Devices, assays and methods of testing preeclampsia

Abstract

Described herein are lateral flow devices, kits, systems and methods for measuring levels of sFlt-1 in a plasma or serum sample and prognosticating a women's risk of having sPE.

Description

DEVICES, ASSAYS AND METHODS OF TESTING PREECLAMPSIACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application Nos. 63 / 617,152 and 63 / 617,154, both filed on January 3, 2024, the entirety of which is hereby incorporated by reference.REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable form named “065472_000923WOPT_SequenceListing.xml”, having a size in bytes of 4,377 bytes, and created on January 2, 2025. The information contained in this computer readable form is hereby incorporated by reference in its entirety.FIELD OF INVENTION

[0003] This invention relates to immunochromatography or lateral flow devices and assays (e.g., point-of care devices and assays) to detect markers indicative of preeclampsia with severe features in pregnant women as a point-of-care tool or a lab based tool.BACKGROUND

[0004] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0005] Preeclampsia (PE) is a disorder unique to pregnancy characterized by hypertension, proteinuria, severe hepatic dysfunction, and coagulopathy. It is responsible for 40,000 - 80,000 maternal deaths annually worldwide and is associated with an increased risk for medically necessary preterm birth, stillbirth, and intrauterine growth restriction. The burden of disease is felt most heavily in the developing world, where the case fatality rate for eclampsia is an order of magnitude higher than in the developed world. Women with PE are usually asymptomatic until they develop severe disease, at which point they are at risk for serious complications and death.

[0006] Clinical diagnosis currently requires measurement of blood pressure, proteinuria, blood tests for evaluation of complications (aspartate aminotransferase (AST), alanine aminotransferase (ALT), platelet (PLT) count), and regular prenatal care, but these standard approaches to monitoring the mother often produce ambiguous and unpredictable results which do not provide clear guidance on how to manage these patients. The American College of Obstetrics and Gynecology (ACOG) revised the diagnostic criteria for PE to include end-organ damage (renal, brain,liver, or hematological system) in hypertensive women in the absence of proteinuria. Delivery is indicated for all patients with PE at 37 weeks gestational age or beyond. However, in patients with suspected or confirmed diagnosis of PE in the preterm period (<37 weeks), immediate delivery is indicated when PE with severe features or sPE (maternal adverse events) develops or due to fetal deterioration (fetal distress, absent blood flow on umbilical dopplers). The clinical conundrum in these cases is that: (1) many expectant mothers who present with high blood pressure during the preterm period do not (yet) meet criteria for PE with severe features (sPE); therefore, imminent delivery is not indicated; (2) a significant percentage of these women never develop signs or symptoms or complications of sPE, and can safely carry the pregnancy until term; and (3) premature delivery in the absence of signs or symptoms of sPE puts the newborn at significant health risks. The challenge is to identify mothers who are at high risk of developing sPE that requires delivery within 2 weeks as this determines whether the hospitalized mother requires closer observation and possible transfer to a higher level of care facility. Those presenting with signs of normotensive pregnancies do not require stepped-up care and can be expectantly managed according to the ACOG recommended standards of care.

[0007] The progressive nature of PE and variability in rates of progression to sPE make identification of patients requiring additional and / or ongoing clinical evaluation and delivery difficult. Without accurate assessment, patient management decisions such as hospitalization, initiation of antihypertensive medications, initiation of magnesium sulfate for seizure prophylaxis, frequency of lab draws, and fetal monitoring are affected and may contribute to adverse perinatal outcomes including premature iatrogenic delivery.

[0008] A prognostic test that rules out severe PE or maternal adverse outcomes in the shortterm period (2 weeks) would allow for expectant management of patients at low risk for developing complications and delivery only when there is clinical evidence of severe maternal end organ dysfunction as defined by ACOG. Per current ACOG guidelines, expectant management includes frequent blood pressure monitoring, weekly biochemical tests for evidence of sPE (AST, ALT, PIT, serum creatinine), serial ultrasonography to monitor fetal growth, and weekly antepartum testing. Risks associated with expectant management in the preterm period in accordance with the current standard of care include but are not limited to the following: development of severe hypertension, eclampsia, and / or HELLP syndrome; placental abruption; fetal growth restriction; and fetal death. These risks can be significantly lowered with a test that identifies patients at lower risk for development of sPE with greater accuracy. In addition, the availability of biomarkers linked with complications of PE (in conjunction with other laboratory and clinical findings such as alterations in liver enzymes or renal function tests) is also expected to help differentiate which patients admitted to the hospital for a hypertensive disorder of pregnancy are at higher risk for poor outcomes versus thoselikely to remain stable. A test to improve risk-stratification that directs appropriate monitoring can confer substantial clinical and economic benefits.SUMMARY OF THE INVENTION

[0009] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0010] Various embodiments provide a lateral flow device for detection of an analyte comprising one or more circulating fms-hke tyrosine kinase 1 (Flt-1) protein isoforms in a biological sample, wherein the lateral flow device includes:(i) a sample receiving region comprising an absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising: phosphate-buffered saline (PBS), a non-ionic copolymer surfactant, a heterophilic antibody blocking reagent, mouse immunoglobulin G (IgG), and sucrose;(ii) a conjugate region comprising absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising a biotinylated anti-sFLT-1 antibody having an anti-sFLT-1 antibody content at about 30.75-36.75 pg / mL, about 5-15% sucrose, about 1- 3% trehalose, a coloring agent, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-1 / control final conjugate solution comprising a detectably labeled monoclonal anti-VEGFRl antibody having a content of the monoclonal anti-VEGFRl antibody at about 0.05-0.15% w / v, a detectably labeled chicken immunoglobulin Y (IgY) having the chicken IgY content at about 0.0125-0.0375%, about 8-12% w / v sucrose, about 1-3% w / v trehalose, and a latex storage buffer, wherein the biotinylated anti-sFLT-1 antibody and the detectably labeled monoclonal anti-VEGFRl antibody bind to different epitopes within sFLT-1;(iii) a test region comprising nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3-0.5 mg / mL polystreptavidin, 0.5-1.5X PBS, and 4-6% w / v sucrose, and the control line having been saturated with about 0.125-0.375mg / mL donkey anti-chicken antibody, 0.5-1.5X PBS, 0.5-1.5% w / v sucrose, and the coloring agent, thereby having the polystreptavidin immobilized in the test line and the donkey anti-chicken antibody immobilized in the control line,(iv) a wick region comprising an absorbent pad; and(v) a backing card.

[0011] Various embodiments provide a lateral flow device for detection of an analyte comprising one or more circulating fms-like tyrosine kinase 1 (Flt-1) protein isoforms in a biological sample, wherein the lateral flow device includes:(i) a sample receiving region comprising an about 9-11 mm in-length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising: a non-ionic copolymer surfactant, a heterophilic antibody blocking reagent (HBR), mouse immunoglobulin G (IgG), and sucrose, in 0.5-1.5x phosphate-buffered saline (PBS), at a ratio of the non-ionic copolymer surfactant : the HBR (if present) : the mouse IgG : the sucrose being about 0.5- 1.5% w / v non-ionic copolymer surfactant : about 1.67-3.67 mg / ml heterophilic blocking reagent (HBR) if present : about 0.5-1.5 mg / ml mouse IgG : about 0.25-0.75% w / v sucrose;(ii) a conjugate region comprising an about 11-13 mm in-length absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising a biotinylated anti-sFLT-1 antibody having an anti-sFLT-1 antibody content at about 30.75-36.75 pg / mL, about 5-15% sucrose, about 1-3% trehalose, a coloring agent at about 40-60 pg / mL, and a biotinylated antibody diluent, optionally the coloring agent comprising FD&C blue #1 Powder, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising a detectably labeled monoclonal anti- VEGFR1 antibody having a content of the monoclonal anti-VEGFRl antibody at about 0.05-0.15% w / v, a detectably labeled chicken immunoglobulin Y (IgY) having the chicken IgY content at about 0.0125-0.0375%, about 8-12% w / v sucrose, about 1-3% w / v trehalose, and a latex storage buffer, wherein the detectable labels independently comprise a particulate label, a metal colloid label, or a fluorescent label, optionally the particulate label comprising a colored latex bead about 400 nm in diameter, and wherein the biotinylated anti-sFLT-1 antibody and the detectably labeled monoclonal anti- VEGFRl antibody bind to different epitopes within sFLT-1;(iii) a test region comprising an about 24-26 mm in-length nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3-0.5 mg / mL polystreptavidin, 0.5- 1.5X PBS, and 4-6% w / v sucrose, and the control line having been saturated with about 0.125- 0.375mg / mL donkey anti-chicken antibody, 0.5-1.5X PBS, 0.5-1.5% w / v sucrose, and the coloring agent at 40-60 pg / mL, wherein the polystreptavidin is immobilized in the test line and the donkey antichicken antibody is immobilized in the control line,(iv) a wick region comprising an about 20-22 mm in-length absorbent pad; and(v) a backing card having about 55-65 mm in length;wherein each region is in capillary contact with at least one other region thereby permitting a sample fluid to flow from the sample receiving region to the test region, and wherein the sample receiving region overlaps with the conjugate region, the conjugate region overlaps with the test region and the test region overlaps with the wick region.

[0012] In some embodiments of the lateral flow device(i) the sample receiving region comprises an about 10 mm length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising about lx PBS, about 1% w / v non-ionic copolymer surfactant HO(C2H4O)a(- C3H6O)b(C2H4O)aH wherein a is 99-101 and b is 55-57, about 2.67 mg / ml heterophilic blocking reagent (HBR) (HBR Plus), about 1 mg / ml mouse IgG, and about 0.5% w / v sucrose;(ii) the conjugate region comprises an about 12 mm absorbent pad having with the biotinylated antibody solution in the first portion of the conjugate region, the biotinylated antibody solution comprising the biotinylated anti-sFLT-1 antibody having the anti-sFLT-1 antibody content at about 33.75 pg / mL, about 10% w / v sucrose, about 2% w / v trehalose, the coloring agent at about 50 pg / mL, and the biotinylated antibody diluent, and the anti-sFLT-l / control final conjugate solution in the second portion of the conjugate region, the second portion of the conjugate region being different from the first portion of the conjugate region, and the anti-sFLT-l / control final conjugate solution comprising the anti-sFLT-1 / control final conjugate solution comprising the latex bead-conjugated monoclonal anti-VEGF R1 antibody having the monoclonal anti-VEGF R1 antibody content at about 0.1% w / v, the latex bead-conjugated chicken IgY having the chicken IgY content at about 0.025% w / v, about 10% w / v sucrose, about 2% w / v trehalose, and the latex storage buffer;(iii) the test region comprises an about 25 mm in-length nitrocellulose CN95 membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.4 mg / mL polystreptavidin, about IX PBS, about 5% w / v sucrose, and the control line having been saturated with about 0.25 mg / mL donkey anti-chicken antibody, about IX PBS, about 1% sucrose, about 50 pg / mL FD&C Blue powder,(iv) the wick region comprises an about 21 mm in-length absorbent pad; and(v) the backing carding having about 60 mm in length and optionally having cuts located 9, 16 and 46mm from a proximal end of the backing card, said proximal end being at or near the sample receiving region.

[0013] In some embodiments of the lateral flow device, the biotinylated antibody diluent comprises about 0.5-1.5% w / v bovine serum albumin and 0.5-1.5X PBS, and / or wherein the latexstorage buffer comprises about 0.5-1.5% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 25-75 mM boric acid.

[0014] In some embodiments of the lateral flow device, the biotinylated antibody diluent comprises about 1% w / v bovine serum albumin and IX PBS, and wherein the latex storage buffer comprises about 1% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 50 mM boric acid.

[0015] In some embodiments of the lateral flow device of claim 1, the latex bead-conjugated mAb anti-VEGF R1 comprises Human VEGFRl / Flt-1 Antibody (Clone #49560) and about 400nm- in-diameter Red carboxylated polystyrene latex particles conjugated at a weight ratio of from 100: 1 to 10: 1.

[0016] In some embodiments of the lateral flow device, the latex bead-conjugated mAb anti- VEGF R1 comprises about Human VEGFRl / Flt-1 Antibody (Clone #49560) and 400nm red carboxylated PS latex particles conjugated at a weight ratio of about 40: 1.

[0017] In some embodiments of the lateral flow device, the latex bead-conjugated Chicken IgY comprises Chicken IgY and about 400 nm Red Carboxylated Latex particles conjugated at a weight ratio of from 100: 1 to 10: 1.

[0018] In some embodiments of the lateral flow device, the latex bead-conjugated Chicken IgY comprises Chicken IgY and red carboxylated latex particles about 400 nm in size conjugated at a weight ratio of about 40: 1.

[0019] In some embodiments of the lateral flow device, the latex storage buffer comprises about 1% w / v of Casein in an alkaline borate buffer, the alkaline borate buffer containing about 50 mM of boric acid and pH adjusted to 8.6.

[0020] Further embodiments provide a kit, comprising: a lateral flow device disclosed herein; and a chase buffer. In some embodiments, the chase buffer comprises the non-ionic copolymer surfactant, potassium chloride, PBS, and a preservative biocide, optionally the preservative biocide comprising 2-Methyl-4-isothiazolin-3-one.

[0021] In some embodiments, a kit comprises a lateral flow device disclosed herein, a chase buffer, and both or either one of a low external control or a high external control. A low external control comprises sFLT-1 at a first predetermined concentration, and a high external control comprises sFLT-1 at a second predetermined concentration, the first predetermined concentration being lower than the second predetermined concentration, and wherein optionally the sFLT-1 comprises a polypeptide having an amino acid sequence of Ser27-His687 of SEQ ID NO:1.

[0022] In some embodiments of the kit, the low external control comprises about 1.5-2.5 ng / mL of sFLT-1 and the high external control comprises about 3.5-5.0 ng / mL sFLT-1.

[0023] In some embodiments, the low external control comprises about 2.4 ng / mL of sFLT-1 and the high external control comprises about 3.6 ng / mL sFLT-1.

[0024] In further embodiments, the kit further includes a quality control diluent. For example, the quality control diluent comprises 0.5-1.5X PBS, 0.09-1.15% w / v 2-Methyl-4-isothiazolin-3-one, about 4-6% w / v Sucrose, about 0.5-1.5% w / v Trehalose, about 40-50 mg / mL bovine serum albumin (BSA), about 90-110 mM ethylenediaminetetraacetic acid (EDTA), and about 18-22 USP / mL Lithium Heparin salt. In some embodiments, the quality control diluent comprises IX PBS, 0.105% w / v 2- Methyl-4-isothiazolin-3-one, about 5% w / v Sucrose, about 1% w / v Trehalose, about 45 mg / mL BSA, about 100 mM EDTA, and about 20 USP / mL Li Heparin salt.

[0025] Also provided are analyzers for analyzing a lateral flow device, wherein an analyzer includes: an opening to receive a lateral flow device; a light source optionally being an illumination light-emitting diode (LED); a camera, lens, fdter assembly to record the image of a lateral flow strip in the lateral flow device; a battery or electric power cord; a display on the outside of the analyzer; and a printed circuit or a computer readable medium comprising computer executable instructions to process the image, integrate the test line (TL) signal, perform background subtraction, calculate background-subtracted TL signal, convert the TL signal into sFLT plasma or serum concentrations.

[0026] In some embodiments, an analyzer further includes one or more of a pSD card port; a battery port; a USB port; and a printer.

[0027] Systems are also provided, which include a lateral flow device and an analyzer, wherein the analyzer is configured to measure intensity of the test line or the test line and the control line of the lateral flow device.

[0028] In some embodiments, the system further includes an analyzer control cassette configured to test the analyzer’s electronics and photometric detection system.

[0029] Methods are also provided for detecting a level of soluble fms-hke tyrosine kinase 1 (sFlt-1) in a plasma or serum sample with a lateral flow device disclosed herein, the method comprising: applying (a) a mixture containing or consisting of a plasma or serum sample and a chase buffer, or (b) the plasma or serum sample and the chase buffer concurrently or sequentially, to the sample receiving region of the lateral flow device; measuring the level of the latex beads at the first location in the test region; and calculating the level of sFlt-1 based on the measured level of the latex beads.

[0030] In some embodiments of the methods, measuring the level of the latex beads is performed at about 20-30 minutes after applying the mixture or the plasma or serum sample to thesample receiving region. In some embodiments of the method, measuring the level of the latex beads is performed at about 25 minutes after applying the mixture or the plasma or serum sample to the sample receiving region.

[0031] In some embodiments, the plasma or serum sample is from a pregnant woman. In some embodiments, the plasma or serum sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant.

[0032] In some embodiments of the method, measuring the level of the latex beads at the first location in the test region, or calculating the level of sFlt-1 based on the measured level of the latex beads, or both is performed with an analyzer disclosed herein.

