, as the "adsorbent," "binding moiety," or "capture reagent," an antibody to one or more of the biomarkers such as hemoglobin. The antibody is mixed with a sample suspected of containing the biomarkers and monitored for biomarker-antibody binding. The biomarker antibody is labeled with a radioactive or enzyme label. In a preferred embodiment, the biomarker antibody is immobilized on a solid matrix such that the biomarker antibody is accessible to biomarker in the sample. The sample then is brought into contact with the surface of the matrix, and the surface is monitored for biomarker-antibody binding.

4. Screening Methods

In certain embodiments, the present invention provides a method of identifying a compound that ameliorates or treats preeclampsia. For examples, such method comprises: (a) administering a candidate compound to a subject; (b) comparing the level of free hemoglobin in a test CSF sample obtained from the subject with the level of free hemoglobin in a control CSF sample, wherein if the free hemoglobin level is lower in the test CSF sample than in the control CSF sample, the candidate compound is a compound that ameliorates or treats preeclampsia. Optionally the free hemoglobin level in CSF is measured using an immunological assay (e.g., an ELISA), a protein chip assay, or surface-enhanced laser desorption/ionization (SELDI). This method includes measurement of hemoglobin a chain, hemoglobin β chain, and modified forms of hemoglobin such as glycated isoforms of hemoglobin a chain and glycated isoforms of hemoglobin β chain. Preferably, the subject is a female human such as a pregnant woman.

There are numerous approaches to screening for therapeutic agents in preeclampsia therapy, which target one or more of the biomarkers (e.g., the CSF hemoglobin level). For example, high-throughput screening of compounds or molecules can be carried out to identify agents or drugs which ameliorates or treats preeclampsia. Test agents to be assessed can be any chemical (element, molecule, compound, drug), made synthetically, made by recombinant techniques or isolated from a natural source. For example, test agents can be peptides, polypeptides, peptoids, sugars, hormones, or nucleic acid molecules (such as antisense or RNAi nucleic acid molecules). In addition, test agents can be small molecules or molecules of greater complexity made by combinatorial chemistry, for example, and compiled into libraries. These libraries can comprise, for example, alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers and other classes of organic compounds. Test agents can also be natural or genetically engineered products isolated from lysates or growth media of cells - bacterial, animal or plant — or can be the cell lysates or growth media themselves. Presentation of test compounds to the test system can be in either an isolated form or as mixtures of compounds, especially in initial screening steps. Compounds identified through the screening methods can then be tested in animal models of cervical cancer to assess their anti- preeclampsia activity in vivo.

EXEMPLIFICATION

Complex proteomic analysis has recently been used to identify proteomic fingerprints for specific disorders (Papadopoulos et al., Lancet 2004, 363: 1358-1363; Puchades et al., MoI Brain Res 2003, 118:140-146; Davidsson et al., MoI Brain Res 2002, 109:128-133; Buhimschi et al., WO 04/043238; Weiner et al., Am J Obstet Gynecol 2004, in press). Two-dimensional gel protein analysis and SELDI-TOF (surface enhanced laser desorption/ionization time-of-flight) mass spectroscopy analysis have identified proteins in cerebrospinal fluid (CSF) that appear to be selectively expressed in patients with Alzheimer's disease (Pucliades et al., MoI Brain Res 2003, 118:140-146) and frontotemporal dementia (Davidsson et al., MoI Brain Res 2002, 109:128-133). However, there are no previous reports of CSF proteomic analysis in preeclampsia. Applicants hypothesize that the CSF of women with S preeclampsia have specific proteomic fingerprints that are related to the severity of the disease. Such discriminatory protein biomarkers may lead to a better understanding of the pathophysiology of this disease, and may be of both diagnostic and prognostic value. This study uses new proteomic technology (SELDI-TOF) to interrogate the CSF of women with preeclampsia.

