HK1240319A1 - Biomarkers for assessment of preeclampsia - Google Patents

Description

Biomarkers for assessing preeclampsia

Cross Reference to Related Applications

The present application claims priority from U.S. application No.14/341, 024, BIOMARKERS for serving OF foregilmpsia filed on 25/7/2014, the disclosure OF which is incorporated herein by reference in its entirety.

Technical Field

Some embodiments herein relate to the field of fetal/maternal health screening tools, and more specifically to biomarkers for assessing preeclampsia.

Background

Preeclampsia is a potentially life-threatening complication specific to pregnancy and occurs in up to 7% of all pregnancies. Hypertensive disorders, including preeclampsia, are the second leading cause of maternal mortality worldwide and contribute to 10% to 25% of all maternal deaths. Unfortunately, clinical manifestations of preeclampsia may appear late in the disease and may be associated with adverse maternal and neonatal outcomes. Robust biomarkers for screening, diagnosis and monitoring, particularly with respect to severe preeclampsia, are necessary for proper control of preeclampsia and to mitigate adverse outcomes. This is especially true in developing countries, where the disease burden is greatest and medical intervention is often ineffective due to late appearance. Furthermore, the incidence of preeclampsia has increased since 1990, which may be directly related to the increase in obesity. Early and robust diagnostic tests are urgently needed to provide appropriate screening categories (triages) for professional medical facilities and control of preeclampsia.

Drawings

Some embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. Some embodiments are illustrated by way of example, and not by way of limitation, in the figures of the accompanying drawings.

Fig. IA-ID are tables illustrating the following, according to various embodiments: the GlyFn serum biomarker concentrations in the longitudinal cohort according to preeclampsia status and third trimester (fig. 1A), the total fibronectin serum biomarker concentrations in the longitudinal cohort according to preeclampsia status and third trimester (fig. 1B), the average weekly variation of GlyFn concentrations in all cohorts according to weekly and preeclampsia status (fig. 1C), and the average weekly variation of total serum fibronectin concentrations in all cohorts according to weekly and third trimester (fig. 1D);

fig. 2 is a table illustrating maternal characteristics in terms of preeclampsia status and cohort, according to various embodiments;

fig. 3 is a graph illustrating that GlyFn levels are significantly higher in patients with preeclampsia at early gestation (1st trimester), mid gestation (2 d trimester), and late gestation (3rd trimester) where GlyFn levels are measured in sera from 45 normotensive control subjects (circles and solid lines) and 62 preeclamptic subjects (plus and dashed lines), according to various embodiments;

figures 4A and 4B are tables showing serum biomarker concentrations in longitudinal cohorts according to preeclampsia status and third trimester of pregnancy (figure 4A), and graphs showing GlyFn concentrations throughout pregnancy in all cohorts (figure 4B), according to various embodiments;

figures 5A and 5B are tables showing serum biomarker concentrations in normotensive cohorts and clinical preeclampsia cohorts (figure 5A), and graphs showing mid-and late-gestation GlyFn concentrations in normotensive control and clinical preeclampsia patients (figure 5B), according to various embodiments;

figures 6A and 6B are tables showing the mean weekly variation of GlyFn concentrations as a function of weekly and preeclamptic status (figure 6A), and receiver operational profiles showing the performance of the classification of late gestation preeclampsia of biomarkers in all cohorts (figure 6B), according to various embodiments;

fig. 7 is a table showing the performance of classification of late gestational preeclampsia of biomarkers in all cohorts according to various embodiments;

fig. 8 is a table showing GlyFnPOC values for predicting preeclampsia at different prevalence estimates, according to various embodiments;

FIG. 9 is a table showing the relationship of GlyFn levels in late gestation with clinical features and outcome in normotensive and clinical preeclampsia, according to various embodiments; and

fig. 10A and 10B illustrate schematic diagrams of one example of a lateral flow immunoassay (fig. 10A) and a lateral flow testing device (fig. 10B) that can be used according to various embodiments disclosed herein.

Detailed Description

In the following detailed description, reference is made to the accompanying drawings which form a part hereof, and in which is shown by way of illustration some embodiments which may be practiced. It is to be understood that other embodiments may be utilized and structural or logical changes may be made without departing from the scope. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of some embodiments is defined by the appended claims and their equivalents.

Various operations may be described as multiple discrete operations in turn, in a manner that is helpful in understanding some embodiments; however, the order of description should not be construed as to imply that these operations are order dependent.

The description may use perspective-based descriptions such as up/down, back/front, and top/bottom. Such descriptions are merely used to facilitate the discussion and are not intended to limit the application of the disclosed embodiments.

The terms "coupled" and "connected," along with their derivatives, may be used. It should be understood that these terms are not intended as synonyms for each other. Rather, in particular embodiments, "connected" may be used to indicate that two or more elements are in direct physical or electrical contact with each other. "coupled" may mean that two or more elements are in direct physical or electrical contact. However, "coupled" may also mean that two or more elements are not in direct contact with each other, but yet still co-operate or interact with each other.

For the purposes of this description, a phrase in the form "A/B" or "A and/or B" means (A), (B), or (A and B). For the purposes of this description, a phrase in the form of "at least one of A, B and C" means (A), (B), (C), (A and B), (A and C), (B and C), or (A, B and C). For the purposes of this description, a phrase in the form of "(a) B" means (B) or (AB), i.e., a is an optional element.

The description may use the terms "embodiment" or "some embodiments," each of which may refer to one or more of the same or different embodiments. Furthermore, the terms "comprising," "including," "having," and the like, as used with respect to embodiments, are synonymous.