[0033] Methods of prognosticating a woman’s risk of having preeclampsia with severe features (sPE) are also provided, which include: assaying a plasma or serum sample obtained from the women to detect a level of soluble fms- like tyrosine kinase 1 (sFlt-1) with a lateral flow device through a method comprising: pre-mixing the plasma or serum sample with a chase buffer to obtain a mixture sample and applying the mixture sample to the sample receiving region of the lateral flow device; measuring the level of the red latex beads at the first location in the test region; calculating the level of sFlt-1 based on the measured level of the red latex beads; and prognosticating the women’s risk of having sPE as high when the level of sFlt-1 is higher than a reference level or prognosticating the women’s risk of having sPE as low when the level of sFlt-1 is less than the reference level, wherein the reference level is 2.0-6.0 ng / mL, wherein a high risk of having sPE is having sPE within three weeks from having her plasma or serum sample assayed.

[0034] In some embodiments, measuring the level of the red latex beads is performed at about 20-30 minutes after applying the mixture sample to the sample receiving region.

[0035] In some embodiments, measuring the level of the red latex beads is performed at about 25 minutes after applying the mixture sample to the sample receiving region.

[0036] In some embodiments, the plasma or serum sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant.

[0037] In some embodiments, measuring the level of the red latex beads at the first location in the test region, or calculating the level of sFlt-1 based on the measured level of the red latex beads, or both is performed with an analyzer disclosed herein.

[0038] In some embodiments, the reference level is 3.0 ng / mL.

[0039] In some embodiments, a high risk of having sPE is having sPE within two weeks from having her plasma or serum sample assayed.

[0040] In some embodiments, a high risk of having sPE is having sPE within one weeks from having her plasma or serum sample assayed.

[0041] Methods of treating or managing preeclampsia or eclampsia for a patient in need thereof are also provided, which include: prognosticating the women’s risk of having sPE, and initiating one or more evaluation or preparedness for the woman whose risk of having sPE is prognosticated as being high, or providing standard of care expectant management to the woman whose risk of having sPE is prognosticated as being low.

[0042] In some embodiments, one or more evaluation or preparedness comprises step-up care for the woman, providing neonatal intensive care unit (NICU) consult, increase frequency of tests to assess severity of the preeclampsia or eclampsia, increase frequency of fetal assessments, planning for steroids or magnesium treatment.

[0043] In some embodiments, the treatment methods further include administering steroids or magnesium to the women when the risk of having sPE is prognosticated as being high.

[0044] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention.BRIEF DESCRIPTION OF THE FIGURES

[0045] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0046] Figure 1 depicts an exemplary patient management using the lateral flow test in accordance with various embodiments of the present invention.

[0047] Figure 2 depicts an exemplary lateral flow device (also called test cassette) in accordance with various embodiments of the present invention.

[0048] Figure 3 depicts an exemplary Analyzer (also called test cassette reader) (left) and micro secure digital (pSD) card (right) in accordance with various embodiments of the present invention.

[0049] Figure 4 depicts an exemplary analyzer control cassette in accordance with various embodiments of the present invention.

[0050] Figure 5 depicts an exemplary diagram of the test strip regions in the lateral flow device in accordance with various embodiments of the present invention.

[0051] Figure 6A depicts a perspective view of an exemplary analyzer inserted with an exemplary lateral flow device. Figure 6B depicts a side cross-section vie showing internal schematic of the analyzer and the lateral flow device in accordance with various embodiments of the present invention.

[0052] Figure 7 depicts an exemplary analyzer reported test results in accordance with various embodiments of the present invention.

[0053] Figure 8 depicts an exemplary analyzer image analysis and data processing in accordance with various embodiments of the present invention.

[0054] Figure 9 depicts 2D PNG Image Transformation to ID Signal Tracing and Background Removal in accordance with various embodiments of the present invention.

[0055] Figure 10 depicts an exemplary calibration curve for a lot of cassettes used in the PRAECIS derivation cohort study in accordance with various embodiments of the present invention.

[0056] Figure 11 depicts a derivation of sFLT-1 prognostic cut-off (in ng / mL) using sensitivity (+ line) and specificity (o line) in accordance with various embodiments of the present invention.

[0057] Figure 12 depicts derivation of sFLT-1 prognostic cut-off (in ng / mL) using PPV (o line) and NPV (+ line) in accordance with various embodiments of the present invention.

[0058] Figure 13 depicts ROC curves for the SFLT-1 LFAtest and for other clinical covariates in Example 10.

[0059] Figure 14 depicts the Kaplan-Meier plot of time to delivery, stratified by SFLT-1 LFA test result, in Example 10.DESCRIPTION OF THE INVENTION

[0060] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2nded. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U. S. Patent No. 5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holhger P. (2000) Methods Enzymol, 326, 461-479; Holhger P. (2005) Nat. Biotechnol. Sep;23(9): 1126-36).

[0061] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0062] As used herein the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 5% of that referenced numeric indication, unless otherwise specifically provided for herein. In various embodiments, the term“about” when used in connection with a referenced numeric indication can mean the referenced numeric indication plus or minus up to 4%, 3%, 2%, 1%, 0.5%, or 0.25% of that referenced numeric indication, if specifically provided for in the claims.

[0063] A “subject” means a human or an animal. In various embodiment, the subject is a woman (human). In various embodiments, the subject is a pregnant human. In further aspects, the subject is a pregnant woman. In additional aspects, the subject is a pregnant woman (human) between 23 weeks and 35 weeks of pregnancy, who has not given birth. In further aspects, the subject is a pregnant woman (human) with a hypertensive disorder such as preeclampsia, chronic hypertension, or gestational hypertension. In further aspects, the subject is a woman (human) at 23-35 weeks of gestation with a hypertensive disorder such as preeclampsia, chronic hypertension, or gestational hypertension. In yet another aspect, the subject is a woman having recently given birth, such as in the past month or months. In some embodiments, the subject is a post-partum human. In some embodiments, the subject is a pregnant human or a post-partum human. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, “patient”, “individual” and “subject” are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples.

[0064] A “patient in need of’ or “subject in need” of treatment for a particular disease, disorder, or condition may be a subject suspected of having that disease, disorder, or condition, diagnosed as having that disease, disorder, or condition, already treated or being treated for that disease, disorder, or condition, not treated for that disease, disorder, or condition, or at risk of developing that disease, disorder, or condition. In various embodiments, a patient in need thereof is a human patient.

[0065] “Sample” or “biological sample” in various embodiments refers to bodily fluid, tissue or a specimen obtained from a subject. Exemplary biological samples used in the present invention (e.g., for the sFlt-1 assay in the lateral flow devices (or also referred to as immunochromatographic devices)) include but are not limited to plasma, serum, whole blood, saliva, urine, and mucus. In various embodiments, the sample is plasma. In various embodiments, the biological sample is serum. In various embodiments, the sample is whole blood. In various embodiments, saliva samples are clarified saliva. Clarified saliva refers to whole saliva that has been filtered through a swab or other form of mechanical filtration, or that has been frozen and / or centrifuged to pelletize mucins. In various embodiments, the biological sample for use with the devices is a plasma. In various embodiments, the biological sample for use with the devices is EDTA plasma, which is a fluid separated (typically viacentrifugation) from blood that has been treated with ethylenediaminetetraacetic acid (EDTA) Biological samples may be freshly obtained, or frozen and then thawed before testing. In various embodiments, the devices utilize freshly obtained plasma (without refrigeration or freezing, and within 6 hours, 12 hours or 1 day after isolation from a mammalian body); or methods of using the devices include contacting freshly obtained plasma with the devices, generally the sample receiving region of the devices. In some embodiments, the devices utilize freshly obtained EDTA plasma. In other embodiments, the devices utilize freshly obtained saliva; and methods of using the devices include contacting freshly obtained saliva with the devices, e.g., the sample receiving region of the devices. In certain embodiments, the blood or a component thereof is plasma and one or more methods described herein comprises removing a volume of the subject’s blood and separating the blood into plasma and cellular components.

[0066] The term “antibody” refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region, referred to herein as the “Fc fragment” or “Fc domain”. Antigen-binding fragments may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, inter aha, Fab, Fab’, F(ab’)2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The Fc domain includes portions of two heavy chains contributing to two or three classes of the antibody. The Fc domain may be produced by recombinant DNA techniques or by enzymatic (e.g., papain cleavage) or via chemical cleavage of intact antibodies. An antibody can be a chimeric, humanized or human antibody. An antibody can be an IgGl, IgG2, IgG3 or IgG4 antibody. In some aspects, an antibody herein has an Fc region that has been modified to alter at least one of effector function, half-life, proteolysis, or glycosylation.

[0067] The term “antibody fragment,” refers to a protein fragment that comprises only a portion of an intact antibody, generally including an antigen binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab’ fragment, which is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd’ fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment which consists of a VH domain; (vii) isolated CDR regions; (viii) F(ab’)2 fragments, a bivalent fragment including two Fab’ fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv); (x) “diabodie” with two antigen binding sites, comprising a heavy chainvariable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain; (xi) “linear antibodies” comprising a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. An antibody or antibody fragment can be scFvs, camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks) and Fab, Fab’ or F(ab’)2 fragment.

[0068] “Specifically immunoreactive,” “selectively immunoreactive,” “selectively binds” or “specifically binds” in various embodiments refers to the ability of an antibody or antibody fragment thereof described herein to bind to a target, such as an analyte in the biological sample, with a KD 10’5M (10000 nM) or less, e.g., 10'6M, 10'7M, 10'8M, 10'9M, IO’10M, 10’11M, IO’12M, or less. In some embodiments, “specifically immunoreactive” to one antigen or a target molecule of an antibody or antibody fragment indicates that the antibody or antibody fragment does not bind or binds to a nonantigen at a level that is at least two-, three-, or four-order of magnitude lower compared to when it binds to the intended antigen or target molecule. Specific binding can be influenced by, for example, the affinity and avidity of the polypeptide agent and the concentration of polypeptide agent. The person of ordinary skill in the art can determine appropriate conditions under which the polypeptide agents described herein selectively bind the targets using any suitable methods, such as titration of a polypeptide agent in a suitable cell binding assay.

[0069] Membrane materials generally contain porous structures. In some embodiments, the porous structure of the membrane is large / void enough to allow transport of sample fluid or test buffers, as well as the migration of analyte, antibodies (including detectable label-modified antibodies), and bound complexes between analyte and antibodies, at least driven by a capillary force. In some embodiments, the porous structure of the membrane has layers where at least the exterior layer(s) are “tight” enough to trap or enclose the analyte, antibodies (including detectable label- modified antibodies) and bound complexes within the membrane. Membrane materials and / or structures are selected to afford desirable speed to result, assay sensitivity, reproducibility at the threshold (cut-off value for the analyte) and in a dynamic range.

[0070] Fms related receptor tyrosine kinase 1 (FLT-1) is also known as vascular endothelial growth factor receptor 1 (VEGFR-1). The VEGFR-1 is also secreted in the extracellular matrix as a soluble isoform (sVEGFR-1 or sFLT-1), which derives from alternative splicing of the VEGFR-1 mRNA.

[0071] Results from the sFLT - 1 assay described herein will allow physicians to prognosticate and risk stratify hospitalized women diagnosed with a hypertensive disorder of pregnancy (HDP) earlier, and more accurately, than currently available laboratory tests used to risk-stratify patients (e.g., liver function tests, platelet counts, renal function tests). Accurate risk stratification will allowclinicians to monitor high-risk patients more closely and diagnose sPE earlier, thus reducing risk to both mother and child.

[0072] Women who suffer from HDP are typically admitted to the hospital and managed using current standard of care (SOC) tests including clinical (e.g., blood pressure and symptoms) and biochemical tests (e.g., liver enzymes, renal function tests, platelet counts) which are used for monitoring the development of sPE and fetal adverse outcomes. However, the SOC tests are poor predictors for the progression to sPE adverse outcomes with < 50% PPV. Many studies show that angiogenic factors, such as sFLT-1 concentrations, antedate both clinical and biochemical tests and provide improved prognostic information for progression to sPE and adverse outcomes.

[0073] In various embodiments, improved risk stratification in hospitalized patients based on sFLT-1 test results results in two patient groups: 1) patients with hypertensive disorder at low risk for development of sPE and delivery within two weeks of testing and, 2) patients with hypertensive disorder at high-risk for development of sPE and delivery within two weeks of testing. Benefits for identification of patients at low risk of developing sPE (adverse maternal outcomes) or not requiring delivery within 2 weeks include an allowance of expectant management and prolongation of pregnancy duration. Expectant management involves observing clinical signs, symptoms, and findings, actively managing conditions such as hypertension, and planned delivery at 37 weeks of gestation. Benefits of identifying women at high risk for developing sPE or requiring delivery within two weeks include increased observation of the mother and fetus, referral to a higher-level facility with level III / IV NICU and optimizing the timing of administration of corticosteroids to improve neonatal lung maturity and / or magnesium sulfate for neuroprophylaxis (both treatments optimally beneficial when timed appropriately). These actions increase the likelihood of positive clinical outcomes for the mother and fetus.

[0074] In various embodiments, sFLT-1 assay described herein is an in vitro diagnostic device for the qualitative detection of soluble Fms-like tyrosine kinase-1 protein (sFLT-1) protein in EDTA plasma or serum samples and is intended to improve the risk stratification of hospitalized singleton pregnant women with hypertensive disorder of pregnancy, between 23 0 / 7 weeks to 34 6 / 7th weeks of gestation, for progression to preeclampsia with severe features (sPE) within two weeks from presentation, over the current standard of care. In various embodiments, the sFLT-1 assay utilizes a sFLT-1 Test Kit (e.g., test cassette figure 2), chase buffer, lyophilized Quality Controls, and pSD card and an Analyzer (e.g., figure 3, 6A, 6B), and Analyzer Control Cassette (e.g., pre-printed Analyzer Electronic Control Cassette (EQC)) (e.g., figure 4). The pSD card contains lot-specific calibration information and QC allowable values is to be uploaded to the Analyzer by the user prior to first use of a given lot of test cassettes.

[0075] Representative contents of the test kit are provided in Table 1.Table 1

[0076] FIG. 2 shows a perspective view of an exemplary lateral flow device 100, containing a test strip housed in a generally rigid housing 190. The lateral flow device 100 includes a sample port 192, which is an opening preferably tapering in towards the sample receiving region of the test strip housed inside; a lateral flow assay (LFA) window 194, wherein a portion of the test strip comprising the test region is visible through the LFA window 194; and an encoded label (e.g., quick response (QR) code 180), which is affixed or printed on surface of the generally rigid housing 190. Test line 132 and control line 134 of the test region are visible.

[0077] Figure 4 shows a perspective view of an exemplary analyzer control cassette 200, which contains a test strip housed in a generally rigid housing 290, wherein the test strip contains thereon pre-printed test line 232 and pre-printed control line 234. The analyzer control cassette 200 further includes an encoded label (e.g., QR code 280) affixed or printed on the surface. Manufacturer- printed test lines and control lines in analyzer control cassettes are used to ‘pre-test’ the analyzer each time the analyzer is powered on to confirm performance of the analyzer’s light source (e.g., LED light) and camera which are used to capture images and quantify test results. If the test line value scanned with the analyzer on the analyzer control cassette, after background adjustment and preferably taking an average from multiple scans, is not different or is within acceptable range from the standard target value, the analyzer is validated on its proper performance and may be used with a test cassette; and if the value differs from the standard target value, re-testing the analyzer with the analyzer control cassette is need, or further examination of the LED and camera of the analyzer may be required.

[0078] Figure 5 shows a diagram of a side view of an exemplary test strip, sometimes also called a lateral flow device (with or without a housing) or a test device. A test strip has a longitudinal axis having a length, 1, defined from a proximal end of the test strip (at or near which the sample is applied) to a distal end of the test strip (wherein after the sample is applied, fluid flowthrough the test strip is in the proximal-to-distal direction); a width axis having a length, w, defined perpendicular to the longitudinal axis from a first lateral edge of the test strip to a second lateral edge of the test strip, wherein 1 is greater than w and the longitudinal and width axes define a plane, p; and a height axis having a length, h, which is perpendicular to the plane p and refers to overall thickness of the test strip. In figure 5, a test strip has a longitudinal length 1 = 60 mm, which comprises porous or bibulous lateral flow materials in different regions, backed by a backing card 150 of about 60 mm long in thelongitudinal axis. At the proximal end of the test strip in figure 5 is a sample receiving region 110, which may include a first absorbent pad of about 10 mm long in the longitudinal axis. Overlapping with the sample receiving region 110 by about 4 mm in the longitudinal axis towards the distal end is a conjugate region 120, which comprises a first portion 122 and a second portion 124, wherein the two portions generally do not overlap and are separated along the longitudinal axis of the conjugate region 120. In figure 5, a second absorbent pad of about 12 mm long may be placed along the longitudinal axis in the conjugate region 120, and the first portion 122 (e.g., about 5 mm long) of the absorbent pad in the conjugate region 120 is saturated with or contains a biotinylated anti-sFLT-1 antibody, whereas the second portion 124 (e.g., about 7 mm long) of the absorbent pad in the conjugate region 120 is saturated with or contains a red latex bead-conjugated anti-VEGFRl antibody. It is preferable that the first portion 122 is upstream relative to the second portion 124, because such configuration allows for optimal flow and ensures that the analyte interacts with the primary antibody (herein biotinylated anti- sFLT-1 antibody) before it can interact with the secondary antibody (herein red latex bead-conjugated anti-VEGFRl antibody). Overlapping with the conjugate region 120 by about 2 mm in the longitudinal axis towards the distal end in figure 5 is a test region 130, which may include a test membrane (e.g., nitrocellulose membrane) of about 25 mm long; and the nitrocellulose membrane contains a test line 132 and a control line 134, each about 1 mm or 1.5 mm long and being separated from each other. Overlapping with the test region 130 by about 2 mm in the longitudinal axis towards the distal end in figure 5 is a wick region 140, and a third absorbent pad of about 21 mm may be placed in the wick region 140. At least a portion of the test region 130 including the test line 132 and the control line 134 is a visible portion to a user or a camera as a lateral flow assay (LFA) window 194, positioned at a vertical distance above plane p and at opposing side to the backing card 150. Typically the LFA window 194 is long enough in the longitudinal axis of the test strip to include ‘background’ portions at least two times, three times, four times, five times, six times, or more of the lengths of the test line 132 and the control line 134, in the longitudinal axis, which do not contain the compounds that the test line 132 or the control line 134 has for association with biotinylated antibody conjugate or control immunoglobulin; and the LFA window 194 is not so long as to include the conjugate region 120 or the wick region 140.