10 1. Patient Characteristics

Of the 22 women recruited, seven had been diagnosed clinically with severe preeclampsia, eight with mild preeclampsia, and the remaining seven were normotensive controls. Indicators for severe preeclampsia included symptoms (n=l), blood pressure criteria (n=2), HELLP syndrome (n=l), pulmonary edema (n=l), or a IS combination of these features (n=2) (Table 1). AU diagnoses were confirmed by review of the medical records. There was no significant difference between women with severe preeclampsia, mild preeclampsia, and controls as regards maternal age (average, [95 percent CI]: 36.7 [33.6-37.8], 35.6 [34.4-36.8], and 34.1 [31.4-36.9] years, respectively; one-way ANOVA, p=0.638), gravidity (median, [range]: 2 [1-3], 20 2.5 [1-5], and 2 [1-5], respectively; one-way ANOVA, p=0.87), parity (median, [range]: 1 [0-2], 1 [0-3], and 1 [0.2], respectively; one-way ANOVA, p=0.70), and gestational age at delivery (average, [95 percent CI]: 33.7 [31.7-35.7], 34.1 [31.1- 37.1], and 36.0 [33.3-38.7] weeks, respectively; one-way ANOVA, p=0.501). Table 1: Patient demographics

Patient Maternal G P Diagnosis Criteria for Gestational Mode Indication Mg2+ no. age severe age at of for prior to (years) preeclampsia delivery delivery cesarean spinal (wks) 1 37 1 0 Severe preeclampsia Elevated LFTs 34 Primary CD Breech Yes 2 35 2 1 Severe preeclampsia Pulmonary edema 34 NVD Twins Yes 3 36 3 1 Severe preeclampsia Pulmonary edema, 30 Primary CD NRFT Yes HELLP 4 37 3 2 Severe preeclampsia HELLP, BP 31 Repeat CD Elective No 5 30 3 1 Severe preeclampsia BP 37 Repeat CD Elective No 6 36 2 0 Severe preeclampsia Symptoms 37 Primary CD Failed IOL, Yes twins 7 39 1 0 Severe preeclampsia BP 33 Primary CD Triplets No 8 29 1 0 Mild preeclampsia — 39 Primary CD FTP No 9 33 1 0 Mild preeclampsia — 29 Primary CD Breech, No NRFT 10 37 3 2 Mild preeclampsia — 36 Repeat CD Prior classical No CD 11 37 1 0 Mild preeclampsia — 36 Primary CD Breech No 12 37 4 3 Mild preeclampsia — 38 KTVD — No 13 35 5 2 Mild preeclampsia — • 30 Primary CD Transverse lie No 14 34 3 2 Mild preeclampsia — 39 NVD — No 15 36 2 0 Mild preeclampsia — 31 Primary CD NRFT No 16 31 2 1 Control — 38 NVD — — 17 37 2 1 Control — 28 NVD — — 18 29 1 0 Control — 39 Primary CD Breech — 19 37 2 1 Control — 36 Repeat CD Elective — 20 32 3 2 Control — 37 Repeat CD Elective — 21 34 1 0 Control — 37 Primary CD Placenta previa — 22 39 5 1 Control — 37 Primary CD Placenta previa —

BP = blood pressure; CD = cesarean delivery; FTP = failure to progress; G = gravidity; HELLP = hemolysis, elevated liver functions, low platelets; IOL = induction of labor; LFTs = liver function tests; NRFT = non-reassuring fetal testing; P = parity; SVD = spontaneous vaginal delivery.

2. Design and Performance of the Preeclampsia Proteomic Biomarker (PPB) Score to

Discriminate Cases of Severe preeclampsia

The distribution of patients with and without severe preeclampsia and their respective PPB score is shown in Figure 3A. The single outlier (see arrow in Figure 3A) was a woman categorized clinically as having severe preeclampsia by pulmonary edema only. Our post-proteomic review of the medical record revealed that, according to the obstetric care provider, the pulmonary edema likely resulted from iatrogenic

fluid overload. Figure 3B illustrates a ROC analysis of the PPB score, which

demonstrates that a cut-off of>1 in the PBB score is able to separate patients classified as having severe preeclampsia from both mild preeclampsia and

normotensive controls with 85.7% sensitivity and 100% specificity (area under the

ROC curve = 0.919; 95% CI [0.721-0.989]).

3. Identification of the Discriminatory Proteomic Biomarkers

One-dimensional SDS-PAGE electrophoresis followed by in-gel tryptic

digestion of excised bands was performed as described above. Figure 4A shows a representative SDS-PAGE gel. Protein bands of interest were designated A through F. Band E was present in patients with severe preeclampsia but not controls, and corresponded in location with the discriminatory proteomic peaks of interest identified by SELDI-TOF (Figure 4B). In-gel tryptic digestion of the bands of interest yielded specific peptide profiles when analyzed by SELDI-TOF (Figure 4C). The small protein peaks identified in the negative control lane (water instead of CSF) represent trypsin autolysis products. The identity of each protein band was confirmed by comparing the corresponding peptide map with a database of tryptic digest peptide maps of known proteins as reported by Profound (http://prowl.rockefeller.edu). The results of this in silico analysis are shown in Figure 4D. These data indicated that band E corresponded to the hemoglobin α chain.