Disclosed herein, in various embodiments, are methods for assessing risk of, predicting, diagnosing, and monitoring preeclampsia in a subject. The methods disclosed herein may also be used to distinguish between mild preeclampsia and severe preeclampsia. Also disclosed are methods of predicting or diagnosing low birth weight and/or HELLP syndrome, life-threatening complications of preeclampsia involving hemolysis, elevated liver enzymes, and low platelet count. In various embodiments, the methods may comprise measuring the level of glycosylated fibronectin (GlyFn) in a biological sample (e.g., a serum sample) from the subject. Although serum samples are described herein, one skilled in the art will appreciate that the disclosed methods may be adapted for use with other biological samples (e.g., whole blood, plasma, urine, saliva, or other bodily fluids).

Fibronectin (Fn) is a rich protein with a broad spectrum of functions. Exemplary human fibronectinAccession number is Genbank accession No. p02751. Fn gene coding sequences and some isoforms of different lengths as a result of alternative splicing and proteolysis. Most of the Fn present in serum or plasma, known as plasma Fn (pfn), is produced and secreted by hepatocytes in soluble form, while the so-called cells Fn (cfn) are produced by many cell types, including fibroblasts, endothelial cells, and smooth muscle cells. The main difference between pFn and cFn is the presence of alternatively spliced additional domains a and B (ECA/B), which are absent in pFn but variably present in cFn. It is becoming increasingly clear that cFn is also found in the circulation, especially in a variety of pathological conditions including diabetes and inflammation. pFn and cFn both exhibit complex glycosylation patterns, and elevated levels of specific glycosylated forms of Fn (Fn-SNA) in maternal serum have been shown to be predictive of gestational diabetes. However, prior to the present disclosure, it was not known that elevated GlyFn correlates with preeclampsia.

As shown herein, while GlyFn may be used to assess preeclampsia and related conditions, total fibronectin levels are not correlated with preeclampsia risk. Fig. 1A-1D are tables illustrating the following, according to various embodiments: the GlyFn serum biomarker concentrations in the longitudinal cohort according to preeclampsia status and third trimester (fig. 1A), the total fibronectin serum biomarker concentrations in the longitudinal cohort according to preeclampsia status and third trimester (fig. 1B), the average weekly variation of GlyFn concentrations in all cohorts according to weekly and preeclampsia status (fig. 1C), and the average weekly variation of total serum fibronectin concentrations in all cohorts according to weekly and preeclampsia status (fig. 1D). Thus, the rate of change of total fibronectin levels (fig. 1B) and total fibrin levels (fig. 1D) was not predictive of preeclampsia status. In contrast, as discussed in more detail below, both the level of GlyFn (fig. 1A) and the weekly rate of change of gestational GlyFn level (fig. 1C) can be used to assess, predict and/or diagnose preeclampsia and related conditions.

In various embodiments, the disclosed methods comprise obtaining a biological sample, such as a serum sample, a whole blood sample, a plasma sample, or a saliva sample, from a pregnant subject. The level of glycosylated fibronectin (GlyFn) in the sample is then determined using any of several possible methods, and the level of GlyFn in the sample is compared to a control value (e.g., a reference value representing the level of GlyFn that is commonly seen in subjects who will not continue to develop preeclampsia). In various embodiments, the subject may be classified as not having preeclampsia or as being at low risk for developing preeclampsia if the level of GlyFn in the sample is determined to be similar to a control value (e.g., when there is no statistically significant difference between the sample GlyFn value and a reference value, or when the sample GlyFn value is determined to be within a range defined as "normal"). However, when the sample GlyFn value is determined to be elevated relative to the control value (e.g., elevated to a statistically significant degree or outside a predetermined "normal" value range relative to the control value), then the subject can be determined to have, or be at high risk for developing, preeclampsia.

More specifically, in some embodiments, methods for determining the risk of preeclampsia in a subject early in pregnancy are disclosed. In these embodiments, the control value may be a reference value (or range of "normal" values) representing the level of GlyFn in a sample from a subject in an early stage of pregnancy that does not continue to develop preeclampsia. In some embodiments, an "increase" in the level of GlyFn in a sample relative to an early pregnancy control value can be at least 15% increase, e.g., 20% increase, 30% increase, 40% increase, 50% increase, 60% increase, 70% increase, 80% increase, 90% increase, 100% increase, or even higher, e.g., 125% increase or 150% increase. For example, the normal (non-preeclampsia) range of GlyFn in a sample from a subject at an early stage of pregnancy may be 10 to 150 μ g/ml, and abnormal (e.g., preeclampsia) levels of GlyFn may be greater than 150 μ g/ml, such as about 175 μ g/ml or higher.

In other embodiments, the method may be a method of determining the risk of preeclampsia in a subject in the middle of gestation. In these embodiments, the control value may be a reference value (or range of "normal" reference values) representing the level of GlyFn in a sample from a subject in mid-pregnancy that does not continue to develop preeclampsia. In some embodiments, an "increase" in the level of GlyFn in a sample relative to a mid-gestation control value can be at least a 15% increase, e.g., a 20% increase, a 30% increase, a 40% increase, a 50% increase, a 60% increase, a 70% increase, an 80% increase, a 90% increase, a 100% increase, or even higher, e.g., a 125% increase or a 150% increase. For example, the normal (non-preeclampsia) range of GlyFn in samples from subjects in the middle of gestation may be 10 to 150 μ g/ml, and abnormal (e.g., preeclampsia) levels of GlyFn may be greater than 150 μ g/ml, such as about 175 μ g/ml or higher.

In still other embodiments, the method may be a method of determining the risk of preeclampsia in a subject in late gestation. In these embodiments, the control value may be a reference value (or range of "normal" reference values) representing the level of GlyFn in a sample from a subject in a later stage of pregnancy that does not continue to develop preeclampsia. In some embodiments, an "increase" in the level of GlyFn of a sample relative to a late-gestation control value may be at least a 30% increase, e.g., a 40% increase, a 50% increase, a 60% increase, a 70% increase, an 80% increase, a 90% increase, a 100% increase, a 125% increase, a 150% increase, or even higher, e.g., a 200% increase or a 300% increase. For example, the normal (non-preeclampsia) range of GlyFn in samples from subjects at early gestation may be 10 to 150 μ g/ml, and abnormal (e.g., preeclampsia) levels of GlyFn may be greater than 150 μ g/ml, e.g., about 200 μ g/ml or higher.