[0079] Figure 4 is a top view of an analyzer control cassette 200, which contains a generally rigid housing 290 having an LFA window through which pre-printed test line 232 and pre-printed control line 234 are visible, and a QR code 280 affixed or printed on the surface of the housing 290.

[0080] Figures 3, 6A and 6B are different views of an analyzer 300, also called a test strip reader or test device reader or test cassette reader. Accessible from exterior of the analyzer 300 includes an opening 310 having a coiled spring 312 embedded therewith, allowing for a lateral flow device to be inserted by compressing the spring 312 and sliding the lateral flow device into the analyzer 300. Typically, a sample port 192 of the lateral flow device is accessible to a user and left outside ofthe opening 310, whereas a LFA window 194 of the lateral flow device is inserted into the analyzer 300. Also accessible from exterior of the analyzer 300 are a battery port 350 for housing a battery 352 or electric power cord; a pSD card port 380 to receive a pSD card 382; a USB port 390, for connection to a printer or another electronic device or outlet; one or more of an input button 362; and a display 360 (e.g., color liquid crystal display (LCD)). The interior of the analyzer 300 includes a light source 330 (e.g., illumination light-emitting diode (LED)); a camera, lens, fdter assembly 340; a battery 352; and a computer readable medium such as a printed circuit board 370. Typically the light source 330 is positioned at a location configured and arranged to permit the light source 330 to illuminate the visible portion of the lateral flow device with electromagnetic radiation and thereby generate a detectable signal. The camera, lens, filter assembly 340 includes (1) a camera or image sensor, positioned on the circuit board 370 that is approximately centered on the width axis of the lateral flow device, wherein the camera or image sensor is configured to capture the detectable signal from the visible portion of the lateral flow device when illuminated by the electromagnetic radiation during generation of the detectable signal; (2) a lens configured to focus an image of the visible portion of the lateral flow device onto the camera or image sensor, wherein the lens typically extends below the circuit board 370 toward plane p; and optionally (3) a filter configured to remove reflected excitation wavelengths and / or to select the measured emission wavelengths. Typically, a computer readable medium such as the printed circuit board 370 is or includes a processing component operably connected to the light source 330 and the camera or image sensor and configured to (i) control the illumination of the vision portion of the lateral flow device by the light source 330, (ii) receive an electrical signal from the camera or image sensor resulting from the detectable signal, and (iii) convert the electrical signal into an assay result indicative of the presence or amount of the FLT-1 in the sample. Typically the display 360 is operably connected to the computer readable medium to display the assay result. In some aspects, the analyzer further includes a barcode scanner. Further features of the analyzer are described in US20230135843, which is incorporated by reference.

[0081] In various embodiments, the sFLT-1 Test Kit pSD card (Figure 3) contains lotspecific calibration information which is to be uploaded to the Analyzer (Figure 3) prior to use of a given test cassette lot. The analyzer will prevent use of a given test lot if the associated lot-specific information has not been uploaded and will prompt the user to insert the pSD card or use a different lot of test cassettes. A pre-printed Analyzer Electronic Control Cassette (EQC) (Figure 4), preferably provided with the Analyzer, is inserted into the analyzer when the instrument is turned on and used to test the analyzer’s electronics and photometric detection system (LED illuminator and camera) to ensure it is operating as expected. Alternatively, a pre-printed Analyzer EQC may be also provided with the test kit.

[0082] In various embodiments, each sFLT-1 test cassette features a QR Code (Figure 2) or is printed or affixed with an encoded label, which stores encoded information related to the test, suchas the test ID, the lot number for the test device, the expiration date for the test device, a calibration information for the test procedure, and / or an indication of the test performed through use of the test device (e.g., sPE indication test). The encoded label may be a QR code, a 2-D bar code, a 1-D bar code, or text. In some embodiments, the encoded label is a QR code. Upon insertion of the test cassette into the Analyzer (e.g., prior to sample application), the QR Code is read, and the analyzer scans the test window for the position of the test window and the colored Control Line dye indicator (e.g., blue when the Control Line contains brilliant blue TCP as the coloring agent) positioned at the Control Line to verify that the Control Line is located in the expected region of interest (ROI), that the cassette is properly inserted, and that the reagent strip is correctly aligned.

[0083] In various aspects, specimen preparation for testing includes (1) Specimen dilution, (2) Insertion of testing cassette, and (3) addition of diluted specimen to testing cassette. An operator will prepare the specimen for testing by pipetting the chase buffer (e.g., 80 pL) into the dilution tube, then pipetting the plasma or serum sample (e.g., 15 pL) into the chase buffer in the dilution tube and gently mixing, thereby obtaining a diluted sample (also called a mixture or sample mixture). In some aspects, at least one dilution tube is pre-loaded with a pre-determined amount of the chase buffer (e.g., 80 pL), preferably in a sterile package; and upon opening the package, the operator may directly add the plasma or serum sample to the chase buffer in the dilution tube, so as to obtain a diluted sample (or sample mixture). After the cartridge is inserted and the test is initiated by the operator using the graphical user interface (GUI), the analyzer instructs the operator to apply the diluted sample to the Sample Application Port (Figure 2) (corresponding to a sample receiving region). The analyzer internal test timer is initiated once the analyzer camera detects flow within the test window (corresponding to a test region). As the mixture flows through the conjugate pad, it solubilizes the absorbed antibody conjugates, and the sFLT-1 antigen in the patient’s sample binds to biotinylated anti-sFLT-1 antibody conjugate (capture antibody) and anti-sFLT-1 antibodies conjugated to a detectable label such as Red Latex Beads or colored latex bead, a particulate, a metal colloid, or a fluorophore (detection reagent). Both antibodies are directed against different epitopes within the sFLT-1 antigen thereby forming a sandwich. The conjugate pad also contains chicken IgY antibodies conjugated to the detectable label (e.g., Red Latex Beads) (control reagent). The sFLT-1 antibody / antigen complex and control reagent then flow to the test membrane (also referred to as a test region; Figure 5). The test line, containing immobilized polystreptavidin, captures the sFLT-1 antibody / antigen complex by binding the biotinylated capture antibody. Nitrocellulose membranes have high affinity for polystreptavidin, thereby resulting in immobilization of polystreptavidin in the test line of the test region when the test membrane is coated / presaturated with polystreptavidin in the test line area. The remaining mixture flows across the control line and the control reagent is captured by immobilized donkey anti-chicken IgY antibodies. The sample continues to flow until immunochromatographic development of the assay is complete (about 25 minutes). Upon completionof the test, the analyzer acquires a final image of the test window which is analyzed for the final Test Line and Control Line signals. In the event the control line does not show a positive signal, the test result should be discarded, as the control line herein containing donkey anti-chicken IgY antibodies should be a positive control when coupled with the detectably labeled chicken IgY from the second portion of the conjugate pad.

[0084] In various embodiments, the Analyzer acquires images of the test window using the green channel of its camera to maximize contrast by the red latex beads from their white background. Colored latex beads are typically particulates of polystyrene matrix with organic dye(s) incorporated. In some embodiments, latex beads of another color than red may be used to conjugate anti-sFLT-1 antibody (‘capture’) or chicken IgY. The densitometric quantification of the detectable label (e.g., colored latex beads or red latex beads) within the ROIs is quantified across the full width of the test membrane (e.g., 140 pixels). ROIs will include the test line (e.g., 10 pixel width), control line (e.g., 10 pixel width), and several areas used to quantify background (e.g., 20 pixel width each). The integrated signals at each point along the membrane will be smoothed using a KZ filter with a 6 pixel window width. After smoothing, the test line (TL) and control line (CL) integrated signals are processed to remove background signal based upon the background color in regions just outside the TL and CL ROI. The presence of the detectable label signal (e.g., red signal from red latex beads) at the CL are evaluated by the Analyzer to determine if the test is valid (tests with inadequate red signal at the CL will be rejected as invalid; a “test invalid” message will be displayed on the analyzer with further instructions for the end user).

[0085] The TL total integrated signal is quantified after KZ filter smoothing and background removal. The total integrated signal is then converted into sFLT-1 concentrations (in ng / mL) using a lot-specific standard curve uploaded onto the analyzer using the lot-specific pSD card (Figure 3) included in each box of test cassettes. Tests where the sFLT-1 concentration is greater than 3.0 ng / mL and the control line produces a valid result are considered positive and patients are considered at higher risk of developing sPE, especially developing sPE within 2 weeks of sample collection time.

[0086] In some embodiments, a lateral flow device includes a housing, which is generally rigid and comprises a base and a lid with a sample receiving aperture (sample port) and a test aperture (LFA window). By way of example only, a housing may be formed by introducing a moldable material into a mold assembly to form the lid and the base. While the lid and the base may be formed with discrete molds, the lid and the base may also be formed as a unitary part. To facilitate fit of the lid and the base, the base and the lid may be formed as a unitary part connected by one or more flexible hinge regions configured to allow the lid to mate to the base. A skilled artisan will understand that a number of polymers may be used to form the housing, including thermoplastics, some thermosets, and elastomers. Common thermoplastics include PMMA, cyclic olefin copolymer, ethylene vinyl acetate, polyacrylate, polyaryletherketone, polybutadiene, polycarbonate, polyester, polyetherimide,polysulfone, nylon, polyethylene, and polystyrene. Common thermosets include polyesters, polyurethanes, duroplast, epoxy resins, and polyimides. This list is not meant to be limiting. Functional filler materials such as talc and carbon fibers can be included for purposes of improving stiffness, working temperatures, and part shrinkage.Indications for Use

[0087] The sFLT-1 Test is a lateral flow immunoassay intended for the qualitative determination of soluble Fms-like tyrosine kinase-1 (sFLT-1) in EDTA plasma or serum of women during the third trimester of pregnancy using the Analyzer.

[0088] The sFLT-1 Test is a prognostic test intended to improve the risk stratification of hospitalized singleton pregnant women with hypertensive disorder of pregnancy, between 23 0 / 7 weeks to 34 6 / 7th weeks of gestation, for progression to preeclampsia with severe features (sPE) within two weeks from presentation, over the current standard of care.

[0089] The sFLT-1 Test is intended to be performed in the clinical laboratory by trained professionals as a prognostic test.

[0090] Elevated concentrations of soluble fms-like tyrosine kinase 1 (sFlt-1) or membranebound Fit- 1 proteins (mFlt-1) in salivary, plasma, serum or whole blood, urine, or another blood fluid are associated with an increased risk or presence of preeclampsia in pregnant mothers. One or more isoforms of Flt-1 (e.g., Flt-1 proteins produced from different mRNA isoforms) can be measured in the devices or methods described in the present invention, including but are not limited to those encoded by mRNAs of sFlt-1 -il3 -short, sFltl-il3-long, sFltl -il4, sFltl-el5a, sFltl-el5b and / or mFlt- 1. Further descriptions of Flt-1 isoforms are provided in Placenta, volume 30, issue 3, pages 250-255, March 2009, and in Scientific Reports, volume 7, Article number: 12139 (2017), which is hereby incorporated by reference. For example, Genbank accession number AF063657 provides a nucleotide (mRNA) and amino acid sequences of human Flt-1. Additional examples of sFlt-1 mRNA splice variants are seen in Genbank accession numbers U01134, BC039007, All 88382, N47911, AA035437, BF061039, BG435852; and Fltl (previous nomenclature: VEGFR-1) is seen in Genbank accession number NM002019. sFlt-1 is a soluble form of Flt-1, which lacks the transmembrane and cytoplasmic domains of the full-length Flt-1 (also known as vascular endothelial growth factor receptor 1, VEGFR- 1). sFlt-1 binds to VEGF with high affinity but does not stimulate mitogenesis of endothelial cells. A human soluble FLT-1 (sFLT-1) according to GenBank accession no. AAC50060 has an amino acid sequence of:1 mvsywdtgvl Icallsclll tgsssgsklk dpelslkgtq himqagqtlh Iqcrgeaahk 61 wslpemvske serlsitksa cgrngkqfcs tltlntaqan htgfysckyl avpts kkket 121 esaiyifisd tgrpfvemys eipeiihmte grelvipcrv tspnitvtlk kfpldtlipd181 gkriiwdsrk gfiisnatyk eiglltceat vnghlyktny Ithrqtntii dvqistprpv241 kllrghtlvl nctattplnt rvqmtwsypd eknkrasvrr ridqsnshan ifysvltidk301 mqnkdkglyt crvrsgpsf k svntsvhiyd kafitvkhrk qqvletvagk rsyrlsmkvk361 afpspewwl kdglpateks aryltrgysl iikdvteeda gnytillsik qsnvfknlta 421 tlivnvkpqi yekavssfpd palyplgsrq iltctaygip qptikwfwhp cnhnhsearc 481 dfcsnneesf ildadsnmgn riesitqrma iiegknkmas tlvvadsris giyiciasnk 541 vgtvgrnisf yitdvpngfh vnlekmpteg edlklsctvn kflyrdvtwi llrtvnnrtm 601 hysiskqkma itkehsitln Itimnvslqd sgtyacrarn vytgeeilqk keitirgehc 661 nkkavfsris kfkstrndct tqsnvkh ( S Q ID NO : 1 ) .

[0091] Without wishing to be bound by a particular theory, sFlt-1 acts as a “physiologic sink” to bind to and deplete the trophoblast cells and maternal endothelial cells of functional growth factors required for the proper development and angiogenesis of the fetus and / or the placenta. In some aspects, a lateral flow device described herein is configured for detecting sFlt-1, including one or more of its isoforms or mRNA splicing variants, or its fragments in a biological sample of plasma, serum, or both. In some aspects, a lateral flow device described herein is not configured for detecting full-length Flt- 1 in a biological sample of plasma or serum. In some aspects, a lateral flow device described herein is configured for detecting Fit- 1 , sFlt-1 (including one or more isoforms), or both, in a biological sample of whole blood.

[0092] In some embodiments, symptoms of / diagnostic criteria for pre-eclampsia include: (1) a systolic blood pressure (BP) >140 mmHg or a diastolic BP >90 mmHg on two occasions at least 4 hours apart after 20 weeks gestation; or systolic blood pressure of > 160 mmHg or diastolic blood pressure > 110 mm Hg; and (2) new onset proteinuria (>300 mg of protein in a 24 hour urine collection (or this amount extrapolated from a times collection), or random urine protein / creatinine ratio >0.3, or dipstick reading of 2+ (used only if other quantitative methods not available); or (3) in the absence of proteinuria, new-onset hypertension with the new onset of any of the following: thrombocytopenia: platelet count less than 100 / I O9 / L; renal insufficiency: serum creatinine concentrations greater than 1.1 mg / dL or a doubling of the serum creatinine concentration in the absence of other renal disease; impaired liver function: elevated blood concentrations of liver transaminases to twice normal concentration; pulmonary edema; and new-onset headache unresponsive to medication and not accounted for by alternative diagnoses or visual symptoms. In some embodiments, the symptoms of pre-eclampsia include 2 or more of the 3 aforementioned parameters. In some embodiments, the symptoms of pre-eclampsia include all 3 of the aforementioned parameters. In some embodiments, the symptoms of pre-eclampsia include renal dysfunction and glomerular endotheliosis or hypertrophy. In some embodiments, symptoms of eclampsia further include any of the following symptoms due to pregnancy or the influence of a recent pregnancy: seizures, coma, thrombocytopenia, liver edema, pulmonary edema, or cerebral edema.