On-chip antibody capture assays illustrate the SELDI-TOF spectra from a patient with severe preeclampsia and a control patient (Figure 5). Representative SELDI spectra using anti-hemoglobin antibody and sheep IgG (negative control) are shown. The SELDI spectrum of CSF from patients with severe preeclampsia obtained with the dry on-chip profiling protocol is also shown as a reference. A series of peaks are present in the SELDI spectrum obtained using anti-hemoglobin antibody (but not negative control) that effectively recapitulates the PPB score as seen with the WCX2 dry on-chip protocol. From these data, the present invention concluded that peaks #2 and #4 from Figure 2 represent the a- (15126 Da [SwissProt]) and /3-chains (15867 Da) of human hemoglobin, respectively. It is likely that peaks #3 and #5 from Figure 2 represent a post-translational modification (glycation) of the a- and /3-chains of hemoglobin, respectively.

4. Correlation with Biochemical and Cytological Analysis of Cerebrospinal Fluid

There was no difference in the total protein concentration of the CSF between patients with severe preeclampsia, mild preeclampsia, and normal controls (average [95 percent CI]: 0.60 [0.53-0.67], 0.59 [0.53-0.66], and 0.66 [0.62-0.71] mg/mL, respectively; one-way ANOVA, p=0.256). There was also no difference in protein concentration between patients with a PPB score of 0-1 (average [95 percent CI]: 0.63 [0.59-0.67] mg/mL, n=16) and those with PPB of 2-4 (average [95 percent CI]: 0.58 [0.51-0.65] mg/mL, n=6), t-test, ρ=0.211.

There was no difference in CSF leukocyte counts between patients with severe preeclampsia, mild preeclampsia, and normal controls (median [range]: 1 [0-3], 0 [0- 5], and 0 [0-1] cell/mm3, respectively; one-way ANOVA, p=0.434). However, the erythrocyte count was significantly higher in the CSF of patients with severe preeclampsia as compared with mild preeclampsia and normal controls (median [range]: 123 [0-301], 0.5 [0-8], and 2 [1-10] cell/mm3, respectively; one-way ANOVA, p=0.001). Patient #2 who had no erythrocytes in her CSF was the same outlier patient with a PPB score of 0 (Figure 3A) who has been categorized clinically with severe preeclampsia by pulmonary edema only that was attributed in large part to iatrogenic fluid overload.

5. Quantitation Of Free Hemoglobin In Cerebrospinal Fluid

Hemoglobin levels in the CSF of women with severe preeclampsia as measured by ELISA were significantly higher than in mild preeclampsia or normotensive controls (average [95 percent CI]: 5.99 [2.37-8.36], 0.16 [-0.16 to 0.49], and 0 [0-0] μg/mL, respectively; one-way ANOVA, p<0.001) (Figure 6A). Again, the one patient without detectable hemoglobin in her CSF (arrow in Figure 6A) was patient #2 (Table 1) who had been diagnosed with severe preeclampsia by pulmonary edema only that was attributed in large part to iatrogenic fluid overload. In contrast to hemoglobin (our protein biomarker), levels of cystatin C (an identified non-biomarker protein) in the CSF were not significantly different between the three groups (average [95 percent CI]: 4.01 [3.41-4.60], 4.44 [3.84-5.05], and 4.68 [4.44-4.91] μg/mL, respectively; one-way ANOVA, p=0.234) (Figure 6B). Levels of free hemoglobin in the CSF correlated with the number of erythrocytes (r=0.910, pO.OOl) and with the PPB score (r=0.923, p<0.001), but not with the WBC count (r=0.220, p=0.325) or total protein content in CSF (r=-0.349, p=0.111).