Further embodiments may be a method of assessing the risk of low birth weight, a method of assessing the risk of HELLP syndrome or a method of diagnosing preeclampsia in a subject. For example, in some embodiments, the subject may be in the late gestation stage of pregnancy, and a level of GlyFn in a serum sample from the subject equal to or greater than about 100 μ g/ml, e.g., about 110 μ g/ml, about 120 μ g/ml, about 130 μ g/ml, about 140 μ g/ml, about 150 μ g/ml, about 160 μ g/ml, about 170 μ g/ml, about 180 μ g/ml, about 200 μ g/ml, or even more, may indicate that the subject has preeclampsia. In various embodiments, a level of GlyFn in a serum sample from the subject equal to or greater than about 250 μ g/ml, e.g., about 275 μ g/ml, about 300 μ g/ml, about 325 μ g/ml, about 350 μ g/ml, about 375 μ g/ml, about 400 μ g/ml, about 425 μ g/ml, about 450 μ g/ml, about 475 μ g/ml, about 500 μ g/ml, or even more, may indicate that the subject is at risk of low birth weight (less than gestational age or SGA) infants or developing HELLP syndrome. In some particular embodiments, a level of GlyFn equal to or greater than about 500 μ g/ml or even higher in a serum sample from the subject may indicate that the subject has a low birth weight (SGA) infant or a high risk of developing HELLP syndrome.

Still other embodiments are methods of distinguishing between mild preeclampsia and severe preeclampsia. In these embodiments, the method may further comprise obtaining at least one additional serum sample from the subject at least one week after obtaining the first serum sample, e.g., a series of weekly samples between weeks 33 and 38 of gestation. In various embodiments, the method may comprise determining the level of glycosylated fibronectin (GlyFn) in the second serum sample, and comparing the level of GlyFn in the second serum sample to a previously determined level of GlyFn in the first serum sample. In various embodiments, a weekly increase in the level of GlyFn in the second (or subsequent) serum sample, as compared to the level of GlyFn in the first (or previous) serum sample, may indicate that diagnosis of preeclampsia is warranted. More specifically, a weekly increase of about 15 μ g/ml to 125 μ g/ml, such as about 25 μ g/ml, about 35 μ g/ml, about 45 μ g/ml, about 55 μ g/ml, about 65 μ g/ml, about 75 μ g/ml, about 85 μ g/ml, about 95 μ g/ml, about 105 μ g/ml, about 115 μ g/ml or about 125 μ g/ml, may indicate that the subject has mild preeclampsia. Likewise, in various embodiments, a weekly increase of more than about 150 μ g/ml, such as about 175 μ g/ml, about 200 μ g/ml, about 225 μ g/ml, about 300 μ g/ml, about 325 μ g/ml, about 350 μ g/ml, about 375 μ g/ml, about 400 μ g/ml, or about 425 μ g/ml or more, may indicate that the subject has severe preeclampsia.

Statistical methods for determining whether the abundance of a protein of interest increases or decreases relative to a reference sample are well known in the art and described below. In various embodiments, the determination of the level of GlyFn in a biological fluid (e.g., whole blood, plasma, serum, saliva, or urine) can be performed using a variety of methods known to those skilled in the art. In various embodiments, in a direct comparative analysis, the reference and test samples can be processed in exactly the same manner in order to correctly represent the relative abundance of GlyFn and obtain accurate results.

For example, in various embodiments, the separation can be performed by 2D-gel electrophoresis based on the charge and molecular weight of the proteins present in the biological sample. For example, separation can be first performed by the charge of the protein using isoelectric focusing (one-dimensional gel electrophoresis), for example using a commercially available Immobilized PH Gradient (IPG) strip. In various embodiments, the second dimension may be an SDS-PAGE analysis, wherein a focused IPG band may be used as a sample. After separation by two-dimensional gel electrophoresis, the proteins can then be visualized with conventional dyes (e.g., coomassie blue or silver stain) and using known techniques and equipment (e.g., Bio-RadGS800 densitometer and PDQUEST)^TMSoftware) imaging.

In some embodiments, individual spots can then be excised from the gel, destained and trypsinized, and the peptide mixture analyzed by Mass Spectrometry (MS). Alternatively, in some embodiments, the peptides may be isolated by, for example, capillary High Pressure Liquid Chromatography (HPLC), and may be analyzed by MS, either alone or in pools (pools). If desired, in some embodiments, the amino acid sequence of the peptide fragment and the protein from which it is derived can be determined. Although all or some of the proteins present in the proteomic profile can be identified and sequenced, this is generally not necessary for diagnostic use of the methods disclosed herein.

As generally discussed above, in various embodiments, preeclampsia or its risk may be diagnosed based on characteristic similarities or differences between a reference sample and a test sample. For example, in various embodiments, if the proteomic profile is presented in the form of a mass spectrum, the expression signature can be a peak representing a GlyFn that is qualitatively or quantitatively different from the mass spectrum of the corresponding normal sample. Thus, any statistically significant change in the amplitude or shape of an existing peak may reflect a change in GlyFn levels relative to the control.

Other embodiments may utilize protein arrays to monitor GlyFn levels, thereby enabling high throughput analysis. Protein arrays are known to those skilled in the art and are typically formed by immobilizing proteins (e.g., antibodies specific for a protein of interest (e.g., GlyFn)) on a solid surface (e.g., glass, silicon, nitrocellulose, or PVDF) using any of a variety of covalent and non-covalent attachment chemistries well known in the art. The array can be probed with fluorescently labeled proteins from two different sources (e.g., normal and test samples) and the fluorescence intensity can reflect the expression level of the target protein (e.g., GlyFn).