[0093] In some embodiments, devices disclosed herein are used to identify preeclampsia with severe features (defined as preeclampsia with severe hypertension (> 160 mmHg systolic or > 110 mmHg diastolic on two occasions at least 4 hours apart) or hypertension with any of the following features: thrombocytopenia (< 100 / I O9platelets / L), renal insufficiency (serum creatinineconcentrations > 1.1 mg / dL or a doubling of the serum creatinine concentration in the absence of other renal disease), cerebral or visual symptoms, impaired liver function (elevated blood concentrations of liver transaminases to more than twice the upper limit normal concentration, e.g., ALT or AST > 80 (U / L), or severe persistent right upper quadrant or epigastric pain unresponsive to medications) or pulmonary edema.

[0094] In some embodiments, devices disclosed herein are used to identify adverse outcomes related to preeclampsia. Adverse maternal outcomes include: elevated liver function tests (aspartate aminotransferase (AST) or alanine aminotransferase (ALT) (> 80 U / L)), low platelet count (<100K / uL), placental abruption (clinical or pathological diagnosis), pulmonary edema, cerebral hemorrhage, convulsion (in the absence of a preexisting seizure disorder), acute renal insufficiency (creatinine >1.1 mg / dL), or maternal death. The adverse fetal / neonatal outcomes include small for gestational age birth weight (<10th percentile for gestational age) with or without abnormal umbilical artery Doppler (absent or reverse flow), fetal death, and neonatal death.Devices

[0095] Various embodiments of present invention provide integrated devices are provided for collection of a biological sample and measurement of sFlt-1 in a plasma or serum sample which can be used by medical caregivers at the point-of-care, out-patient use, in-patient use, or sent to a laboratory.

[0096] In some aspects, the devices are used for collection of a biological sample.

[0097] Various embodiments of the devices include a layer or layers of porous structured materials (e.g., membranes), which allows for transport of molecules through advection, diffusion or a capillary force by fluid, and one or more reagents entrapped or embedded in the porous structured materials, which allows for interaction with one or more target molecules from the biological sample and detection of the presence and / or quantity of the target molecules. In certain aspects, the transport of molecules is along the porous structured materials from one end to the other, the reagents include antibodies or antibody fragment to allow for specificity in the interaction with target molecules, and the detection of the presence and / or quantity of the target molecules is through a detectable label, hence the devices configured for lateral flow immunochromatographic assays.

[0098] In various embodiments, the sFLT-1 can be any one or more isoforms of Flt-1 or fragments thereof, including but are not limited to those encoded by mRNAs of sFlt- 1 -il3 -short, sFltl - il3-long, sFltl-il4, sFltl-e!5a, sFltl-e!5b.

[0099] Various embodiments of the invention provide for a lateral flow device for detection of an analyte comprising one or more circulating fms-like tyrosine kinase 1 (Flt-1) protein isoforms in a biological sample, wherein the lateral flow device comprises:(i) a sample receiving region comprising an about 9-11 mm (length-wise in the direction of lateral flow, i.e., longitudinal axis of the lateral flow device) absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising: about 0.5-1.5x phosphate-buffered saline (PBS), about 0.5-1.5% non-ionic copolymer surfactant (e.g., HO(C2H4O)a(-C3HsO)b(C2H4O)aH wherein a and b are integers (e.g., a is 94-106 and b is 50-62), and optionally, wherein a is 99-101 and b is 55-57), about 1.67-3.67 mg / ml heterophilic blocking reagent (HBR) (e.g., HBR Plus), about 0.5-1.5 mg / ml mouse IgG, and about 0.25-0.75% sucrose;(ii) a conjugate region comprising an about 11-13 mm long absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising about 30.75-36.75 ug / mL biotinylated anti-sFLT-1 antibody (e.g., Biotinylated DuoSet anti-sFLT-1 Capture Antibody), about 5-15% sucrose, about 1-3% trehalose, about 40-60 ug / mL coloring agent (e.g., FD&C blue #1 Powder), and a biotinylated antibody diluent, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising about 0.05-0.15% detectably labeled anti- VEGFR1 antibody (e.g., mAb anti-VEGF R1 DuoSet capture conjugated to about 400nm red latex beads), about 0.0125-0.0375% detectably labeled chicken IgY (e.g., chicken IgY conjugated to about 400nm red latex beads), about 8-12% sucrose, about 1-3% trehalose and a latex storage buffer; preferably the Biotyinylated DuoSet anti-sFLT-1 Capture Antibody and the detectably labeled mAb anti-VEGF R1 DuoSet capture bind to different epitopes of sFLT-1, so as to form a ‘sandwich’ complex: Biotinylated anti-sFLT-1 complexed to sFLT-1 complexed to detectably labeled mAb anti- VEGF Rl;(iii) a test region comprising an about 24-26 mm in-length nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3-0.5 mg / mL polystreptavidin, 0.5- 1.5X PBS, 4-6% sucrose, and the control line having been saturated with about 0.125-0.375mg / mL donkey anti-chicken antibody, 0.5-1.5X PBS, 0.5-1.5% sucrose, 40-60 pg / mL coloring agent (e.g., FD&C Blue powder), preferably the polystreptavidin being immobilized in the test line and the donkey anti-chicken antibody being immobilized in the control line;(iv) a wick region comprising an about 20-22 mm in-length absorbent pad; and(v) a backing card having about 55-65 mm in length, preferably in a position substantially underlaying the sample receiving region, the conjugate region, the test region, and the wick region;wherein each region is in capillary contact with at least one other region thereby permitting a sample fluid to flow from the sample receiving region to the indication region, wherein the sample receiving region overlaps with the conjugate region, the conjugate region overlaps with the test region and the test region overlaps with the wick region.

[0100] Heterophilic blocking reagent (HBR) primarily contains purified immunoglobulins of a specific animal source (e.g., murine origin) with specific binders that neutralize heterophilic antibodies, effectively preventing interference by heterophilic antibodies in immunoassay results. Heterophilic antibodies are endogenous antibodies found in patients’ serum / plasma which can bind to immunoglobulins of other species, including the species used to generate the antibodies used as reagents for immunoassays. These antibodies can interfere in immunoassay, causing a spurious elevation of measured value that is independent of the true analyte concentration, thus potentially misclassifying samples. Therefore, HBR is used in preferred embodiments as a blocking reagent in the lateral flow device. Compared to conventional, passive blocking agents that use nonspecific substance (e.g., mouse IgG, mouse serum, nonspecific monoclonal antibodies, aggregated IgG, etc.), HBR accomplishes the blocking by steric hinderance, and is effected by specific binders, such that with HBR, less protein is required for blocking more false positives, and HBR blocks all anti-species (anti-rabbit, anti-goat, anti-sheep, as well as anti-mouse) and all anti-subtypes of mouse monoclonals (anti-mouse IgGl, anti-mouse IgG2, etc.) for HBR of murine origin, for example. For example, Scantibodies Laboratory provides several HBRs: HBR, HBR 1, HBR-1, HBR-2, HBR-3, HBR-6, HBR-9, HBR-11, and HBR-Plus.

[0101] In some embodiments, HBR is included in the sample pad block solution. In some embodiments, the sample pad block solution includes both an HBR and mouse IgG as blocking agents against heterophilic antibodies. In other embodiments, the sample pad block solution does not include HBR. In other embodiments, another heterophilic blocking agent such as those described in WO2016154250 is included in the sample pad block solution.

[0102] FD and C Blue No.1, also known as acid blue 9, brilliant blue FCF, or erioglaucine, is a compound having CAS number 3844-45-9. Alternative coloring agents may be used including but not limited to allura red AC, erythrosine, saffron, spirulina, sunset yellow FCF, anthocyanin, or canthaxanthin.

[0103] Various embodiments of the invention provide for a lateral flow device for detection of an analyte comprising one or more circulating Fit- 1 protein isoforms in a biological sample, wherein the lateral flow device comprises:(i) a sample receiving region comprising an about 9-11 mm (length-wise in the direction of lateral flow, i.e., longitudinal axis of the lateral flow device) absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising all or two ormore of: a non-ionic copolymer surfactant, an HBR, mouse IgG, and sucrose, in phosphate buffered saline, wherein the non-ionic copolymer surfactant, the HBR, the mouse IgG, and the sucrose are at a ratio, if present, of: about 0.5-1.5 w / v % non-ionic copolymer surfactant (e.g., HO(C2H4O)a(-C3lEO)b(C2H4O)aH wherein a and b are integers (e.g., a is 94-106 and b is 50-62), and optionally, wherein a is 99-101 and b is 55-57) to about 1.67-3.67 mg / ml heterophilic blocking reagent (HBR) (e.g., HBR Plus) to about 0.5-1.5 mg / ml mouse IgG to about 0.25-0.75 w / v % sucrose;(ii) a conjugate region comprising an about 11-13 mm long absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising biotinylated anti-sFLT-1 antibody (e.g., Biotinylated DuoSet anti-sFLT- 1 Capture Antibody) having an anti-sFLT-1 antibody content of about 30.75-36.75 ug / mL, about 5- 15% sucrose, about 1-3% trehalose, about 40-60 ug / mL coloring agent (e.g., FD&C blue #1 Powder), and a biotinylated antibody diluent, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising a detectably labeled anti-VEGFRl antibody (e.g., mAb anti-VEGF R1 DuoSet capture conjugated to about 400nm red latex beads) having an anti-VEGFRl antibody content of about 0.05-0.15 w / v %, a detectably labeled chicken IgY (e.g., chicken IgY conjugated to about 400nm red latex beads) having a chicken IgY content of about 0.0125-0.0375% w / v, about 8-12 w / v % sucrose, about 1-3 w / v% trehalose, and a latex storage buffer; preferably the Biotyinylated DuoSet anti-sFLT-1 Capture Antibody and the detectably labeled mAb anti-VEGF R1 DuoSet capture bind to different epitopes of sFLT-1, so as to form a ‘sandwich’ complex: Biotinylated anti-sFLT-1 complexed to sFLT-1 complexed to detectably labeled mAb anti- VEGF Rl;(iii) a test region comprising an about 24-26 mm in-length nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3-0.5 mg / mL polystreptavidin, 0.5- 1.5X PBS, 4-6% sucrose, and the control line having been saturated with about 0.125-0.375mg / mL donkey anti-chicken antibody, 0.5-1.5X PBS, 0.5-1.5% sucrose, 40-60 pg / mL coloring agent (e.g., FD&C Blue powder), preferably the polystreptavidin being immobilized in the test line and the donkey anti-chicken antibody being immobilized in the control line;(iv) a wick region comprising an about 20-22 mm in-length absorbent pad; and(v) a backing card having about 55-65 mm in length, preferably in a position substantially underlaying the sample receiving region, the conjugate region, the test region, and the wick region;wherein each region is in capillary contact with at least one other region thereby permitting a sample fluid to flow from the sample receiving region to the indication region, wherein the sample receiving region overlaps with the conjugate region, the conjugate region overlaps with the test region and the test region overlaps with the wick region.

[0104] In various embodiments, the lateral flow device comprises:(i) a sample receiving region comprising an about 9, 10 or 11 mm length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising: about 0.6X, 0.7X, 0.8X, 0.9X, 1.0X, 1.1X, 1.2X, 1.3X, 1.4X, or 1.5X phosphate-buffered saline (PBS), about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, or 1.4%, non-ionic copolymer surfactant (e.g., HO(C2H4O)a(-C3HfiO)b(C2H4O)aH wherein a and b are integers (e.g., a is 94-106 and b is 50-62), and optionally wherein a is 99-101 and b is 55-57), about 1.67 mg / ml, 1.87 mg / ml, 2.07 mg / ml, 2.27 mg / ml, 2.47 mg / ml, 2.67 mg / ml, 2.87 mg / ml, 3.07 mg / ml, 3.27 mg / ml, or 3.47 mg / ml heterophilic blocking reagent (HBR) (HBR Plus), about 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1.0 mg / ml, 1.1 mg / ml, 1.2 mg / ml, 1.3 mg / ml, 1.4 mg / ml, or 1.5 mg / ml mouse IgG, and about 0.35%, 0.45%, 0.55%, 0.65% or 0.75% sucrose;(ii) a conjugate region comprising an about 11, 12 or 13 mm absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising about 31.5 pg / mL, 31.75 pg / mL, 32.75 pg / mL, 33.75 pg / mL, 34.75 pg / mL, 35.75 pg / mL, or 36.75 pg / mL Biotinylated DuoSet anti-sFLT-1 Capture Antibody, about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% sucrose, about 1%, 1.5%, 2% or 3% trehalose, about 40, 45, 50, or 60 pg / mL FD&C blue #1 Powder, and biotinylated antibody diluent, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising about 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, or 0.15% mAb anti-VEGF R1 DuoSet capture conjugated to about 400nm red latex beads, about 0.015%, 0.025%, 0.035% or 0.0375% chicken IgY conjugated to about 400nm red latex beads, about 9%, 10%, 11%, or 12% sucrose, about 1%, 2%, or 3% trehalose and latex storage buffer;(iii) a test region comprising an about 24 mm, 25 mm or 26 mm length nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3 mg / mL, 0.4 mg / mL, or 0.5 mg / mL poly streptavidin, 0.6X, 0.7X, 0.8X, 0.9X, 1.0X, 1.1X, 1.2X, 1.3X, 1.4X, or 1.5X PBS, 5%, 5.5% or 6% sucrose, and the control line having been saturated with about 0.13 mg / mL, 0.14 mg / mL, 0.15mg / mL, 0.25 mg / mL, or 0.375 mg / mL donkey anti-chicken antibody, 0.6X, 0.7X, 0.8X, 0.9X, 1.0X, 1.1X, 1.2X, 1.3X, 1.4X, or 1.5X PBS, 0.65%, 0.75%, 0.85%, 0.95%, 1.05%, 1.15%, 1.25%, 1.35%, 1.45%, or 1.5% sucrose, 40 pg / mL, 45 pg / mL, 50 pg / mL, or 55 pg / mL FD&C Blue powder,(iv) a wick region comprising an about 20 mm, 21 mm or 22 mm length absorbent pad; and(v) a backing card having about 55 mm, 60 mm, 65 mm in length; wherein each region is in capillary contact with at least one other region thereby permitting a sample fluid to flow from the sample receiving region to the indication region, wherein the sample receiving region overlaps with the conjugate region, the conjugate region overlaps with the test region and the test region overlaps with the wick region.

[0105] In various embodiments,(i) the sample receiving region comprises an about 10 mm length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising about lx PBS, about 1% non-ionic copolymer surfactant HO(C2H4O)a(-C3HfiO)b(C2H4O)aH wherein a is 99- 101 and b is 55-57, about 2.67 mg / ml heterophilic blocking reagent (HBR) (HBR Plus), about 1 mg / ml mouse IgG, and about 0.5% sucrose;(ii) the conjugate region comprises an about 12 mm absorbent pad having with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising about 33.75 ug / mL Biotinylated DuoSet anti-sFLT-1 Capture Antibody, about 10% sucrose, about 2% trehalose, about 50 ug / mL FD&C blue #1 Powder, and biotinylated antibody diluent, and an anti-sFLT-l / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising an anti-sFLT-l / control final conjugate solution comprising about 0.1% mAb anti-VEGF R1 DuoSet capture conjugated to 400nm red latex beads, about 0.025% chicken IgY conjugated to 400nm red latex beads, about 10% sucrose, about 2% trehalose and latex storage buffer;(iii) the test region comprises an about 25 mm length nitrocellulose CN95 membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.4 mg / mL polystreptavidin, about IX PBS, about 5% sucrose, and the control line having been saturated with about 0.25 mg / mL donkey anti-chicken antibody, about IX PBS, about 1% sucrose, about 50 ug / mLFD&C Blue powder,(iv) the wick region comprises an about 21 mm length absorbent pad; and(v) the backing carding having about 60 mm in length and optionally having cuts located 9, 16 and 46mm from the bottom edge of the backing card.

[0106] In various embodiments, the biotinylated antibody diluent comprises about 0.5-1.5% bovine serum albumin and 0.5-1.5X PBS.

[0107] In various embodiments, the biotinylated antibody diluent comprises about 1% bovine serum albumin and IX PBS.

[0108] In various embodiments, the latex storage buffer comprises about 0.5 -1.5% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 25-75 mM boric acid.

[0109] In various embodiments, the latex storage buffer comprises about 1% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 50 mM boric acid.