A rough calculation based on the molecular weight of the protein tetrameric moiety of hemoglobin (62 kDa), which would be detected in the ELISA, and the results of our ELISA data, Applicants estimate that women with severe preeclampsia have a concentration of about 100-nM free hemoglobin in their CSF. Based on the positive control sample in which Applicants lysed a known number of erythrocytes and spiked a sample of CSF from a control patient, Applicants deduced that at least 100 erythrocytes/mm3 had to have lysed to yield measurable hemoglobin levels by ELISA and that the CSF of women with severe preeclampsia contains the equivalent of an average of 280 lysed erythrocytes/mm3. Direct spectrophotometry is currently the 'gold standard' for the detection of free hemoglobin in CSF (Figure 7A). CSF of patients with severe preeclampsia had increased absorbance at 414 run (oxyhemoglobin) compared with patients with mild preeclampsia (average [95 percent CI]: 4.3 [-0.41 to 9.0] versus 0.6 [-0.6 to 1.9] xlO'3 absorbance units; Tukey, p=0.017). However, CSF in several of the control patients exhibited higher absorbance values at both 414 and 455 run, probably due to an interfering substance(s) unrelated to hemoglobin. Figure 7B shows an absorbance spectrum from a control sample with PPB 0 and absent hemoglobin by ELISA compared with the absorbance spectrum from an erythrocyte lysate containing 1,000 cells/mm3. As a result of this interference, there was no statistical difference in absorbance at 414 nm between control patients (average [95 percent CI]: 4.3 [-0.4 to 9.0]) and patients with either mild (Tukey, p= 0.139) or severe preeclampsia (Tukey, p=0.584). There was also no difference in absorbance at 455 nm (bilirubin shoulder) among the three groups (one way ANOVA, p=0.475). There was a significant correlation between absorbance at 414 nm and hemoglobin levels measured by ELISA (r =0.643, p=0.001) and the PPB score (r=0.620, p=0.0021). In an ROC analysis, a cut-off of 0.007 absorbance units at 414 nm was able to cluster cases with clinical diagnosis of severe preeclampsia with 71.4 percent sensitivity and 93.3 percent specificity (area under the ROC curve = 0.771; 95 percent CI [0.545-0.920]).

6. Functional Implications of Free Hemoglobin in Cerebrospinal Fluid

To investigate whether the presence of nanomolar amounts of free hemoglobin in the CSF of women with severe preeclampsia may be causally related to the neurologic manifestations commonly seen in this condition, Applicants performed a series of experiments designed to look at known markers of inflammation and oxidative stress in the CSF of such women. IL-6 levels in the CSF of patients with a PPB score of>1 were not statistically different from those with a PPB score <1 (average [95 percent CI]: 24.2 [-5.9 to 54.3] versus 47.9 [8.5-87.3] pg/mL, respectively; t-test, p=0.390). Similarly, measurements of total nitric oxide metabolite concentrations were not different between these two groups (average [95 percent CI]: 1.4 [1.2-1.7] versus 1.5 [1.3-1.6] μM, respectively; t-test, p=0.841). However, the level of protein oxidation was significantly elevated in the CSF of women with a PPB score of>1 as compared with women with a PPB score <1 (average [95 percent CI]: 959.4 [617.4-130.4] versus 638.7 [562.8-714.7] finol of DNP per μg protein, respectively; ttest, p=0.015) (Figure 8).

7. Methods and Materials

1) Patients and Samples

Twenty two pregnant women admitted to the Brigham & Women's Hospital, Boston from January 2001 to December 2003 were recruited randomly based on clinician availability. Laboring women were clustered into three clinical categories based on widely accepted clinical criteria for the diagnosis and categorization of preeclampsia (American College of Obstetricians and Gynecologists. Diagnosis and management of preeclampsia and eclampsia. Practice Bulletin No. 33. Obstet Gynecol 2002; 99:159): severe preeclampsia (n=7), mild preeclampsia (n=8), and normotensive controls (n=7). A 1-2 mL sample of CSF was collected at the time of spinal or combined spinal-epidural anesthesia for delivery. Institutional Review Board approval for this study was obtained. All procedures were performed by a single clinician (LT). None of the patients had received regional anesthesia in the preceding three months. A 0.5-mL aliquot of CSF was sent immediately to the clinical laboratory for cytologic analysis. The remaining sample was divided into 0.5-mL aliquots and stored at -800C prior to proteomic or further biochemical analysis. Manual cell counts were performed on unspun CSF samples by a single laboratory technician (PF), who was blinded to the patient's clinical condition. Results are expressed as cells/mm3.