Various embodiments may also use any of a variety of immunoassay formats for quantifying protein expression levels. Generally, immunoassays can be homogeneous or heterogeneous. For example, in various embodiments, enzyme-linked immunosorbent assays (ELISAs) can be used to quantify protein expression. In one example, in a "sandwich" assay, a solid surface may be coated with a solid phase antibody and the test sample may be reacted with the bound antibody. Any unbound antigen can then be washed away and a known amount of enzyme-labeled antibody can then be reacted. The label can then be quantified as a direct measurement of the amount of the protein of interest present in the sample.

In some embodiments, ELISA may also be used as a competitive assay. For example, in a competitive assay, a test sample containing a protein of interest can be mixed with a precise amount of an enzyme-labeled protein of interest, and both can compete for binding with an antibody attached to a solid surface. In various embodiments, excess free enzyme-labeled protein may be washed away prior to addition of the enzyme's substrate, and the intensity of the color produced by the enzyme-substrate interaction may be used as a measure of the amount of protein of interest in the test sample.

Various other embodiments may quantify the protein of interest using Enzyme amplified immunoassay (EMIT) which may include a test sample, an Enzyme-labeled molecule for the protein of interest, an antibody specific for the protein of interest, and a specific Enzyme chromogenic substrate. In various embodiments, an excess of specific antibody may be added to the test sample, and the protein of interest may then bind to the antibody. In various embodiments, a measured amount of the corresponding enzyme-labeled protein may then be added to the mixture, and the antibody binding sites not occupied by the protein of interest from the test sample may be occupied by molecules of the enzyme-labeled protein. Thus, in various embodiments, enzyme activity may be reduced because only free enzyme-labeled protein may act on the substrate, and the amount of converted substrate may reflect the amount of free enzyme remaining in the mixture. In various embodiments, a high concentration of the protein of interest in the sample can result in a higher absorbance reading.

Various other embodiments include immunoassay kits for quantifying a protein of interest in a test sample. In various embodiments, these kits may comprise, in separate containers, one or more monoclonal or polyclonal antibodies having binding specificity for GlyFn and optionally anti-antibody immunoglobulin (particularly labeled anti-antibody immunoglobulin).

Also disclosed herein are capture devices and sample collection kits for use in the disclosed methods. In some embodiments, the disclosed methods can be performed using a sample capture device, such as a lateral flow device (e.g., a lateral flow test strip) that can allow for quantitation of GlyFn. Lateral flow devices are available in a variety of different configurations, but in one example, the test strip may comprise a flow path from an upstream sample application zone to a test site, e.g., from the sample application zone through the flow zone to a capture zone. In various embodiments, the flow region can contain a flowable marker that can interact with a protein of interest, and the capture region can contain reagents that bind to the protein of interest for detection and/or quantification. In other embodiments, an exemplary sample collection kit may comprise an absorbent medium (e.g., filter paper) that may comprise a label for placing a test sample on the medium. Such kits may also comprise a lancing device for obtaining a blood sample from a subject, and optionally a mail box (mailer) for sending the test sample to a doctor or laboratory for analysis. Such sample collection kits may be used, for example, during standard prenatal testing (e.g., eight weeks, twelve weeks, sixteen weeks, twenty four weeks, twenty-eight weeks, thirty weeks) or a follow-up one week thereafter, and/or sample collection may be performed while obtaining blood for other standard prenatal testing.

The following examples are provided for illustrative purposes and should not be construed as being limiting in any way.

Examples

Example 1: object selection

Study participants were recruited from both patient populations. The vertical cohort consisted of 60 women who were continuously sampling throughout the pregnancy, taking the first sample between 6 and 14 weeks of gestation, and obtaining additional samples at each of the third trimester. At various gestational ages, 45 women remained normotensive and 15 had developed preeclampsia. The clinical preeclampsia cohort of 47 patients diagnosed with preeclampsia at various gestational ages was analyzed to measure the rate of change of GlyFn levels during their preeclampsia. Preeclampsia status is defined as systolic pressure ≥ 140mmHg or diastolic pressure ≥ 90mmHg, proteinuria ≥ 300 mg/day. 207 serum samples were analyzed using a plate assay, and 86 serum samples were also analyzed using a GlyFn point-of-care (POC) device. 26 participants included in the analysis had one measurement, 62 had two measurements, and 19 had three measurements.

Study participants were recruited from the gynecology and obstetrics department of the Olympic university Hospital, Olympic, Finland, and the Finnish pregnant and lying-in women cohort serum bank, national institute of health and welfare, in 2004 to 2006. The study protocol was approved by the ethics committee of the university of allusion hospital, providing informed consent for all participants.

Example 2: analyte determination

Maternal blood was spun, aliquoted, and stored at-80 ℃ until the following assay was performed.

GlyFn is determined: the reaction-Bind plates (Thermo Scientific, Rockford, IL) were coated with Fc fragment-specific goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, Pa.; Cat #115-005-071) in carbonate buffer pH 9.6 and incubated overnight at 4 ℃ before washing with PBS-0.05% Tween 20. The plates were blocked with 3% bovine serum albumin in Phosphate Buffered Saline (PBS) pH 7.2 and held at room temperature for 1 hour. The plates were then washed with PBS-0.05% tween 20 buffer and anti-GlyFn monoclonal antibody was added and incubated for 45 min at room temperature. Although a specific antibody is used in this example, one skilled in the art will appreciate that any antibody specific for glycosylated (rather than unglycosylated) fibronectin may be used. In addition, other specific binding agents that specifically bind glycosylated (rather than non-glycosylated) fibronectin (e.g., lectins) can be used in similar assays to detect and measure GlyFn. In general, the antibodies used in the methods and devices of the present disclosure can be monoclonal or polyclonal. By way of example only, monoclonal antibodies can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (Nature 256: 495-497, 1975) or derivatives thereof. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (Antibodies, laboratory Manual, CSHL, New York, 1988).