[0110] In various embodiments, the mAb anti-VEGF R1 DuoSet Capture conjugated to 400nm red latex beads are prepared from about 8-12 mg of about 400nm Red carboxylated polystyrene (PS) latex particles, 0.125-0.375 mg of Human VEGFRl / Flt-1 Antibody (Clone #49560) (about 0.125- 0.375 mg Ab / mL of 1% Conj.; about 40: 1) [mg Ab / mL], about 0.05-0.15MMES Buffer pH 6.0, about 40-60 mM of borate buffer, about 14-16 mg / mL of ED AC, about 40-60 mg / mL of NHS, about 0.5- 1.5X PBS, and about 40-60 nM and 0.5-1.5% of latex storage buffer. In some embodiments, the colored latex bead-conjugated mAb anti-VEGFRl antibody has a weight ratio of mAb anti-VEGFRl antibody : colored latex bead being about 40 : 1. In some embodiments, the red latex bead-conjugated mAb anti-VEGFRl antibody has a weight ratio of mAb anti-VEGFRl antibody : red latex bead being from 100: 1 to 10 : 1.[OHl] In various embodiments, the mAb anti-VEGF R1 DuoSet Capture conjugated to 400nm red latex beads comprises, or are prepared from, about 8 mg, 9 mg, 10, 11 mg, or 12 mg of about 400nm Red carboxylated PS latex particles, 0.125 mg, 0.2mg, 0.25 mg, or 0.375 mg of Human VEGFRl / Flt-1 Antibody (Clone #49560) (about 0.125 mg, 0.2mg, 0.25 mg, or 0.375 mg Ab / mL of 1% Conj.; about 40: 1) [mg Ab / mL], about 0.05 M, 0.075 M, 0.1 M or 0.15M MES Buffer pH 6.0, about 40 mM, 45 mM, 50 mM, 55 mM or 60 mM of borate buffer, about 14 mg / mL, 15 mg / mL, or 16 mg / mL of EDAC, about 40 mg / mL, 50 mg / mL, 55 mg / mL, or 60 mg / mL of NHS, about 0.6X, 0.7X, 0.8X, 0.9X, LOX, 1.1X, 1.2X, 1.3X, 1.4X, or 1.5X PBS, and about 40 mM, 45 mM, 50 mM, 55 mM or 60 mM and 0.5%, 1.0%, 1.2%, or 1.5% of latex storage buffer.

[0112] In various embodiments, the mAb anti-VEGF R1 DuoSet Capture conjugated to 400nm red latex beads comprises about 10 mg of 400nm Red carboxylated PS latex particles, 0.25mg of Human VEGFRl / Flt-1 Antibody (Clone #49560) (0.25 mg Ab / mL of 1% Conj.; 40: 1) [mg Ab / mL], about 0.1M MES Buffer pH 6.0, about 50 mM borate buffer, about 15 mg / mL of EDAC, about 50 mg / mL of NHS, about IX PBS, and about 50nM and 1% of latex storage buffer.

[0113] In various embodiments, the Chicken IgY Conjugated to 400nm red latex beads comprises or are prepared from about 8-12 mg of about 400 nm Red Carboxylated Latex, 0.125-0.375 mg of Chicken IgY, 0.05-0.15M MES Buffer pH 6.0, about 40-60 mM Borate Buffer pH 8.6, about 13-17 mg / mL EDAC, about 40-60 mg / ML NHS, 0.5-1.5X PBS, and Latex Storage Buffer.

[0114] In various embodiments, the Chicken IgY Conjugated to 400nm red latex beads comprises or are prepared from about 9 mg, 10 mg, 11 mg, or 12 mg of about 400 nm Red Carboxylated Latex, 0.125 mg, 0.2 mg, 0.25 mg, or 0.375 mg of Chicken IgY, 0.05 M, 0.1 M, 0.12 M, or 0.15M MES Buffer pH 6.0, about 40 mM, 50 mM, 55 mM or 60 mM Borate Buffer pH 8.6, about 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, or 17 mg / mL EDAC, about 40 mg / mL, 45 mg / mL ,50 mg / mL, 55 mg / mL, or 60 mg / mL NHS, 0.5X, 0.75X, LOX, 1.1X, 1.2X, 1.3X, 1.4X or 1.5X PBS, and Latex Storage Buffer.

[0115] In various embodiments, the Chicken IgY Conjugated to 400nm red latex beads comprises about 10 mg of about 400 nm Red Carboxylated Latex, 0.25 mg of Chicken IgY, 0.1M MES Buffer pH 6.0, 50mM Borate Buffer pH 8.6, about 15 mg / mL EDAC, about 50 mg / ML NHS, IX PBS, and Latex Storage Buffer. In some embodiments, the colored latex bead-conjugated chicken IgY has a weight ratio of colored latex bead : chicken IgY being about 40 : 1. In some embodiments, the red latex bead-conjugated chicken IgY has a weight ratio of red latex bead : chicken IgY being from 100: 1 to 10 : 1.

[0116] In various embodiments, the anti-sFLT-l / control final conjugate solution comprises the detectably labeled mAb anti-VEGF R1 and the detectably labeled chicken IgY at a weight ratio from 10:1 to 2: 1, stored in the latex storage buffer in the presence of sucrose and trehalose. In various embodiments, the anti-sFLT-l / control final conjugate solution comprises the red latex bead- conjugated mAb anti-VEGF R1 and the red latex bead-conjugated chicken IgY at a weight ratio of about 4: 1.

[0117] In various embodiments, latex storage buffer comprises about 50 mM of Borate pH 8.6, and about 1% of Casein.

[0118] In various embodiments, the sample receiving region comprises an absorbent pad or sample pad based on a material that preferably is a hydrophilic material which facilitates absorption and transport of a fluid sample. Cotton fibers, rayon fibers, glass fibers, or a combination thereof are exemplary materials to form a sample receiving component / region. Further embodiments provide the sample receiving components / regions are preferably presaturated, coated or treated with a block solution containing a salt (e.g., PBS), a surfactant (e.g., non-ionic copolymer such as HO(C2lLO)a(- C3H6O)b(C2H4O)aH wherein a is 99-101 and b is 55-57), a carrier protein (e.g., mouse IgG), and / or a heterophilic antibody blocking reagent.

[0119] In various embodiments, the conjugate region includes a material in the form of a conjugate pad which facilitates transport of free antibodies or antibody fragments or a complex formed by analyte-antibody or analyte-antibody fragment to a nearby or underlying chromatographic substrate, allowing interaction or binding between analyte and antibody (or antibody fragment). In some aspects, the conjugate pad has overlapping contact in one area with the sample pad. In other aspects, the conjugate pad does not form an individual region, as a conjugate region is merged withthe sample receiving region; and hence, the device may include a sample pad and a conjugate pad in a combined sample receiving (and development) region.

[0120] In various embodiments, the test region includes a chromatographic substrate, such as a chromatographic membrane (also called test membrane). For example, microporous nitrocellulose membrane (CN95) is a suitable material for test region. CN140 membrane and CN150 membrane are alternative materials for the test region.

[0121] In some embodiments, a sample pad includes polyester fibers such as Alhstrom grade 6614 pad; and the conjugate region comprises an absorbent pad or conjugate pad based on chopped glass with fiber such as Alhstrom grade 8980, and the wick region comprises an absorbent pad based on Alhstrom grade 243. Alhstrom grade 6614 pad has a basis weight of 75 g / m2, caliper of 0.42 mm, a wicking rate of 5s / 2cm, and a water absorption capacity of 57 mg / cm2. Alhstrom grade 8980 pad has a basis weight of 80 g / m2, caliper of 0.42 mm, a wicking rate of 2.2s / 4cm, and a water absorption capacity of 56 mg / cm2. Other grades of sample pads include grade 121 based on binder-free microglass, grade 141 based on binder-free microglass, grade 142 based on microfiber glass with binder, grade 8950 / 8951 / 8964 / 8980 based on chopped glass with binder, grade 601 / 238 / 237 / 319 based on cotton, grade 1281 based on cotton / rayon blend, and grade 1667 based on blend of fiber.

[0122] In various embodiments, a lateral flow device contains a test strip housed in a generally rigid cassette, wherein the test strip comprises the sample receiving region, the conjugate region, the test region, the wick region, and the backing card, and wherein the cassette contains a sample receiving aperture (sample port) for receiving sample to the sample receiving region of the test strip, and a test window opening for visualizing or detection of the test line and the control line.

[0123] Various embodiments of the invention provide for a kit, comprising: a lateral flow device of the present invention as described herein; and a chase buffer. Various embodiments provide for a kit, comprising: a lateral flow device, a chase buffer, and two vials of FLT-1 or sFLT-1 of known amounts, wherein the known amounts of the FLT-1 or sFLT-1 are for use as a quality control. Various embodiments of the kit further include a microSD card that contains calibration information specific to the lot of the lateral flow device in the kit, and QC allowable values, which is to be uploaded to an Analyzer (test reader device).

[0124] In various embodiments, the chase buffer comprises non-ionic copolymer surfactant (e.g., HO(C2FLO)a(-C3FLO)b(C2H4O)aH wherein a and b are integers (e.g., a is 94-106 and b is SO- 62), and optionally, wherein a is 99-101 and b is 55-57), Potassium Chloride, IX PBS, and 2-Methyl- 4-isothiazolin-3-one solution.

[0125] In various embodiments, the kit further comprises a low external control, a high external control, or both.

[0126] In various embodiments, the low external control comprises recombinant sFLT-1 titrated to produce average test line (TL) values 0% - 20% lower than the average TL value of the pooled plasma LOW RISK Lot Qualifier QC which is comprised by 2.4 ng / mL sFLT-1, and the high external control comprises recombinant sFLT-1 titrated to produce average TL values 0% - 20% higher than the average TL value of the pooled plasma HIGH RISK Lot Qualifier QC which is comprised of 3.6 ng / mL sFLT-1. That is, in some embodiments, the low external control comprises recombinant sFLT-1 titrated to produce an average TL value between 1.92 ng / mL and 2.4 ng / mL, i.e., 20%-0% lower than 2.4 ng / mL sFLT-1. In other embodiments, the low external control comprises recombinant sFLT-1 titrated to produce an average TL value between 1.5 ng / mL and 2.5 ng / mL, e.g., a TL value of sFLT-1 at about 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2., 2.3, 2.4, or 2.5 ng / mL. In some embodiments, the high external control comprises recombinant sFLT-1 titrated to produce an average TL value between 3.60 ng / mL and 4.32 ng / mL, i.e., 0%-20% higher than 3.6 ng / mL sFLT-1. In other embodiments, the high external control comprises recombinant sFLT-1 titrated to produce an average TL value between 3.5 ng / mL and 5.0 ng / mL, e.g., a TL value of sFLT-1 at about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 ng / mL.

[0127] In various embodiments, the kit further comprises a quality control diluent.

[0128] In various embodiments, the quality control diluent comprises 0.5-1.5X PBS, 0.09- 1.15% 2-Methyl-4-isothiazolin-3-one solution, about 4-6% Sucrose, about 0.5-1.5% Trehalose, about 40-50 mg / mL BSA, about 90-110 mM EDTA, and about 18-22 USP / mL Li Heparin salt.

[0129] In various embodiments, the quality control diluent comprises 0.5X, 0.75X, LOX, 1.1X, 1.2X, 1.3X, 1.4X or 1.5X PBS, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14% or 1.15% 2-Methyl- 4-isothiazolin-3-one solution, about 4.5%, 5%, 5.5%, or 6% Sucrose, about 0.5%, 0.6%, 0.7%, 0.75%, 1.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5% Trehalose, about 40 mg / mL, 42mg / mL, 45 mg / mL, or 50 mg / mL BSA, about 90 mM, 92 mM, 95 mM, 100 mM, 105 mM, or 110 mM EDTA, and about 18, 19, 20, 21 or 22 USP / mL Li Heparin salt.

[0130] In various embodiments, the quality control diluent comprises IX PBS, 0.105% 2- Methyl-4-isothiazolin-3-one solution, about 5% Sucrose, about 1% Trehalose, about 45 mg / mL BSA, about 100 mM EDTA, and about 20 USP / mL Li Heparin salt.

[0131] Various embodiments of the invention provide for an analyzer for analyzing a lateral flow device, comprising: an opening to receive a lateral flow device of the present invention; a barcode scanner; an illumination light-emitting diode (LED); a camera, lens, filter assembly to record the image of a lateral flow strip in the lateral flow device;a battery; a display on the outside of the analyzer; and a computer readable medium comprising computer executable instructions to process the image, integrate the test line (TL) signal, perform background subtraction, calculate background- subtracted TL signal, convert the TL signal into sFLT plasma or serum concentrations.

[0132] Various embodiments of the invention provide for an analyzer for analyzing a lateral flow device, comprising: an opening to receive a lateral flow device of the present invention; an illumination light-emitting diode (LED); a camera, lens, filter assembly to record the image of a lateral flow strip in the lateral flow device; a battery or electric cord; a display on the outside of the analyzer; and a printed circuit, wherein a computer readable medium comprising computer executable instructions is operable with the analyzer to process the image, integrate the test line (TL) signal, perform background subtraction, calculate background-subtracted TL signal, convert the TL signal into sFLT plasma or serum concentrations.

[0133] In various embodiments, the analyzer further comprises one or more of a pSD card port; a battery port; a USB port; or a printer.

[0134] In various embodiments, a camera - also referred to as a photodetector - may be in the form of an imaging sensor. Detectable signal resulting from illumination of the portion of the lateral flow test strip visible through the LFA window may be captured for analysis by the camera (or photodetector).

[0135] In various embodiments, a lens is provided as a component of an optical stack comprising the camera, lens, fdter assembly, because imaging sensors may have an image area that is on the order of a few millimeters while the desired field of view is much larger. As the required distance from an object and the desired field of view (typically the size of the object with additional buffer space) are known quantities, the lens focal length can be selected by the skilled artisan. A lens mount can be provided to mount the lens in the optical stack.

[0136] In various embodiments, an optical stack can provide a filter, or the camera, lens, filter assembly includes a filter. This filter may be selected to remove the excitation wavelength in a fluorescence-based assay, to select a desired emission wavelength to reach the imaging sensor, or both, and may be provided as a single filter or multiple filters in a stack. A filter mount can be provided to position the filter in the optical stack.

[0137] In various embodiments, the analyzer further comprises a processor or processing component, operably linked to the light source or illuminating LED and the camera of the camera, lens, filter assembly, and configured to i) control the illumination of the visible portion of the test strip by the light source, (ii) receive an electrical signal from the at least one image sensor resulting from the detectable signal, and (iii) convert the electrical signal into an assay result indicative of the presence or amount of the analyte of interest in the sample. In various aspects, the processor is operable via the computer executable instructions. The captured image can be analyzed e.g., by the processor in the analyzer or a separate analysis computer (e.g., a smartphone or other computing device). Identification of the test and / or control lines in the image can be determined using a peak search algorithm on the captured image.

[0138] In various aspects, the display is operably connected to the processor to display the assay result.

[0139] Various embodiments of the invention provide for a system, comprising a kit of the present invention as described herein; and an analyzer of the present invention as described herein, configured to measure the intensity of the test line, control line, or both on the lateral flow device.

[0140] In various embodiments, the system further comprises an analyzer control cassette configured to test the analyzer’s electronics and photometric detection system.Orientations

[0141] In some embodiments, liquid in the biological samples carrying analytes along with a chase buffer moves from the chromatographic substrate to a wick or absorbent pad.

[0142] In some embodiments, the indication region / component (also referred to as the test region) of the devices can be configured to direct flow of a liquid through the indication region / component in a generally horizontal orientation, e.g., substantially along a single horizontal plane from a first end of the test region / component to the second end of the test region / component.

[0143] Further embodiments of the devices are provided in a kit with reagents provided below, such as one or more of antibodies against target molecules and against control, chase buffer, additives, containers, and instruction manuals.

[0144] Cut-off values, also referred to designated clinical threshold, for the semi-quantitative assay in the devices are designated for assigning a positive value when the concentration of the analyte in a sample exceeds the cut-off value to indicate a predisposition or high likelihood to develop the symptom or condition that the analyte is associated with, or for assigning a negative value when the concentration of the analyte in a sample is below the cut-off value to indicate a low likelihood or no sign of developing the symptom or condition that the analyte is associated with.

[0145] In some embodiments, the measuring of the analyte includes using a densitometer. In various embodiments, the cut-off value chosen for a densitometer can be chosen to correlate the densitometer units to a cut-off value that is a concentration of any one or more isoforms of Fit- 1 or fragments thereof (soluble or membrane bound). In various embodiments, the cut-off value chosen for a densitometer can be chosen to correlate the densitometer units to a cut-off value that is a concentration of any one or more isoforms of Fit- 1 or fragments thereof (soluble or membrane bound), and the development / run time on the lateral flow device of the present invention; for example, about 20-30 minutes; particularly, it can be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes. Particular embodiments can be about 25 minutes.

[0146] Devices disclosed herein are provided to allow for sensitivity and specificity of the assay and to be able to distinguish around a defined positive / negative threshold reproducibly in a semi- quantitative (threshold) assay format.

[0147] The devices in various embodiments are self-contained, disposable, single-use devices. Under various conditions, the devices are used for conducting an assay and providing visual result within 30 minutes, 25 minutes, 20 minutes, 15 minutes, 14 minutes, 13 minutes, 12 minutes, 11 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes or 1 minute. In various instances, the visual indication and result of the assay on the devices are stable for at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes after the assay is completed, such that in methods of using the devices to determine likelihood or unlikelihood of having preeclampsia or sPE, results in the indication region can be read or visually examined immediately after the assay is completed or, even re-read or re-examined in about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes after the assay is completed. For example, the assay is completed in 25 minutes, and the results are stable for at least 30 minutes thereafter. In many instances, the devices have a shelf-life of about one month, two months, three months, four months, or longer, or at least two months, when stored at room temperature, preferably in a sterile package. In other embodiments, the devices have a shelf-life of about five months, 6 months, 7 months 8 months, 9 months 10 months, 11 months, 12 months or longer. In other instances, the devices are also stable and can be stored in refrigeration (e.g., at 4 °C) for about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 months, or at least for 12 months. In other instances, the devices are also stable and can be stored in refrigeration (e.g., at 4 °C) for about 1 year, 1.5 years, 2 years, 2.5 years, 3 years or longer.