2) Proteomic Analysis

(a) Optimization of conditions for protein profiling of CSF using SELDI-TOF mass spectrometry - Applicants have previously used this same experimental paradigm to identify clinically relevant proteomic biomarkers in the amniotic fluid of patients with intra-amniotic inflammation (Buhimschi et al., WO 04/043238).

The first step in this analysis involved pooling 10-μL biological samples from the two clinically extreme groups, in this instance CSF from patients with severe preeclampsia (n=7) and controls (n=7). Serial dilutions of these two pooled CSF samples (1 :2 to 1 : 100) were incubated onto various ProteinChip® array surfaces (Ciphergen Biosystems, Fremont, CA) and under various binding conditions as previously described (Buhimschi et al., WO 04/043238; Weiner et al., Am J Obstet Gynecol 2004, in press). A dilution of 1:3 in binding buffer provided optimal signal- to-noise (S/N) ratios for detection of the ubiquitous non-biomarker peaks (data not shown), and was used in all subsequent experiments. The various array surfaces tested included reverse phase hydrophobic surface with C-16 long chain aliphatic residues (H4), strong anion exchanger carboxylate residues (SAX2), weak cation exchanger quaternary ammonium (WCX2), and metal affinity (IMAC3). For H4 chip surfaces, optimization involved additional hydrophobic binding/washing gradients from 10 to 75 percent acetonitrile. For SAX2, WCX2, and IMAC3 arrays, binding was tested at various pH (4.0, 6.0, 7.4, 8.0, and 10.0). For IMAC3, Applicants also tested affinity binding to metal anions (Zn2+, Cu2+, Ni2+, Cd2+, and Ga3+) by overlaying the array surface with two applications of 100-mM ZnSO4, CuSO4, NiSO4, CdSO4, or GaNO3, respectively. After a 1-hour incubation, the CSF sample was aspirated and the spots washed individually with 6 volumes of 10-μL of respective binding buffer, air-dried, and then overlaid with matrix solution diluted in 0.5 percent trifluoroacetic acid/50 percent acetonitrile. The matrix (energy absorbing molecule) consisted of either 1-μL of a 20 percent saturated solution of α-cyano-4-hydroxycinnamic acid (CHCA) or two sequential applications of 1-μL 50 percent saturated solution of sinnapinic acid (SPA). The chips were again air-dried and then read in a Protein Biology System® IIC SELDI-TOF mass spectrometer (Ciphergen Biosystems) using the ProteinChip® software v. 3.1. The resulting protein profile contained a fingerprint of the proteins optimally bound to the respective spots of the array separated by their mass/charge ratio (m/z). By varying the array surfaces, binding and washing conditions, metal affinities, and energy absorbing molecules, Applicants were able to create a multi- dimensional separation of proteins in a complex biological fluid. This approach is superior to experiments in which biological samples are simply dried on the array spots, resulting in a one-dimensional separation of the proteome.

Systematic manual analysis of SELDI tracings identified a region of interest at 14 to 17 kDa on the proteomic array, which resolved optimally on the IMAC3/Zn2+ arrays overlaid with SPA. Several visually conspicuous peaks were present in this mass region that appeared to differentiate between the "diseased" (severe preeclampsia) and "normal" sample pools. However, two separate pH binding/washing conditions (pH 4.0 and 7.4) were required to optimally visualize all of the discriminatory biomarker peaks in this region.

The second step consisted of running each of the 22 CSF samples individually under conditions established using the pooled specimens (above). Briefly, 4-μL of each sample was diluted 1 :3 with binding buffer (either phosphate buffered saline [PBS, pH 7.4] or 100-mM sodium acetate buffer [pH 4.0]), and 5-μL of each diluted CSF sample was applied to duplicate IMAC3 arrays pretreated with two sequential applications of 100-mM mM ZnSO4 for 15 minutes. Following a 1-hour incubation, unbound proteins were removed by washing each spot with the respective binding buffer. After air-drying, each spot was covered with two sequential layers of 1-μL 50 percent saturated SPA solution and the arrays read in the SELDI reader. This procedure required approximately 2 hours.