The sample and the standard protein (human Fn, R isolated from serum)&D Systems catalog #1918-FN-02M), washed, and biotinylated anti-human FN polyclonal antibody was added. Labeling was performed using high sensitivity streptavidin-horseradish peroxidase (HRP) (Thermo Scientific; catalog No. 21130). After 45 min incubation, the plates were then washed with PBS-0.05% Tween 20 buffer, developed with 100. mu.l of K-Blue TMB substrate (Neogen, Glasgow; Cat #304177), and added by 2N H₂SO₄And (4) quenching.

GlyFn POC assay: a fluorescence immunoassay was developed that included an automated cassette Reader (LRE Medical poc Reader, lremidication, Oceanside, CA) and a disposable single-use plastic assay cassette that used standard immunoassay techniques to specifically and quantitatively detect GlyFn in serum samples. Polyclonal anti-Fn antibody and Tide Fluor used in the above plate assay^TMA5 WS succinimidyl ester fluorescent tag (AAT Bioquest, Sunnyvale, CA; Cat #2281) was conjugated and used as a detection antibody. The monoclonal anti-GlyFn antibody used in the above plate assay was used as a capture antibody and immobilized on a solid phase (test zone). Goat polyclonal anti-rabbit IgG, Fc antibody (Jackson immunoresearch laboratories, inc.; catalog No. 111-045-046) was immobilized in a separate capture zone to serve as a reference for the test zone and provide assurance that the device performed properly.

Serum was diluted in assay buffer and applied to test strips. The serum flows down the diagnostic channel by capillary action, allowing the fluorescent detection antibody to enter the suspension. The GlyFn in the specimen binds to the fluorescent antibody to form a multivalent complex captured by the antibody immobilized in the test zone. The cartridge is inserted into the cartridge reader and after 10 minutes quantitative measurements of glyFn concentration in the range of 10g/mL to 2000g/mL are shown and/or printed out on the meter screen.

Levels of sFlt1 were determined by ELISA. Since the assay requires a large amount of serum, 13 participants were unable to determine the analyte. PlGF levels were determined using a commercial kit (R & D Systems Human PlGFQuantikine ELISA kit; catalog # DPG 00). Due to the lack of serum samples, 57 subjects performed the analysis. Plates were read at 450nm using an Epoch plate reader (BioTek, Winooski, VT) and data were processed using Gen5 software version 1.10.8 and analyzed as described below.

Example 3: statistical analysis

Normotensive, longitudinal preeclampsia, and clinical preeclampsia samples were analyzed separately and only combined or compared where indicated. Maternal characteristics between the study groups were compared using Kruskal-Wallis non-parametric ANOVA for continuous variables and Fisher's exact test for categorical variables. The age-matched measurements for each subject in gestational period three were used to compare the level of GlyFn between longitudinal participants with and without preeclampsia using the non-parametric wilcoxon t-test (non-parametric). To analyze the sFlt1, PlGF, and sFlt1/PlGF ratios, samples were grouped into late gestation and the normotensive group was compared to the clinical preeclampsia cohort by the non-parametric Wilcoxon t test. To assess changes in GlyFn levels in participants with mild preeclampsia and severe preeclampsia, a subset of patients with two replicate measurements for 33 to 38 weeks was used to calculate the average weekly change.

For each subject of all cohorts, a Receiver Operating Characteristic (ROC) curve was generated using the probabilities predicted by logistic regression models from 14 to 40 weeks gestation using a single age-matched measurement. Simple logistic regression was used to calculate the area under the ROC curve (AUROC) and the corresponding 95% confidence interval. Sensitivity and specificity were reported based on the selected threshold, and 95% confidence limits calculated by scoring with continuity correction were reported. A statistical test of the differences in the ROC curves was calculated using the comparison matrix of differences. Hypothetical predictive values and 95% Confidence Intervals (CI) were calculated using a population prevalence of 3%, 5%, or 7% using standard logit methods.

Post hoc analysis was performed relative to GlyFn values with maternal and fetal clinical characteristics and outcome. In all cohorts, GlyFn was compared with gestational age at birth, birth weight, systolic and diastolic blood pressure. In clinical preeclamptic participants, GlyFn was compared to gestational age at onset of preeclampsia, uric acid, ALAT, proteinuria, HELLP syndrome, gestational age-less and placental insufficiency. For continuous variables, Pearson correlation coefficient (Pearson correlation coefficient) and linear regression slope are calculated. For interpretability, the linear regression slope was calculated to reflect the change in GlyFn of 100 μ g. For the categorical variables, the Fisher exact test was performed by classifying participants as those with or without GlyFn levels ≧ 500 μ g.

Comparison of GlyFn plate assays to GlyFn POC was performed on samples assayed by both methods to assess the ability of the POC test to distinguish between participants with and without preeclampsia and to assess the ability of GlyFn to monitor the progression of preeclampsia in the middle and late gestation. A correlation coefficient is calculated to compare the two measurements. ROC curves were generated for GlyFn plates and POC data, respectively, for classifying PE versus control, control versus mild PE, and mild versus severe PE, and AUROC for each was compared between GlyFn plates and POC assays. The reported P values are two-sided, with P < 0.05 considered statistically significant. The statistical analysis was performed using SAS software of SAS system of Windows, version 9.3.