[0148] The devices in various embodiments have a dimension or size that is portable. In one embodiment, the devices or at least the sample receiving region of the devices are configured to placement into the mouth of a human, and / or underneath the tongue, such that saliva is in contact with the sample receiving region of the devices.

[0149] Various aspects of the devices utilize a plasma or serum sample of a volume of about 10 pL, 15 pL, 20 pL, 30 pL, 40 pL, 50 pL, 100 pL, 200 pL, 300 pL, 400 pL, 500 pL, 1 mL, 2 mL, 3 mL, 4 mL, 5 mL or 10 mL of a sample. In some aspects, a device requires 10-20 pL, 20-30 pL, 30-40 pL, 40-50 pL, 50-100 pL, 100-200 pL, 200-300 pL, 300-400 pL, 400-500 pL, 500 pL-1 mL, 1-2 mL, 2-3 mL, 3-4 mL, 4-5 mL, or 5-10 mL. In some aspects, a lateral flow device disclosed herein requires 10-20 pL of plasma sample mixed with about 60-80 pL diluent. In some aspects, a lateral flow device disclosed herein requires about 15 pL of plasma sample mixed with about 80 pL diluent or chase buffer.

[0150] Further embodiments provide methods of manufacturing a lateral flow device for detecting fms-hke tyrosine kinase 1 (Flt-1) protein fragments or isoforms including sFltl isoforms. The methods include: (1) providing a base and providing a substrate positioned above the base, the substrate defining: a sample receiving region, an indication region (also referred to as a test region), and optionally a development region (also referred to as a conjugation / conjugate region) positioned between (or overlapping with each of) the sample receiving region and the test region, wherein the development region comprises capture antibody and label antibody against sFLT-1 and allowing for binding and complexing to form a ‘sandwich’: capture antibody - sFLT-1 - label antibody, and wherein each region comprises a porous material and is in capillary contact with at least one other region, thereby permitting a fluid to wick from the sample receiving region to the test region; (2) immobilizing a modification (e.g., streptavidin or avidin) capable of binding the capture antibody at a first location (in various instances denoted as a ‘test line’) in the test region, wherein the capture antibody comprises a monoclonal or polyclonal antibody specifically immunoreactive with sFlt-1, or an antigen-binding fragment of the antibody, conjugated with for example biotin, wherein the modifications on the test line and on the capture antibody can be members of any binding pairs; (3) providing a label antibody comprising a detectable label and an antibody or fragment thereof capable of binding the sFlt-1, preferably binding at a different epitope than the capture antibody, wherein the capture antibody, sFlt-1 in a sample which is applied to the device, and the detection antibody form a complex (‘sandwich’), and wherein the complex is capable of being transported from the development region to the test region.

[0151] In further aspects, the methods of manufacturing a lateral flow device further include (4) providing at a second location (in various instances denoted as a ‘control line’) in the indication region a second capture reagent, (5) providing a second detection reagent capable of being transported to the indication region, (“detection” reagent referring to having a detectable label,) wherein the second detection reagent is capable of binding a house-keeping molecule in the fluid sample or the second detection reagent is an immunoglobulin of a different species than the sample source, and that the second detection reagent is pre-saturated in at least a portion of the development region, wherein the second detection reagent is not cross-reactive with Flt-1, with the label antibody for Flt-1, or with thecapture antibody for Flt-1, and (6) further providing an end flow region (also referred to as a wick region) comprising a porous material and positioned such that a fluid is conducted from the sample receiving region through the indication region.Detection and / or Diagnostic Assays and Methods

[0152] Assays are provided for detecting Flt-1 in a plasma or serum sample obtained from a subject using the devices disclosed herein. The assays include contacting plasma or serum sample from the subject with the sample receiving region of the devices, and determining the presence or absence of a signal in the indication region where anti-sFlt-1 antibody is immobilized, wherein the presence of a signal in the indication region where anti-sFlt-1 antibody is immobilized detects the presence of sFlt-1 in the sample, and the absence of a signal in the indication region where anti-sFlt-1 antibody is immobilized indicates sFlt-1 is not detected in the sample.

[0153] Assays are provided for detecting and quantifying sFlt-1 in a plasma or serum sample obtained from a subject using the devices disclosed herein. The assays include contacting the plasma or serum from the subject with the sample receiving region of the devices, and quantifying the level a signal in the indication region where anti-sFlt-1 antibody is immobilized.

[0154] In various embodiments, the assays are intended for a pregnant woman, a pregnant woman at risk of hypertensive disorder, a pregnant woman at risk of preeclampsia and / or eclampsia, a pregnant woman having hypertensive disorder, or a pregnant woman having preeclampsia or eclampsia. In various embodiments, the assays are intended for postpartum women. In various embodiments, the assays are intended for a pregnant woman between 23 0 / 7 weeks to 34 6 / 7 weeks of gestation. In various embodiments, the assays are intended for a pregnant woman between 23 0 / 7 weeks to 34 6 / 7 weeks of gestation with hypertensive disorder.

[0155] In various embodiments, the assays are intended for postpartum women who are about 1 week, 2 weeks, 3 weeks, and / or 4 weeks postpartum. In various embodiments, the assays are intended for postpartum women who are about 1 month 2 months and / or 3 months post-partum. In various embodiments, the assays are intended for postpartum women who are more than 3 months postpartum.

[0156] Various embodiments provide the devices afford an “assay sensitivity” that is 100% or at least 99%, 98%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% or 70%. “Assay sensitivity” can be defined as the percentage of true positive incidence over a total incidence of true positive and false negative; for example, in detecting sFlt-1. Various embodiments provide the devices afford an “assay specificity” that is 100% or at least 99%, 98%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60% or 50%. “Assay specificity” can be defined as the percentage of true negative incidence over a total incidence of false positive and true negative. Various embodiments provide the devices afford an assay positive predictive value that is 100% or at least99%, 98%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60% or 50%. Assay positive predictive value can be defined as the percentage of true positive incidence over a total incidence of true positive and false positive. Various embodiments provide the devices afford an assay negative predictive value that is 100% or at least 99%, 98%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60% or 50%. Assay negative predictive value can be defined as the percentage of true negative incidence over a total incidence of true negative and false negative.

[0157] Various embodiments of the invention provide for a method of detecting a level soluble fms-hke tyrosine kinase 1 (sFlt-1) in a plasma or serum sample with a lateral flow device of the present invention as described herein, the method comprising: applying a plasma or serum sample to the sample receiving region of the lateral flow device; applying a chase buffer to the sample receiving region of the lateral flow device; measuring the level of the red latex beads at the first location in the indication region; calculating the level of sFlt-1 based on the measured level of the red latex beads.

[0158] In various embodiments, measuring the level of the red latex beads is performed at about 20-30 minutes after applying the plasma or serum sample to the sample receiving region. In various embodiments, measuring the level of the red latex beads is performed at about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes after applying the plasma or serum sample to the sample receiving region. In various embodiments, measuring the level of the red latex beads is performed at about 25 minutes after applying the plasma or serum sample to the sample receiving region.

[0159] In various embodiments, the biological sample is from a pregnant woman. In various embodiments, the biological sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant. In various embodiments, the biological sample is from a pregnant woman who is between 23 0 / 7 weeks to 30 6 / 7 weeks pregnant. In various embodiments, the biological sample is from a pregnant woman who is between 31 0 / 7 weeks to 34 6 / 7 weeks pregnant.

[0160] In various embodiments, measuring the level of the red latex beads at the first location in the indication region, or calculating the level of sFlt-1 based on the measured level of the red latex beads, or both is performed with an analyzer of the present invention as described herein.

[0161] Various embodiments provide for a method of prognosticating a woman’s risk of having preeclampsia with severe features (sPE), comprising assaying a plasma or serum sample obtained from the women to detect a level soluble fms-hke tyrosine kinase 1 (sFlt-1) with a lateral flow device of the present invention as described herein, the method comprising: applying (a) a mixture of a plasma or serum sample with a chase buffer, i.e., a mixture sample, or (b) the plasma or serum sample and the chase buffer, sequentially or concurrently, to the sample receiving region of the lateral flow device, preferably (a) is applied; measuring the level of the red latex beads at the first location (in various instances denoted as a test line) in the indication region (also referred to as a testregion); calculating the level of sFlt-1 based on the measured level of the red latex beads; and prognosticating the women’s risk of having sPE as high when the level of sFlt-1 is higher than a reference level or prognosticating the women’s risk of having sPE as low when the level of sFlt-1 is less than the reference level, wherein the reference level is 2.0-6.0 ng / mL, and wherein a high risk of having sPE is having sPE within three weeks from having her plasma or serum sample assayed. In various embodiments, the reference level is about 2.0 ng / mL, 2.2 ng / mL, 2.4 ng / mL, 2.6 ng / mL, 2.8 ng / mL, 3.0 ng / mL, 3.2 ng / mL, 3.4 ng / mL, 3.6 ng / mL, 3.8 ng / mL, 4.0 ng / mL, 4.2 ng / mL, 4.4 ng / mL, 4.6 ng / mL, 4.8 ng / mL, 5.0 ng / mL, 5.2 ng / mL, 5.4 ng / mL, 5.6 ng / mL, 5.8 ng / mL or 6.0 ng / mL. In various embodiments, the reference level is about 2.0-3.0 ng / mL. In various embodiments, the reference level is about 2.0-4.0 ng / mL. In various embodiments, the reference level is about 2.0-5.0. In various embodiments, the reference level is about 2.91, 2.92, 2.93, 2.94, 2.95, 2.96, 2.97, 2.98, 2.99, 3.0, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, or 3.1 ng / mL. In various embodiments, the reference level is about 3.0 ng / mL

[0162] In various embodiments, measuring the level of the red latex beads is performed at about 20-30 minutes after applying the plasma or serum sample to the sample receiving region. In various embodiments, measuring the level of the red latex beads is performed at about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes after applying the plasma or serum sample to the sample receiving region. In various embodiments, measuring the level of the red latex beads is performed at about 25 minutes after applying the plasma or serum sample to the sample receiving region.

[0163] In various embodiments, the biological sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant. In various embodiments, the biological sample is from a pregnant woman who is between 23 0 / 7 weeks to 30 6 / 7 weeks pregnant. In various embodiments, the biological sample is from a pregnant woman who is between 31 0 / 7 weeks to 34 6 / 7 weeks pregnant.

[0164] In various embodiments, measuring the level of the red latex beads at the first location in the indication region, or calculating the level of sFlt-1 based on the measured level of the red latex beads, or both is performed with an analyzer the present invention as described herein.

[0165] In various embodiments, the reference level is 3.0 ng / mL.

[0166] In various embodiments, a high risk of having sPE is having sPE within two weeks from having her plasma or serum sample assayed. In various embodiments, a high risk of having sPE is having sPE within one week from having her plasma or serum sample assayed.Treatment Methods

[0167] Various embodiments provide methods for administering a therapy for treating and / or managing preeclampsia. Various embodiments provide methods for administering a therapy for treating a preeclampsia-related disorder to a pregnant human in need thereof. Various embodimentsprovide methods for administering a therapy for treating a preeclampsia-related disorder to a postpartum human in need thereof. The methods comprise detecting an amount of sFlt- 1 in a biological sample obtained from the patient using a lateral flow device described herein, and administering a dosage amount of the therapy to the pregnant human or to the postpartum human.

[0168] Some embodiments provide methods for administering a therapy for treating and / or managing preeclampsia or a preeclampsia-related disorder to a pregnant human in need thereof, which comprise detecting an amount of Flt-1 (e.g., sFlt-1) in a biological sample obtained from the patient above a reference level with a lateral flow device described herein, and administering a dosage amount of the therapy to the pregnant human detected with the amount of Flt-1 (e.g., sFlt-1) above the reference level.

[0169] Further embodiments provide methods for administering a therapy for treating and / or managing preeclampsia or a preeclampsia-related disorder to a pregnant human in need thereof, which include administering a dosage amount of the therapy to the pregnant human detected with an amount of Flt-1 (e.g., sFlt-1) in a biological sample obtained from the pregnant human above a reference level with a lateral flow device described herein.

[0170] Various embodiments provide for a method of treating or managing preeclampsia or eclampsia for a patient in need thereof, comprising: prognosticating the women’s risk of having sPE by the method of the present invention as described herein, and initiating one or more evaluation or preparedness for the woman whose risk of having sPE is prognosticated as being high, or providing standard of care expectant management to the woman whose risk of having sPE is prognosticated as being low.

[0171] In various embodiments, the one or more evaluation or preparedness comprises step- up care for the woman, providing neonatal intensive care unit (NICU) consult, increase frequency of tests to assess severity of the preeclampsia or eclampsia, increase frequency of fetal assessments, planning for steroids or magnesium treatment.

[0172] In various embodiments, the method further comprises administering steroids or magnesium to the women when the risk of having sPE is prognosticated as being high. In various embodiments, the method further comprises administering a treatment such as an anti -Flt-1 antibody and / or therapeutic apheresis to lower the sFltl to the women when the risk of having sPE is prognosticated as being high.EXAMPLES

[0173] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.Example 1 sFLT-1 Test Kit

[0174] As noted in Table 1 above, the sFLT-1 Test Kit may be comprised of the following components: Test Cassettes Chase Buffer Lyophilized Quality Controls (High and Low (1 vial each)) pSD card (lot-specific). The product Instructions for Use is also provided with each test kit.

[0175] Materials of construction for the test cassette are identified in Table 2; component numbers align with the numbering in Figure 5 above. Formulations for the custom reagents used in the test cassette are provided in Table 3. Formulations for the chase buffer and lyophilized Quality Controls are provided in Table 4 and Table 5, respectively.Table 2: sFLT-1 Test Cassette Materials of ConstructionTable 3: Custom Reagent FormulationsTable 4: sFLT-1 Test Chase Buffer FormulationTable 5: sFLT-1 Test Lyophilized High- and Low-Quality Control Formulations* In some embodiments, the Low / High external control utilizes R & D Systems PN 321 -FL, which is a recombinant human VEGFRl / Flt-1 Fc chimera protein, derived from Spodoptera frugiperda, Sf 21 (baculovirus), comprising from N-terminus to C-terminus: human VEGFR1 (Ser27-His687)accession no. AAC50060 - IEGRMD (SEQ ID NO:2) - human IgGl (Prol00-Lys330) - 6-His (SEQ ID NO:3) tag.Lyophilized Quality Controls

[0176] The sFLT-1 Test Kit contains two (2) lyophilized Quality Controls (see Table 5 for formulation) which is to be run by the operator with each new lot of test cassettes and may also be run according to internal facility policies and procedures.

[0177] Quality Control materials are titrated with sFLT - 1 protein targeting concentrations that are 20% above and below the prognostic cut-off of the test. These concentrations are based on recommendations derived from CLSI guideline document EP12-A2 “User protocol for evaluation of qualitative test performance”. Optimal negative and positive quality control materials should be titrated to achieve concentrations that are close enough to the prognostic cut-off of the test that they can sensitively detect if the cut-off of the test system has drifted or shifted significantly away from its original value. At the same time, they should be titrated such that they are not so close to the cut-off as to cause a high proportion of qualitatively false values because of intrinsic imprecision of the test. For example, it is suboptimal if more than 5% of the LOW RISK QC or more than 5% of the HIGH RISK QC test results give the wrong value because of random imprecision.

[0178] The lot-specific expected values will be provided in the lot-specific pSD card included in each test kit and will be uploaded to the analyzer by the operator prior to use of a new lot of test cassettes. The analyzer will report both qualitative results for both control levels to the operator. Values that are more than two (2) standard deviations higher or lower than the expected value (< mean - 2SD or > mean + 2SD) will be deemed unacceptable.

[0179] If either of the results are found to be unacceptable, the QC process is to be repeated twice before patient testing can proceed. If QC results are acceptable both times on repeat testing (2 out of 3 acceptable values), the analyzer will be unlocked for patient testing. If either of the replicate QC tests are still unacceptable (if 0 / 3 or 1 / 3 results are acceptable), the instrument will remain locked for patient testing using the specific lot of test cassettes.

[0180] In the event of QC failure, the operator may test patient samples using a different lot of test cassettes by repeating the QC process.Analyzer

[0181] The Analyzer (Figure 3) is a small, benchtop analyzer that measures the intensity of bands on a lateral flow strip; an internal schematic of the analyzer is provided in Figure 6.