The third step was designed to optimize the experimental procedure and minimize the redundancy of two separate incubation conditions while maintaining the same accuracy. In brief, 5-μL of the individual undiluted CSF samples were rapid- dried onto spots of WCX2 protein chip arrays by placing the arrays onto a metal block heated to 370C, overlaid with two applications of 50 percent saturated SPA, and read in the SELDI reader (Ciphergen Biosystems). This dry on-chip protocol procedure, which required a total of 15 minutes, satisfactorily identified all discriminatory and non-discriminatory protein biomarkers identified initially on the IMAC3/Zn2+ arrays. A representative proteomic analysis is shown in Figure 1.

(b) Characterization and analysis of a discriminatory proteomic profile in the CSF of patients with preeclampsia - Having optimized the rapid dry on-chip protocol and SELDI analysis (above), individual CSF samples were analyzed in a blinded fashion by a single investigator (IAB). Samples were randomly assigned to the arrays. One spot on each array was used as a negative control (5-μL PBS alone). SELDI tracings for individual samples were analyzed using the Ciphergen ProteinChip software v. 3.1. Protein peaks were identified manually in each SELDI tracing within the 14 to 17 kDa region using the centroid peak detection tool built into the Ciphergen software. After baseline subtraction and normalization for total ion current, several parameters for each peak — including intensity (peak height at centroid), S/N ratio, mass (m/z-1), and area under the peak— were exported to an Excel spreadsheet for further analysis. The presence or absence of a peak in a given sample was determined objectively after comparison with background noise levels for each designated mass obtained from the tracings with PBS alone. The value of 1.5 x (average + 2 standard S deviations) of S/N ratio for each designated mass on tracings with PBS alone was taken as a cut-off to establish the presence or absence of a peak on SELDI tracings obtained with CSF samples. Using this approach, five distinct protein peaks (designated #1 through #5) were noted in the 14 to 17 kDa region of the CSF proteomic spectrum (Figure 2). As shown in Figure 2, peak #1 is present in all CSF 0 samples but not in the negative control (binding buffer), and was therefore used as a reference peak. Peaks #2 though #5, on the other hand, were present only in the CSF of patients with severe preeclampsia and not in the CSF of patients with mild preeclampsia or normotensive controls. These were therefore regarded as our four discriminatory protein peaks (biomarkers) of interest. Prior experience with proteomic 5 technology led one of the investigators (IAB) to suggest that peaks #3 and #5 likely represent posttranslational modifications (glycation) of the parent peaks #2 and #4, respectively.

To determine the utility of the four discriminatory proteomic biomarkers to separate patients with severe preeclampsia from those with mild preeclampsia or 0 normotensive controls, a preeclampsia proteomic biomarker (PPB) scoring system was developed using Boolean indicators in which a score of 0 was assigned if the peak was absent or a score of 1 if the peak was present. The PPB score therefore ranges from 0 (all peaks absent) to 4 (all peaks present). This approach (mass restrictive scoring) has been also used previously to identify discriminatory proteomic biomarker profiles for the diagnosis ofinflammation/infection in amniotic fluid (Buhimschi et al., WO 04/043238; Weiner et al., Am J Obstet Gynecol 2004, in press).

3) Identification of the Discriminatory Proteomic Biomarkers

(a) In-gel typtic digests - To identify the discriminatory protein biomarkers, one-dimensional SDS-PAGE electrophoresis was followed by in-gel tryptic digestion of excised bands as previously described (Buhimschi et al., WO 04/043238). Briefly, 0.1 -ml CSF from 2 samples with the highest S/N for the biomarker peaks and 2 control samples with absent biomakers but highest S/N for the reference peak were boiled with 25-μL loading buffer (200-mM Tris-HCl [pH 6.8], 12 percent SDS, 0.4 percent bromophenol blue, 40 percent glycerol) under non-reducing conditions for 5 minutes, and loaded onto an 18 percent SDS-PAGE gel. Low molecular weight markers (Ultralow Color marker, Sigma, St Louis, MO) and mass spectrometry molecular weight standards (0.5-nM mixture of equine myoglobin and cytochrome C [Ciphergen Biosystems]) were included. After staining with 0.1 percent Comassie Blue R250 (Sigma) in 40 percent methanol/10 percent acetic acid for 1 hour and destaining with 40 percent methanol/10 percent acetic, bands of interest were cut out, minced, and incubated with 50-μL of proteomics sequencing grade trypsin (Sigma) containing 0.2-μg en2yme in 0.4-mM HCl/25-mM ammonium bicarbonate at 37°C for 16h. Thereafter, 1.5-μL of each digest was dried onto WCX2 spots, overlaid with 1- μL of 10 percent saturated CHCA solution, and peptide maps read manually in the SELDI instrument (Ciphergen Biosystems). The identity of each protein band was confirmed by comparing the corresponding peptide profile with a database of tryptic digest peptide maps of known proteins as reported by Profound (http://prowl.rockefeller.edu) with matching into SwissProt (http://www.expasy.us).