Example 4: GlyFn for assessment of preeclampsia

Figure 2 is a table illustrating maternal characteristics according to preeclampsia status and cohort, according to various embodiments. Patients in the clinical preeclampsia group delivered earlier (p < 0.01) and had lower neonatal birth weights (p < 0.01; FIG. 2). There was no difference between maternal age (p ═ 0.14) and unproductive (0.31) between cohorts. The mean gestational age at which preeclampsia was diagnosed in the longitudinal preeclampsia group was significantly later than the clinical preeclampsia cohort.

Fig. 3 is a graph showing a comparison of longitudinal normotensive and preeclamptic groups, showing that GlyFn levels in preeclamptic patients are significantly higher than controls within each of the third trimester of pregnancy, and fig. 4A and 4B are tables showing the following, in accordance with various embodiments: serum biomarker concentrations in the longitudinal cohort according to preeclampsia status and third trimester (fig. 4A), and a plot of GlyFn concentrations throughout gestation in the longitudinal cohort (fig. 4B). Comparison of the longitudinal normotensive and preeclamptic groups revealed that GlyFn levels were significantly higher in preeclamptic patients within each of the third trimester of pregnancy than in the control (p < 0.01, FIGS. 3, 4A and 4B).

Fig. 5A and 5B are tables showing serum biomarker concentrations in normotensive and clinical preeclampsia cohorts (fig. 5A), and graphs of mid-and late-gestation GlyFn concentrations in normotensive control and clinical preeclampsia patients (fig. 5B), according to various embodiments. To assess changes in serum biomarkers in late gestation, levels of GlyFn, sFLt1, PlGF, and sFLt1/PlGF ratios from age-matched samples of blood pressure normal controls and clinical preeclampsia cohorts were compared. All serum biomarkers differed significantly (p < 0.01) between participants with or without preeclampsia.

Fig. 6A and 6B are tables showing the mean weekly variation of GlyFn concentration as a function of week and preeclampsia status (fig. 6A), and receiver operational profiles showing the performance of the classification of late gestation preeclampsia of biomarkers in all cohorts (fig. 6B), according to various embodiments. Repeated measurement analysis of biomarker changes over time found no significant change in GlyFn (p ═ 0.83) throughout the gestational period in the control. In patients with preeclampsia, the weekly change between 33 and 38 weeks was 81.7(SE 94.1) μ g/ml for participants with mild preeclampsia and 195.2(SE 88.2) μ g/ml for participants with severe preeclampsia.

Fig. 7 is a table showing the advanced preeclampsia classification performance for all biomarkers in cohort, and fig. 8 is a table showing GlyFn POC values for predicting preeclampsia at different prevalence estimates, according to various embodiments. The clinical utility of these biomarkers for the detection of preeclampsia was tested by ROC curves. AUROC for GlyFn, sFlt1, PlGF, and aFlt1/PlGF ratios are shown in FIG. 7. Since the sFlt1 assay required significant serum levels, the assay was limited to 15 controls during 20 to 39 weeks gestation and 39 preeclamptic participants who had sufficient serum for the sFlt1 assay. AUROC for GlyFn is 0.99 and tends to be significantly different from that of sFlt1 (AUROC: 0.96, p ═ 0.11) and PlGF (AUROC: 0.94, p ═ 0.10). GlyFn shows a sensitivity of 0.97(0.85 to 1.00) and a specificity of 0.93(0.66 to 1.00) when classified at a threshold of 176.4. mu.g/ml. At this threshold, the population prevalence of preeclampsia was estimated to be 5%, with positive and negative predictive values of 47% (95% CI: 23-72%) and 89% (95% CI: 80-98%), respectively (FIG. 8).

Fig. 9 is a table illustrating the relationship of late gestation GlyFn levels to clinical characteristics and outcome in normotensive and clinical preeclampsia, according to various embodiments. Thus, the results of post hoc analysis of maternal and fetal fates with GlyFn are shown in fig. 9. GlyFn values have a significant linear relationship with gestational age at parturition, birth weight, blood pressure, uric acid and ALAT. For each 100 μ g/ml increase in GlyFn, a 0.59 week (4 days) decrease in gestational age at birth (p < 0.01), 129.4 grams of birth weight decrease (p < 0.01), 1.39mm/Hg systolic (p ═ 0.04), 1-14mm/Hg diastolic (p ═ 0.01), 13.6 μmol/L uric acid increase (p < 0.01), and 5.88U/L ALAT increase (p < 0.01) are predicted. GlyFn is not significantly associated with gestational age (p 0.27) or proteinuria (p 0.68) in the diagnosis of preeclampsia.

For participants with preeclampsia, there was a significant relationship between infants with small gestational age and GlyFn levels ≧ 500 μ g/ml. Of the preeclamptic patients with GlyFn levels < 500. mu.g, 8% (1/32) had infants with a small gestational age, whereas for preeclamptic patients with GlyFn levels ≧ 500. mu.g/ml, 26% (6/24) was small for gestational age (p ═ 0.03). Patients with high GlyFn levels and HELLP or placental insufficiency were not significantly associated with preeclampsia (p ═ 0.13 and p ═ 0.27, respectively); however, a higher percentage of women with GlyFn levels ≧ 500 μ g/ml develop HELLP (26% versus 8%; p ═ 0.13) and placental insufficiency (26% versus 12%; p ═ 0.27).

The results of the GlyFn plate assay were compared to the GlyFn POC assay on a subset of samples. There was a strong correlation between the plates and the POC assay (r ═ 0.76, p < 0.01). ROC curves were generated for both methods and AUROC was similar between plate (AUROC 0.99, 95% CI: 0.99 to 1.00) and POC (AUROC 0.93, 95% CI: 0.85 to 1.00) assays. For POC and plate assays, ROC curves were generated to compare the ability to distinguish mild and severe PE, and POC outperforms plate assays (AUROC 0.78 versus 0.68).