[0182] Specimen preparation and testing include (1) Specimen dilution, (2) Insertion of testing cassette, (3) (optional) Scanning of specimen being tested, and (4) addition of diluted specimen to testing cassette. The operator will prepare the specimen for testing by pipetting the chase buffer (80pL) into the dilution tube, then pipetting the plasma or serum sample (15 pL) into the dilution buffer and gently mixing. After addition of the diluted specimen, the analyzer internal test timer is initiated once the analyzer camera detects sample / buffer mixture flowing within the test window. After 22 minutes the development period is complete, and the analyzer reads the test line results and surrounding background. The measurements are made by means of internal illumination and a small custom camera that records the image of the lateral flow strip. The analyzer firmware processes the image, integrates the test line (TL) signal, performs a background subtraction, and calculates background-subtracted TL signal. The TL signal is then converted into sFLT-1 plasma or serum concentrations (in ng / mL) using the lot-specific calibration settings uploaded from the lot-specific pSD card included with each test kit. Test results that indicate sFLT-1 concentrations above the diagnostic cut-off are reported as HIGH RISK for sPE, and results below the diagnostic cut-off are reported as LOW RISK for sPE; sFLT-1 quantitative concentrations are not reported with results (Figure 7). The analyzer has 3 buttons and a display that allow the user to navigate the results, analyzer status, set administrative options which would include communication options, QC frequency, and lock outs.

[0183] The Analyzer System (Figure 3) is comprised of the following components: Analyzer, Removable battery door, Barcode scanner, Printer, 4 x AAA batteries, External 5V power supply, USB communications port, pSD card port, and Analyzer Control Cassette (Figure 4).

[0184] The analyzer is configurable for use with a barcode scanner and a printer for retrieving test sample information (e.g. , patient ID, specimen ID) and generating a results printout. Furthermore, the analyzer features Bluetooth and Wi-Fi connectivity for communication to peripherals and Lab Information Systems (LIS). An optional Wi-Fi-to-ethemet adapter is made available for laboratories who wish to interface through an ethemet port.

[0185] The analyzer utilizes a UL-listed USB power supply that meets Category II or III overvoltage standards; the analyzer may also be loaded with 4 AAA batteries which can be used for backup power or as the primary energy source while the instrument is unplugged.Software Description

[0186] The analyzer operates with pre-installed firmware that allows for testing, local printing of results, electronic storage of results, and HL7 interfacing and upload of test results to a laboratory information system. The analyzer will be operated by trained personnel within the clinical laboratory setting.On-Analyzer Systems

[0187] The embedded software (Firmware) housed on the analyzer interfaces with several on- analyzer systems, such as a camera sensor, clock circuitry, sample illumination LEDs, and analog sensors corresponding to analyzer self-diagnostics and environment capture. The embedded softwarealso interfaces with user interface elements, such as a display, buttons, and a switch that indicates when a cassette has been inserted.Peripheral Systems

[0188] The embedded software interfaces with several peripheral systems that may be optionally used depending on whether bar-coded specimen labels are being used and whether the testing site has connectivity with a laboratory information system (LIS). These peripherals include a barcode scanner (connected via Bluetooth wireless), pSD card (via pSD card port), and local printer (connected via Wi-Fi, ethemet cable, or USB port).External Systems

[0189] The embedded software may also interface with an external system (connected via WiFi or ethemet cable), such as a Lab Information System (LIS) restricted to a Clinical Laboratory.Analyses

[0190] The analyzer uses an onboard LED (white light) and camera to illuminate the test cassette and to capture the image of the test line (TL) (Figure 8) and control line (CL) (Figure 8) and surrounding background and quantify these images into analyzer densitometric Relative Units (Figure 9). The TL and CL integrated signals are adjusted by subtracting background signal based upon the surrounding background ROIs. The background-subtracted integrated CL value is used as the intratest control to evaluate the correct position of the test cassette, the successful flow of buffer and reagents, and acceptability of the test results. The background-subtracted integrated TL value is converted into sFLT-1 concentrations (in ng / mL) using the calibration settings for that lot of cassettes. sFLT-1 values are interpreted as indicative of High Risk of sPE when sFLT-1 concentrations are > 3.0 ng / mL, and Low Risk of sPE when sFLT-1 concentrations are < 3.0 ng / mL. CL acceptable values and TL calibration settings are uploaded into the analyzer using an pSD card provided with each lot of test cassettes. Specific steps in these analyses are described in Input and Outputs sections below.Inputs

[0191] Test window image capture and test timer initiation: When the test is manually started by the operator, the camera starts real-time monitoring of the test cassette test window to detect when chase buffer first appears in the test window. Appearance of buffer flow within the test window triggers the onboard testing timer, starting the test development period. 22 minutes after the testing timer is initiated, the test is complete, and the camera acquires an image for quantification of TL and CL. The image is processed using the camera’s green channel to maximize sensitivity and contrast to the red latex beads and is saved as a PNG file for processing.

[0192] Transformation of test window 2D image of densitometric Relative Units into ID signal tracing across full length of test cassette. The image of each test strip is 1280 pixels in width and 140 pixels in height. At each position along the full length (X axis) of the test strip, the densitometric Relative Units are integrated across the full width of the test strip. This produces a seriesof 1280 boxes along the full length of the test strip that are each 1 pixel wide (X-axis) by 140 pixels tall (Y-axis). The total number of densitometric Relative Units within each box is summed, and these values are assembled into a signal tracing according to their position on the length of the Test Strip (Figure 9).

[0193] The signal tracing is broken into ROIs for the TL and CL and areas on either side of the TL and CL which are used to calculate surrounding background for subtraction (Figure 8 and Figure 9)

[0194] The signals within the TL peak (10 pixel width) and CL peak (10 pixel width) (Figure 8) and the background ROIs (20 pixel width each) are integrated (area under the curve), and the background is subtracted to obtain the final background-subtracted TL and CL integrated signals.

[0195] Test Line signal lot-specific calibrated conversion to sFLT-1 protein concentrations: Each lot of sFLT-1 test cassettes will be calibrated after test lot production by the manufacturer using human pooled plasma calibrators with known sFLT-1 protein concentrations. Calibration curves for each lot are generated using weighted least-squares best fit first-order linear model of the form (Figure 10):[sFLT-1 concentration in ng / mL] = slope x [background-adjusted TL integrated signal] + y-interceptBackground-subtracted TL values from QC and patient sample test results is converted into sFLT-1 protein concentrations using the calibration curve specific to the lot of test cassettes in use. Lot-specific calibration curves will be uploaded into the analyzer using the pSD card provided with each new lot of test cassettes.Outputs

[0196] Assay results will be available to the operator via the on-board manual display and may be printed (using the printer) or transmitted to a Laboratory Information System or middleware.

[0197] Clinical results will be reported as HIGH RISK (displayed on GUI as “sPE: HIGH RISK”) or LOW RISK (displayed as “sPE: LOW RISK”). The Instructions for Use will include an “Interpretation of Results" section to provide result-dependent guidance on patient management decision-making.Analyzer Photometric QC using Control Cassette

[0198] A pre-printed Electronic Control Cassette (EQC) (Figure 4), containing manufacturer- printed Test Lines and Control Lines, is provided with the analyzer. The EQC is to be inserted each time the analyzer is powered on. Upon insertion, the Control Cassette is scanned to acquire five (5) images from which the average background-adjusted TL integrated value is calculated. If this value differs from the standard target value set at the time the analyzer was put into use, the analyzer willprompt the operator on the screen to re-insert the Control Cassette and photometric EQC testing will be repeated.

[0199] The Control Cassette is used to confirm the performance of the analyzer LED and camera which are used to capture images and quantify test results every time the analyzer is powered on.Example 2Specimen Collection, Preparation, and Storage

[0200] EDTA-plasma or serum samples will be obtained from the patient by a licensed phlebotomist or healthcare practitioner and prepared in the clinical laboratory. In various embodiments, a minimum of 20 pL of plasma or serum is required for testing.

[0201] EDTA plasma or serum may be stored at room temperature for 24 hours or until ready for transport to the laboratory, or at 4°-8°C for up to 7 days.

[0202] Stability studies will be conducted in accordance with CLSI EP25-A and data will be provided in the de novo Classification Request or 510(k) to support these claims.Example 3Kit Storage and Handling

[0203] The sFLT-1 Test Kit test cassettes and chase buffer may be stored at room temperature until expiration.

[0204] The sFLT-1 Test lyophilized quality controls can be stored at room temperature and reconstituted controls can be stored at 4° - 8° C.

[0205] Stability studies to establish these storage and handling conditions for the components of the sFLT-1 Test are in process; stability data will be provided in the de novo Classification Request or 510(k).Example 4Assay Specifications

[0206] Expected and exemplary Assay specifications are provided in 6.Table 6: Expected Assay Specification

[0207] Prognostic accuracy will be calculated using the following formula:Prognostic accuracy = > True positive + true negative)(true positive + true negative + false positive + false negative)

[0208] In this patient population at risk for the development of preeclampsia with severe features, positive predictive values of standard-of-care tests are <50%.Example 5 Cut-Off ValuesEstablishment of sFLT-1 Cut-Off Values

[0209] The cut-off values were established using 215 prospectively collected EDTA- plasma samples from women between 23 0 / 7 weeks to 34 6 / 7th weeks diagnosed with a hypertensive disorder of pregnancy (PE, chronic hypertension (HTN) with or without superimposed PE, or gestational HTN) as defined by the American College of Obstetrician and Gynecologists (ACOG) guidelines.

[0210] Samples were collected from a single site (Cedars-Sinai Medical Center) participating in the PRAECIS Study (clinicaltrials.gov: NCT03815110). The rationale is that it is challenging to identify who among those with hypertensive disorders of pregnancy will develop preeclampsia with severe features, including kidney, liver, pulmonary, or cerebral injury. The only definitive treatment is delivery of the placenta, and therefore the fetus, which can lead to severe morbidity and mortality to the neonate if delivery is very premature. Therefore, risk stratification methods and systems to identify who among women with hypertensive disorders of pregnancy are at risk of developing preeclampsia with severe features within a certain timeframe are needed in order to allocate resources (e.g. betamethasone and magnesium for fetal neuroprotection) accordingly.

[0211] Collected samples, stored at -70°C since time of collection, were thawed at room temperature and run on the sFLT-1 Test by a single user blinded to clinical outcomes. Further details on the samples collected during the study are provided herein.

[0212] A breakdown of patient ages and sPE diagnosis 2 weeks after testing are provided in Table 7 and Table 8 below. The data presented below excludes information on 79 patients who were excluded from the final analysis due to pre-existing sPE at time of testing and 5 missing values.Table 7: Patient Gestational AgeTable 8: Patient Clinical Diagnoses within 2 weeks of Admission

[0213] Based on sFLT-1 concentrations measured by the sFLT-1 test, women who developed preeclampsia with severe features within two (2) weeks after presenting with hypertensive disorder of pregnancy had significantly higher average sFLT-1 concentrations compared to mothers who did not develop sPE (6.48 ng / mL vs. 2.99 ng / mL, p<0.0001). A summary of the data is provided in Table 9 below.Table 9-1: sFLT-1 Concentrations Summary Statistics by Diagnosis of sPE at 2 Weeks

[0214] An assay cut-off of 3.0 ng / mL was selected as it provides the optimal combination of sensitivity and specificity for prediction of sPE within two (2) weeks of testing (Figure 11) while selecting highest negative predictive value (NPV) and clinically meaningful positive predictive value (PPV) (Figure 12).

[0215] We optimized the cut-offs for NPV, as clinically the greatest utility is to rule out disease among the suspected preeclampsia population. To identify the best cut-off values, we used the derivation study samples from PRAECIS study (N=215) and calculated PPV / NPV over a range of cut-offs. At a sFltl cut-off value of 3, NPV was highest at 92%. Sensitivity, specificity, PPV and NPV values for the various cut-offs are shown below.Table 9-2. Cut-off value selection and related sensitivity, specificity, PPV, and NPV values.Cut-off Values

[0216] Based on the data presented above: Patients with sFLT-1 concentrations > 3.0 ng / mL are considered at HIGH RISK for progression to sPE; Patients with sFLT-1 concentrations < 3.0 ng / mL are considered at LOW RISK for progression to sPE.Example 6 StandardizationCalibrators

[0217] Traceability to a reference material is best established using plasma calibrators similar in matrix and composition as the patient samples being testing. The calibrators are comprised of pooled EDTA plasma from remnant specimens collected from 34 human patients (pregnant and non-pregnant) which were mixed to three target sFLT-1 concentrations and frozen at < -70°C for long term use. Pooled plasma calibrators were designed and produced in accordance with ISO 17511:2020; further information on the samples is provided below.

[0218] Pre-centrifuged individual samples (n = 34) were collected and EDTA plasma was separated from cells. Plasma specimens showing visible signs of hemolysis, icterus, or lipemia were discarded. Specimens were centrifuged for 10 minutes to remove platelets, precipitate, or particulate matter. The specimens were tested for sFLT-1 concentrations using the CE-marked Thermo Kryptor sFLT-1 immunoassay performed on a Thermo Kryptor Compact Plus analyzer. The samples were mixed to target sFLT-1 concentrations of approximately 2.0, 4.0, and 6.0 ng / mL. sterile fdtered through a 0.45 pm filter, aliquoted and frozen. Aliquots of each calibrator were thawed and re-tested in four (4) replicates to determine the final assigned calibrator concentrations (Table 10).Table 10: Value Assignment of sFLT-1 assay Pooled Calibrators

[0219] The original lot of plasma calibrators includes 150 separate sets of calibrators stored in two (2) different freezer sites. Freezers are monitored with electronic alarms systems to warn the company in case of catastrophic freezer failure. Each set of calibrators is thawed and used to calibrate each lot of reagents on a biannual schedule (one (1) new lot every 6 months). With this schedule, the original calibrators may be used to maintain traceability for the expected life of the product.Example 7 sFLT-1 TestPrecision: Single Site Single Lot Within-Laboratory Precision and Reproducibility of External Low and High Quality Controls

[0220] Reconstituted quality control samples were used to evaluate sFLT-1 test precision and reproducibility in samples with sFLT-1 concentrations 20% below and 20% above the prognostic cutoff 3.0 ng / mL (2.4 and 3.6 ng / mL, respectively). Samples were tested over five (5) days. On each day, each control was tested in five (5) replicates on two (2) different analyzers (5 days x 2 Analyzers x 5 Replicates per Analyzer). For each sample, average within-run imprecision and total inter-assay reproducibility were calculated. Raw data and summary statistics are shown in Table 11 and Table 12, respectively.Table 11: Within-Laboratory Precision and Reproducibility Raw Data (sFLT-1 in ng / mL)Table 12: Within-Laboratory Precision and Reproducibility (sFLT-1 in ng / mL)Example 8 Clinical StudyPrimary Objective

[0221] The objective of this study is to demonstrate the clinical performance of the sFLT-1 Test to improve risk-stratification for development of preeclampsia (PE) with severe features in pregnant women (23 0 / 7 weeks to 34 6 / 7th weeks gestation) hospitalized for hypertensive disorders of pregnancy (preeclampsia [PE], chronic hypertension [HTN] with or without superimposed PE, or gestational HTN [gHTN]) within two (2) weeks of testing (presentation), over the current standard of care.Clinical Samples

[0222] EDTA plasma samples collected through the PRAECIS study (NCT number: NCT03815110; Sponsor: Cedars-Sinai Medical Center) are utilized for this study.

[0223] The PRAECIS study was a two-part prospective, multicenter, blinded, non- interventional study conducted at 18 sites throughout the United States. The PRAECIS cohort is representative of the hypertensive American pregnant population (46.6 - 52.5% identify as White and 14.5 - 20.1% identify as Black, representing the increased risk of hypertensive disorders among those who identify as Black). Demographics of the study population are as follows:1. Race a. 31.5% identified as Black / African American b. 51.7% identified as White / Caucasian2. Geographic Distribution a. 17% were recruited from the Northeast b. 22% were recruited from the West c. 23% were recruited from the South d. 39% were recruited from the Midwest3. Center Type a. 16% of patients were recruited from community centers b. 84% of patients were recruited from academic centers.4. Clinical Outcomes a. 33.5% of the PRAECIS population developed preeclampsia with severe features compared to approximately 37.2% of hypertensive, hospitalized deliveries in 2014. b. 31.5% identified as Black / African American

[0224] A total of 1014 women who met the criteria listed in Table 13 were enrolled. EDTA plasma samples were obtained from all patients within 24 hours of enrollment for use in the study and a leftover remnant sample was frozen for future use. (EDTA as an anticoagulant is added to the blood before centrifugation to obtain plasma samples. EDTA binds calcium ions in the blood.) Patients were followed for a two (2) week period or until delivery (whichever came first) for development of sPE and for any other adverse maternal or fetal / neonatal outcomes.Table 13: Inclusion / Exclusion Criteria

[0225] Clinical samples collected in part 1 of the study (n = 299) were assigned to the derivation cohort (samples used to establish the assay cut-off value) and samples collected in part 2 of the study (n = 715) were assigned to the validation cohort.