(b) On-chip antibody capture assay - To confirm the identity of the discriminatory CSF protein biomarkers whose probable identity was revealed by in- gel tryptic digests, an on-chip antibody capture assay was performed. In this assay, CSF was incubated onto ProteinChip® arrays after antibodies had been covalently bound to spots, and bound proteins were subjected to SELDI-TOF mass spectrometry. Briefly, epoxy-coated PS20 ProteinChip® arrays were incubated for Ih with 5-μL of 1 mg/mL affinity purified sheep anti-human hemoglobin antibody (Bethyl Laboratories, Montgomery, TX) or 5-μL of lmg/mL sheep IgG (Sigma) as a negative control. The anti-human hemoglobin antibody recognizes both the a- and /3-chains of hemoglobin. After incubation, the arrays were washed with binding buffer (PBS containing 100-mM NaCl and 0.01 percent Triton X-IOO) and blocked with IM Tris (pH 9) for another hour. Thereafter, pretreated spots were incubated with 5-μL of serial dilutions of the CSF samples used for tryptic digests (1:1 to 1:10,000 in binding buffer) for Ih, washed again with binding buffer followed by 10-mM HEPES, allowed to air-dry, covered with two layers of 50 percent SPA5 and read in the SELDI instrument (Ciphergen Biosystems). The SELDI spectra were also compared with those obtained using the WCX2 dry on-chip protocol.

4) Quantitation of Biomarker and Non-Biomarker Proteins in Cerebrospinal S Fluid

(a) ELISA assays - To confirm our observation that PPB biomarkers represent free hemoglobin chains using traditional biochemical and immunologic tools, Applicants performed ELISA assays for human hemoglobin (our biomarkers of interest) and for cystatin C (the reference protein peak also identified in CSF by 0 tryptic digest and matched in SwissProt database).

For hemoglobin ELISA, microtiter plates (Immuno MaxiSorp, Nalge Nunc, Rochester, NY) were coated overnight with capture antibody (10-μg/mL sheep anti- human hemoglobin antibody [Bethyl Laboratories]). The plates were washed, blocked, and incubated with CSF samples at a dilution of 1 : 100 or hemoglobin 5 standards (Bethyl Laboratories) ranging from 6.25 to 1000 ng/mL. Detection was accomplished using a sheep anti-human hemoglobin antibody conjugated to horseradish peroxidase (1:50,000 dilution [Bethyl Laboratories]) and 3,3',5,5',- tetrarnethylbenzidine (Vector Laboratories, Burlingame, CA) as substrate. The color reaction was stopped with 2-M sulfuric acid, and plates were read at 450 nm with 650 nm wavelength correction. The inter- and intra-assay variabilities were less than 5 percent. The sensitivity of the assay was 0.6 ng.

For quantification of cystatin C, a commercially available ELISA kit (Biovendor, Brno, Czech Republic) was used. CSF samples were diluted (1:100) and assayed against human cystatin C standards ranging from 1 to 5000 ng/mL. The reported sensitivity is 0.2 ng and inter- and intra-assay variabilities are less than 7 percent.