Example 5: GlyFn as biomarker of preeclampsia

GlyFn is shown herein to be a powerful biomarker for preeclampsia, and thus, GlyFn may be used in a variety of assessment and diagnostic methods, such as assessing the progression of preeclampsia over time based on elevated levels in maternal serum at an early stage of pregnancy, as well as monitoring the progression throughout pregnancy in order to predict or diagnose preeclampsia in a subject. As disclosed herein, elevated GlyFn levels are associated with important clinical features and outcomes, including early childbirth, reduced birth weight, and elevated blood pressure, uric acid, and ALAT. GlyFn is a uniquely useful analyte for monitoring preeclampsia, as GlyFn levels remain constant in controls throughout pregnancy. The best sensitivity and specificity for predicting preeclampsia was found to be at a cutoff value of GlyFn of 176.4 μ g/ml.

Thus, in various embodiments, measurement of serum GlyFn levels can be used in methods of controlling preeclampsia, methods of predicting poor clinical outcome (low birth weight, HELLP, etc.), and methods of distinguishing between mild and severe eclampsia.

The superior performance inference of this GlyFn fraction reflects that GlyFn is strongly linked to the pathological process that initiates preeclampsia. Without being bound by theory, this in turn may reflect a specific correlation in the development of preeclampsia of Fn splice variants or proteolytic fragments that exhibit unique glycosylation patterns. Interestingly, oxygen levels have recently been reported to regulate the expression of core-1O-glycan Gal β 1-3GalNac epitopes in human placenta; thus, without being bound by theory, placental insufficiency can result in altered glycoprotein levels in preeclampsia.

Without being bound by theory, the association of GlyFn with gestational diabetes and preeclampsia may be the result of the fact that: both disorders are associated with inflammation and endothelial dysfunction. Thus, early pregnancy inflammation and endothelial dysfunction associated with disrupted spiral artery remodeling may be associated with elevated levels of specific forms of glycosylated Fn. The different patterns of GlyFn abundance in these two related disorders remain interesting (e.g., gestational diabetes consistently increases in all trimester pregnancies but gradually increases during preeclampsia), but it may indicate that factors triggering gestational diabetes are established and maintained at a constant level early in gestation, while the onset and development of preeclampsia involves a sustained increase in the production of GlyFn.

The methods disclosed herein enable the use of GlyFn as a biomarker to monitor the severity of preeclampsia and the use of GlyFn to predict the onset of preeclampsia, particularly in early or mid-gestation patients. sFlt1 and PlGF are currently used in investigative studies to diagnose pre-eclampsia, but are not used for early prediction or monitoring of disease progression. The correlation found between GlyFn and clinical outcome is important and unique as it establishes a method for predicting which patients will have poor maternal and/or fetal outcome. This analysis shows that throughout pregnancy (including prior to clinical manifestations of preeclampsia), the GlyFn differs significantly between patients with and without preeclampsia, not shown for other preeclampsia analytes, and supports the concept of early pathogenesis of the disease. As disclosed herein, elevated levels of GlyFn may be used as an early indicator of risk of preeclampsia.

The ability to use this test in the POC format provides practitioners with a means to quickly determine the risk of preeclampsia in their pregnant patients and to determine the risk of poor maternal and fetal fate in those patients with preeclampsia.

Example 6: glycosylated fibronectin lateral flow immunoassay (FNLFIA)

In some embodiments, the GlyFn levels can be assessed using a lateral flow device. Various lateral flow assay methods can be used to test biological samples for the presence or absence or amount of an analyte (e.g., GlyFn). In one example, a "sandwich" assay method uses antibodies immobilized on a solid support that form part of a complex with labeled antibodies to determine the presence of a target analyte by observing the presence and amount of the antibody complex bound to the analyte label. For the purposes of lateral flow immunoassays, the label may be an enzyme, a colored microsphere, a fluorescently labeled microsphere, or other similar detection methods that provide for the detection and/or quantification of analyte binding to the test line may be used.

Conventional lateral flow test strips have a solid support with a sample receiving area and a target capture area on the solid support. A solid support material is a material that is capable of supporting both the sample-receiving zone and the target capture zone and provides capillary flow of the sample from the sample-receiving zone to the target capture zone when the lateral flow test strip is exposed to a suitable solvent or buffer (which serves as the carrier liquid for the sample). General classes of materials that can be used as supports include organic or inorganic polymers as well as natural and synthetic polymers. More specific examples of suitable solid supports include, but are not limited to, glass fibers, cellulose, nylon, sephadex, various chromatography papers, and nitrocellulose. One particularly useful material is nitrocellulose.

Fig. 10A and 10B show schematic diagrams of examples of lateral flow immunoassays (fig. 10A) and lateral flow testing devices (fig. 10B) that can be used according to various embodiments disclosed herein. In one specific non-limiting example of such a device, 200. mu.g/ml rabbit anti-GlyFn was immobilized on the membrane as a test line (0.5. mu.L/strip) and 300. mu.g/ml goat anti-mouse IgG was immobilized as a control line (0.5. mu.L/strip). Mouse anti-Fn-conjugated microspheres (10 μ L of 150 μ g/mL mouse anti-fibronectin, 1mg/mL solids) were dried on conjugate pads that had been treated with a solution (per liter) containing: 3.81g of sodium borate, 2.0g of dextran, 5.0g of BSA, 1.0g of Tween 20 and 0.5g of sodium azide, pH8.0, followed by drying at 50 ℃ for 1 hour.

The samples were then buffered in HEPES running buffer (10mM HEPES, 0.1mM CaCl)₂、155mM NaCl、0.1％NaN₃0.75% tween 20 and 0.01% polyvinyl alcohol) at a ratio of 1: 500. When the sample is applied to the sample pad, capillary flow hydrates the GlyFn-containing sample and interacts with the labeled microspheres, forming GlyFn-labeled microsphere complexes, which migrate further to the test line where they are captured by rabbit anti-GlyFn.