(b) Direct spectrophotometry - Spectrophotometry of individual CSF samples was performed as previously described (Cruickshank, J Clin Pathol 2001, 54:827-830; Watson, Ann Clin Biochem 1998, 35:684-685). Data were compared with results of the PPB scoring system and hemoglobin ELISA. Briefly, 90-μL of each CSF sample was added in duplicate to wells of a microtiter plate, and optical density scanned from 300 to 700 nm using a Spectramax microtiter plate reader equipped with SoftmaxPro v.311 software (Molecular Devices, Sunnyvale, CA). Oxyhemoglobin alone produces an absorption peak at 413-415 nm, bilirubin alone produces a broad peak at 450-460 nm, and bilirubin together with oxyhemoglobin produce a shoulder at 450-460 nm on the downslope of the oxyhemoglobin peak (Beetham, J Neurol Neurosurg Psychiatry 2004, 75:528). For this reason, endpoint absorbance values were collected at 414 nm (for oxyhemoglobin, suggestive of recent bleeding) and at 455 nm (for oxyhemoglobin and bilirubin, suggestive of old bleeding). As a positive control and in an effort to estimate the amount of free hemoglobin in CSF in a semi-quantitative fashion, endpoint absorbance values were compared with values obtained by spiking a sample of CSF from a control patient with the content of a known number of lysed erythocytes per μL, prepared by hypotonic lysis of washed erythrocytes from peripheral venous blood of a healthy donor. Applicants thus obtained a surrogate CSF erythrocyte standard curve containing from 1 to 105 lysed erythrocytes/ μL that was subjected to direct spectrophotometry and ELISA assay as described. By plotting the assayed hemoglobin concentration versus the logarithm of lysed erythrocytes, Applicants were able to estimate roughly the number of erythrocytes that have to lyse into 1-mL of CSF to achieve the hemoglobin levels seen in the CSF of women with severe preeclampsia.

5) Functional Implications of Free Hemoglobin in Cerebrospinal Fluid

To further investigate whether the presence of free hemoglobin in the CSF serves simply as a marker of severe preeclampsia or whether it plays a direct role in the neurologic manifestations commonly seen in this condition, Applicants performed a series of experiments designed to look at known markers of inflammation and/or oxidative stress in the CSF.

(a) Interleukin-6 (IL-6) ELISA - IL-6 is a known marker of inflammation in various biologic models, and has been shown to be elevated in patients with subarachnoid hemorrhage (Takizawa et al., Neurol Res 2001, 23:724-30). IL-6 concentrations in individual samples of CSF from all 22 patients were measured using a commercially available ELISA (Endogen, Rockford, IL) with a reported sensitivity of 1-pg/mL and an inter- and intra-assay variabilities of less than 10 percent. (b) Measurements of vasoactive nitric oxide metabolites - The concentrations of total nitric oxide metabolites (NO2" and NO3") were measured in all CSF samples. Briefly, nitrates in the CSF were reduced to nitrites using acid washed spongy cadmium (Davison and Woof, Analyst 1978, 103:403-6), and total nitrites in the medium were measured based on the Greiss reaction (Green et al., Anal Biochem 1982, 126:131-8) optimized for microtiter plate assay in a prior study (Buhimschi et al., MoI Hum Reprod 2000, 6:404-14).

(c) Estimation of protein carbonylation - Protein carbonylation in individual CSF samples was estimated as a measure of oxidative modification as previously described (Levine et al., Methods Enzymol 1994, 233:346-357). Total protein concentration in the CSF was measured with a bicinchoninic acid/cupric sulfate reagent (BCA kit; Pierce, Rockford, IL). Duplicate CSF samples containing 10-μg of total CSF protein were evaporated to dryness using a SpeedVac and derivatized with 2,4-dinitrophenylhydrazine (DNPH) or derivatization control solution using an OxyBlot kit (Serologicals Corporation, Norcross, GA). Proteins with incorporated dinitrophenyl residues were electrophoresed on 10 percent SDS-PAGE gels, transferred to PVDF membranes, and detected using Western blot analysis after a primary incubation with a rabbit anti-DNP antibody, a secondary incubation with peroxidase-conjugated secondary antibody, and ECL chemiluminescent detection (Amersharm, Piscataway, NJ). Autoradiographs were scanned and analyzed with Image J v. 1.31 digital image-analysis software (NIH, Bethesda, MD). Images were compared with quantitative standards in which known amounts of DNP residues are incorporated in a standard mixture of proteins on a dot blot processed along with the membranes with electrophoresed proteins. The levels of protein oxidation are reported in frnol of DNP per μg protein.

6) Data Presentation and Statistical Analysis

The results are presented as mean and 95 percent confidence interval (continuous variables) or median and ranges (categorical variables). All data sets were tested for normal distribution using Kolmogorov-Smirnov test. Statistical comparisons were performed using t-tests or one-way analysis of variance (ANOVA) followed bypost hoc Tukey test with p values of less than 0.05 indicating a statistically significant difference. Non-normally distributed data sets were logarithmically transformed prior to statistical testing. Receiver operating characteristic curve (ROC) curve analysis was done with MedCalc (Broekstraat, Belgium).

INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.