After capillary migration is complete, the device is scanned and the amount of GlyFn in the sample is determined by quantitative densitometry against a standard curve of purified GlyFn as standard. Although specific lateral flow devices are described herein, those skilled in the art will recognize that lateral flow devices are conventional and variations of the disclosed devices may be used.

Although certain embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that a wide variety of alternate and/or equivalent embodiments or implementations calculated to achieve the same purposes may be substituted for the embodiments shown and described without departing from the scope of the present invention. Those with skill in the art will readily appreciate that embodiments may be implemented in a very wide variety of ways. This application is intended to cover any adaptations or variations of the embodiments discussed herein. Therefore, it is manifestly intended that the embodiments be limited only by the claims and the equivalents thereof.

Claims (23)

1. A method for assessing risk of preeclampsia in a subject, comprising:

obtaining a first biological sample from the subject;

determining a level of glycosylated fibronectin (GlyFn) in the first biological sample; and

comparing the determined level of GlyFn to a control value; wherein a determined increase in the level of GlyFn in the first biological sample relative to the control value indicates that the subject is at increased risk of preeclampsia.

2. The method of claim 1, wherein the biological sample is a serum sample or a saliva sample.

3. The method of claim 2, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject at an early stage of pregnancy.

4. The method of claim 3, wherein the control value is a reference value representing the level of GlyFn in a sample from a subject in the early stage of pregnancy where preeclampsia does not occur.

5. The method of claim 4, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 15% increase.

6. The method of claim 4, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 50% increase.

7. The method of claim 2, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject during mid-gestation.

8. The method of claim 7, wherein the control value is a reference value representing the level of GlyFn in a sample from a subject in mid-pregnancy who does not develop preeclampsia.

9. The method of claim 8, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 15% increase.

10. The method of claim 8, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 50% increase.

11. The method of claim 2, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject in the late gestation period.

12. The method of claim 11, wherein the control value is a reference value representing the level of GlyFn in a sample from a subject in the late stage of pregnancy in which preeclampsia does not occur.

13. The method of claim 12, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 30% increase.

14. The method of claim 12, wherein the increase in the determined level of GlyFn in the first serum sample relative to the control value is at least a 100% increase.

15. The method of claim 2, wherein the method further comprises determining whether the determined level of GlyFn is equal to or greater than 250 μ g/ml, wherein a determined level of GlyFn equal to or greater than 250 μ g/ml indicates that the subject is at risk for low birth weight or HELLP syndrome.

16. The method of claim 2, wherein the method further comprises determining whether the determined level of GlyFn is equal to or greater than 500 μ g/ml, wherein a determined level of GlyFn equal to or greater than 500 μ g/ml indicates that the subject is at high risk for low birth weight or HELLP syndrome.

17. The method of claim 2, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject at an early or mid-gestation period, and wherein a determined level of GlyFn equal to or greater than 175 μ g/ml indicates that the subject has preeclampsia.

18. The method of claim 17, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject in the late gestation period, and wherein a determined level of GlyFn equal to or greater than 200 μ g/ml in the first serum sample from the subject indicates that the subject has preeclampsia.

19. The method of claim 17, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject in the late gestation period, and wherein a determined level of GlyFn in the first serum sample from the subject equal to or greater than 250 μ g/ml indicates that the subject has severe preeclampsia.

20. The method of claim 2, wherein obtaining a first serum sample from the subject comprises obtaining a first serum sample from the subject in the late gestation period, and wherein the method further comprises comparing the determined GlyFn levels in samples from at least two different time points to distinguish between mild preeclampsia and severe preeclampsia.

21. The method of claim 20, wherein the method further comprises:

obtaining a second serum sample from the subject at least one week after obtaining the first serum sample;

determining the level of glycosylated fibronectin (GlyFn) in the second serum sample; and

comparing the determined level of GlyFn in the second serum sample with the determined level of GlyFn in the first serum sample;

wherein an increase in the determined level of GlyFn in the second serum sample from 25 μ g/ml to 125 μ g/ml per week as compared to the determined level of GlyFn in the first serum sample indicates that the subject has mild preeclampsia; and is

Wherein a determined increase in GlyFn levels in the second serum sample by greater than 150 μ g/ml per week as compared to the determined GlyFn levels in the first serum sample indicates that the subject has severe preeclampsia.

22. A method for monitoring preeclampsia in a subject, comprising:

obtaining a first biological sample from the subject;

determining a level of glycosylated fibronectin (GlyFn) in the first biological sample;

obtaining a second biological sample from the subject at least one week after obtaining the first serum sample;

determining a level of glycosylated fibronectin (GlyFn) in the second serum sample; and

comparing the determined level of GlyFn in the second serum sample with the determined level of GlyFn in the first serum sample;

wherein an increase in the determined level of GlyFn in the second serum sample by less than 25 μ g/ml per week as compared to the determined level of GlyFn in the first serum sample indicates that the subject does not have preeclampsia;

wherein an increase in the determined level of GlyFn in the second serum sample from 25 μ g/ml to 125 μ g/ml per week as compared to the determined level of GlyFn in the first serum sample indicates that the subject has mild preeclampsia; and is

Wherein a determined increase in GlyFn levels in the second serum sample by greater than 150 μ g/ml per week as compared to the determined GlyFn levels in the first serum sample indicates that the subject has severe preeclampsia.

23. A test device for performing the method of claim 1, the test device comprising:

shell body

A test strip contained within the housing, the test strip comprising one or more immunoreagents, wherein one of the one or more immunoreagents is an anti-GlyFn antibody;

a means for quantifying the binding of the anti-GlyFn antibody to GlyFn in a biological sample.