[0226] Patient characteristics for all enrolled patients are presented below. The validation sample set is independent of the sample set used to establish the assay cut-off (collected as part of derivation cohort). Of the 715 patients enrolled in the validation cohort, 159 patients were excluded (sPE at admission; therefore inclusion / exclusion criteria were not met) from the sample set for a total of 556 samples remaining in the validation cohort. As the clinical samples were frozen (-20°C or lower) after collection, a study will be performed to demonstrate the equivalence of the analyte stability.Hypertensive Disorders of Pregnancy

[0227] Hypertensive disorders of pregnancy were diagnosed as defined by the following ACOG guidelines:• Preeclampsia (PE): Hypertension (systolic BP > 140 mm Hg or diastolic BP > 90 mm Hg or both (on two occasions at least 4h apart) after 20 weeks’ of gestation with new-onset proteinuria (300 mg / 24hr or protein / creatinine ratio > 0.3); in the absence of proteinuria: hypertension associated with thrombocytopenia (platelets < 100,000 / pL), impaired liver function (elevated transaminases to twice normal), new development of renal insufficiency (elevated serum creatinine >1.1 mg / dL or a doubling of serum creatinine in the absence of other renal disease), pulmonary edema, new-onset cerebral or visual disturbances.• Chronic HTN: Hypertension known to predate conception or detected before 20 weeks’ gestation.• Chronic HTN with superimposed PE defined as one of the following:• Chronic HTN present prior to 20 weeks’ gestation with new-onset proteinuria after 20 weeks’ gestation.• Chronic HTN present prior to 20 weeks’ gestation with proteinuria prior to 20 weeks’ gestation with one or more of the following: 1) sudden exacerbation of HTN or need to escalate antihypertensive drug dose especially when previously well controlled with these medications;2) sudden manifestations of other signs and symptoms of PE as above (e.g., elevated transaminases); 3) platelets < 100,000 / pL; 3) right upper quadrant pain and severe headaches;4) pulmonary congestion or edema; 5) new-onset renal insufficiency in absence of other renal disease; 7) sudden, substantial and sustained increase in protein excretion.• Gestational HTN: New-onset hypertension after 20 weeks’ gestation in the absence of proteinuria.

[0228] Per ACOG Guidelines 2020, the presence of any of these features (in women already diagnosed with PE) would constitute PE with severe features (sPE):• Systolic blood pressure of 160 mm Hg or higher, or diastolic blood pressure of 110 mm Hg or higher on two occasions at least 4 hours apart while the patient is on bed rest (unless antihypertensive therapy is initiated before this time)• Thrombocytopenia (platelet count less than 100,000 / pL)• Impaired liver function as indicated by abnormally elevated blood concentrations of liver enzymes (to twice normal concentration), severe persistent right upper quadrant or epigastric pain unresponsive to medication and not accounted for by alternative diagnoses, or both• Progressive renal insufficiency (serum creatinine greater than 1.1 mg / dL or a doubling of the serum creatinine concentration in the absence of other renal disease)• Pulmonary edema• New-onset cerebral or visual disturbances• Headache unresponsive to medication and not accounted for by alternative diagnoses

[0229] In addition to the criteria listed above, women who met the basic criteria for gestational hypertension or chronic hypertension who developed new evidence of thrombocytopenia, impaired liver function, renal insufficiency, pulmonary edema or visual loss or cerebral disturbance were considered as having superimposed PE with severe featuresPatient Characteristics

[0230] Baseline characteristics for all enrolled patients (n = 1014) are presented in Table 14 below.Table 14: Baseline Patient CharacteristicsSample Characterization Information• The following sample characterization information is available for each sample and will be provided as part of the line data within the de novo Classification Request:• Patient demographics• Medical history• Pregnancy history• Prior hospitalizations for hypertensive disorders of pregnancy• Medication history• Diagnosis of severe PE at enrollment• Maternal blood pressure• Maternal proteinuria• Maternal clinical signs / symptoms• Maternal chemistry, hematology, coagulation panels• Two-week outcomes - severe PE and associated labs and clinical signs / symptoms• Delivery outcomes - indicated or non-indicated• Maternal delivery assessments• Fetal / neonatal outcomes including birth weights and percentiles• Medications administered• Adverse maternal and fetal outcomes• Delivery information• Indication for delivery• Type of delivery• Complications of delivery or postpartum maternal stay• Criteria substantiating diagnosis of sPE, if applicableStudy Endpoints

[0231] Primary Endpoint: Performance (sensitivity [SE], specificity [SP], positive predictive value [PPV], negative predictive value [NPV], and accuracy) of the sFLT-1 Test will be calculated with their respective 95% confidence intervals using the established cut-off.

[0232] Secondary Endpoints: Performance (SE, SP, PPV, NPV, and accuracy) of the sFLT- 1 Test in determining the risk for adverse maternal outcomes (as defined below) within 2 weeks after testing.

[0233] Adverse maternal outcome: A composite of adverse maternal outcomes included severe hypertension (systolic or diastolic blood pressure > 160 mm Hg or > 110 mm Hg, respectively) and one or more of the following: placental abruption, cerebral hemorrhage, seizure without underlying seizure disorder, pulmonary edema, disseminated intravascular coagulation, elevated liver enzymes (AST or ALT above 80 U per L), serum creatinine above 1.4 mg per dl, or platelet count below 100,000 per mL within 2 weeks of enrollment.

[0234] Performance (SE, SP, PPV, NPV, and accuracy) of the sFLT-1 Test in determining the risk for adverse fetal / neonatal outcomes (as defined below).

[0235] Adverse fetal / neonatal outcome: A composite of adverse fetal and / or neonatal outcomes included delivery indicated for hypertensive or placental complications of pregnancy within2 weeks of enrollment, birthweight below the 10thpercentile (small for gestational age, SGA), or fetal or neonatal death.

[0236] Performance (SE, SP, PPV, NPV, and accuracy) of the sFLT-1 Test alone or in combination with the current standard of care per ACOG guidelines in predicting maternal development of sPE.

[0237] Time to delivery in women whose are identified as high risk vs. those who are identified as low risk using the sFLT-1 Test.Statistical Analyses

[0238] All hypotheses will be tested at the 5% significance level and all calculations will be performed using the statistics language R.

[0239] Subgroup Analysis

[0240] The intended use population encompasses women: (a) who are hospitalized with different hypertensive conditions (PE, chronic HTN with or without superimposed PE, and gHTN) and (b) women at different gestational ages (i.e., between 23 0 / 7 and 34677thweeks). Prior clinical studies have shown that women who develop de novo PE have similar angiogenic marker abnormalities as women who develop superimposed PE (i.e., in women with chronic HTN, etc.). Therefore, it is expected that the performance measures of the sFLT-1 Test will be similar among these subgroups. Subgroup analysis will be presented in the de novo Classification Request.Example 9Exemplary Instructions for Use and Workflow

[0241] The sFLT-1 Test results generated herein are intended to assess risk of development of preeclampsia with severe features. The test is read using an associated Analyzer. The test kit contains 30 individually packaged single-use test cassettes and other items required for testing.

[0242] Each device contains a test cassette that, along with the Dilution buffer, contains essential components of the assay (see Table 15). The device is inserted into an Analyzer, in most portion and leaving a sample receiving region of the device outside of the Analyzer for assessing; then diluted EDTA plasma sample (preferably a mixture of a dilution buffer at 80 pl and 15 pl EDTA plasma sample) is added to the sample receiving region of the device; and the Analyzer reads and analyzes the test. Results are displayed on the Analyzer display indicating “High Risk” or “Low Risk” of developing preeclampsia with severe features.Table 15. sFLT-1 Test Kit ContentsFurther Materials and Equipment

[0243] Laboratory grade (types 1 or 2) purified water

[0244] Calibrated pipette and tips for delivering 15pL

[0245] Calibrated pipette and tips for delivering 80pL

[0246] Calibrated pipette and tips for delivering 500pL

[0247] Personal Protective Equipment (Lab coat, gloves, protective eyewear, faceshield, etc.)Optional Equipment

[0248] Tube rack to hold 0.6mL Dilution TubeTest Storage and Handling

[0249] This test should be stored and performed at ambient temperature (18° - 30° C).

[0250] Avoid storage of any materials near heating or cooling vents or in direct sunlightSample Preparation

[0251] Test requires human EDTA plasma collected into evacuated specimen tube containing EDTA anticoagulant, do not use whole blood or other specimen types.

[0252] Freshly collected specimen tubes should be centrifuged at 1000 - 2000 ref for 10 minutes to separate plasma from cellular constituents.

[0253] Fresh plasma supernatant may be sampled directly from primary tube after centrifugation or transferred to another tube for testing.

[0254] Fresh EDTA plasma samples kept at room temperature (preferred specimen type) should be tested within 8 hours of collection.

[0255] Alternately, EDTA plasma specimens may be stored at 4° - 8°C for up to 24 hours and then tested, or frozen at <-70°C until analysis.

[0256] Frozen EDTA plasma specimens should be allowed to thaw at room temperature for 60 minutes, then mixed by gentle inversion 3 times, and centrifuged at 2000 ref for 2 minutes. Thawed samples should be tested within 9 hours after thawing.

[0257] I. Startup

[0258] The first time an analyer is used and whenever a new lot of test cassettes is put into use, the Analyzer is configured for the new lot using the pSD card provided with the test kit.1) Power on the Analyzer by pressing and holding the center button.2) A logo will appear once the unit has been powered on.

[0259] II. Navigating menu screens1) Analyzer menus are navigated using the three buttons below the Analyzer screen: left button scrolls up, right button scrolls down, and middle button selects whatever option is highlighted.

[0260] III. Logging On1) Scroll down and select the account type you wish to log in to.2) When applicable, enter the passcode.3) Optionally hit the center button to exit out of WiFi setup.4) The screen will now appear with the “Insert Ctrl QC” and menu option.5) Insert the EQC cassette; after it is read, if the EQC passes performance specifications, the test screen will read “Ctrl QC Pass” and the test cassette should be removed and put back in its sealed foil packet until next use (do not discard). EQC cassette can be used for up to 1 year before it should be replaced. The Analyzer is now unlocked for use and will move to the Test Menu screen.

[0261] IV. Powering On BlueTooth Printer (optional)1) To use the BlueTooth printer included in the Analyzer kit for printing test results, either pre-charge the printer using its USB power cord included with the printer, or plug it in during use.2) To power on the printer for use, press power button on the printer. Printer will turn on and automatically pair with the Analyzer using Bluetooth communications. The Analyzer will then show screen indicating successful pairing of the BT printer.

[0262] V. Powering On and Use of BlueTooth barcode scanner1) A BlueTooth (BT) barcode scanner included in the Analyzer kit is to be used to scan in specimen identifier barcode on specimen, and to scan in barcodes found on the liquid QC when liquid QC is being run. It may also be used to scan in User login codes as an alternative to manual entry.2) Prior to use, charge the BlueTooth scanner using the USB charging cable included in the kit. The charging cable may be plugged into any USB-compatible phone charger. If the scanner is not adequately charged it will flash red.3) To power up the BlueTooth barcode scanner for use, the white power button on the back of the scanner must first be moved from the Off position to the On position.4) Next, to wake the scanner out of sleep mode and pair it with the Analyzer, press the button on the top of the scanner while holding it within 3 inches of the Analyzer.5) When the blue indicator light at the end of the scanner is showing solid blue, this means the scanner is paired with the Analyzer. When it is flashing this means it is not paired.6) The red light around the scanner button indicates its battery charge. When it is not lit, this indicates it has sufficient charge. When it is flashing red that means it is low on charge. When it is plugged into its USB charger, it will glow constant red. A poorly charged scanner may become unpaired from the Analyzer during use.7) To use the barcode scanner for scanning 1- or 2-dimensional barcodes, simply direct the scanner’s forward face towards the barcode and press the button. You should see a red light appear on the barcode and it will be scanned in and sent to the Analyzer. Take care to make sure there are no other barcodes nearby that the scanner could accidently scan and mistakenly use as you specimen ID.8) BT Scanner states according to LED indicator lightsTable 17.If BlueTooth pairing between the scanner and Analyzer are lost during use:1. Ensure that the scanner is adequately charged2. If step 1 fails, power cycle both the scanner and the analyzer (turn both off, turn on Analyzer, then turn on scanner).3. If step 2 fails, log in to the analyzer as a Service Rep, and select Menu -> Analyzer Settings -> Configure Scanner -> Remove Scannera. After the analyzer display indicates that the scanner has been successfully removed, ensure that the small blue oval LED is blinking on the scanner. Re-pair it by selecting Configure Scanner -> Pair Scanner, then select the scanner when it appears in the search results. The analyzer display will show that the barcode scanner has been successfully added, and the small oval LED on the scanner will turn solid blue. Make sure no other BT scanners are within 10 feet of the Analyzer when manually re-pairing.4. If the steps above fail, contact a service representative.

[0263] VI. Importing New Lot Data and Configuring New Lot for Use

[0264] When using a new lot of sFLT-1 Test Kits with the Analyzer, the configuration file which includes the calibration settings and liquid QC acceptability criteria for the new lot must be imported before the cassettes can be used, the Analyzer will not accept cassettes that have not been configured. The assay configuration file can be imported onto the Analyzer using the microSD card that is provided with the test kit. These cards are provided with each test kit.1) Make sure the Analyzer is powered off.2) Insert the microSD card provided with the lot of cassettes into the back of the instrument.3) Once the microSD card is inserted, power the unit on by pressing and holding the center button.4) Log in to the account type.5) Enter the main menu, and select “Configure Lot”6) Next, select “Import New Lot”7) Then select the lot you wish to add by pressing the center button.8) Analyzer will display a success message when the lot has been successfully imported. Note that High Risk and Low Risk liquid QC must be performed and pass acceptability criteria before the new lot can be used

[0265] VII. Performing Liquid QC

[0266] In order for a new cassette lot to function, both a High Risk and a Low Liquid QC (LQC) sample need to be tested and need to pass the QC procedure.

[0267] Preparation of Liquid QC using the High Risk and Low Risk QC Controls:1. Obtain 1 vial of lyophilized “High Risk QC” and 1 vial of lyophilized “Low Risk QC”.2. Open one of the vials and place the rubber stopper face up on a clean dry surface.3. Using a calibrated pipette, add 500uL of lab grade water to the vial.4. Replace the rubber stopper tightly.5. Let sit for 15 - 30 minutes on the benchtop.6. Invert vial 5 times slowly to mix while avoiding bubbles / foaming.7. Repeat steps 2 - 5 with the second vial of QC Control.Note: If the reconstituted High and Low QC solutions will not be used right away, they can be stored at 4°C for up to 6 hours. The reconstituted QC Controls are now referred to as Liquid QC (LQC).

[0268] Accessing and Running Liquid QC from the Menu1) In order to avoid mixing up the High Risk and Low Risk QC samples during testing, dilute, scan, and test the High Risk and Low Risk QC one at a time and only keep that vial in front of you.2) Dilute liquid QC in preparation for testing:A. Reconstitute the High Risk and Low Risk vials of Liquid QC using procedure described above (Preparation of Liquid QC using the High Risk and Low Risk QC Controls), or recover already reconstituted vial from refrigerator storage.B. Prepare a calibrated pipette for pipetting Liquid QC by setting to 15pL.C. Prepare a calibrated pipette for adding Dilution Buffer by setting to 80pLD. Obtain 1 bottle of Dilution Buffer. Note: each bottle is sufficient to test more than 40 devices.E. Obtain 2 pouched devices. Do not open pouches until immediately prior to testing.F. Obtain 2 dilution tubes from the accessory pack provided in the kit.G. Pipette 15pL of High Risk or Low Risk LQC into the dilution tube.H. Pipette 80pL of Dilution Buffer into the dilution tube containing the LQC.I. Mix the QC with dilution buffer by pipetting up and down slowly 4 times, avoid bubbles or foaming. Change pipette tips in between mixing and adding sample to test cassette. Note: this diluted reagent can be used for up to 6 hours after preparation.3) From the menu, select “Configure Lot”.4) Select “View Existing Lots”5) Select the Lot you wish to run QC on from the list of available lots.6) Select the Type of QC you wish to run (Low or High).7) Use the paired BlueTooth barcode scanner to scan the Liquid QC Vial.8) Tear open 1 pouch.9) Insert the test cassette that will be run for Liquid QC. Place the device into the Analyzer, following the arrow printed on the device to orient the direction of insertion, and push the device until there is a clear stop. The sample port (aka sample receiving region) of the device will be exposed and accessible.10) Analyzer will prompt the user to apply the scanned QC sample (Low Risk or High Risk depending on what was selected and scanned during steps 4 and 5) to the sample port. Usinga new pipette tip, apply 80uL of the appropriately diluted Liquid QC to the sample port. Avoid touching the pipette tip to the surface of the sample port.11) A timer will count down the time remaining for the cassette. DO NOT REMOVE CASSETTE UNTIL TESTING IS COMPLETE.12) Once the QC test has completed, the Analyzer will display the QC result message.13) Repeat steps to test the other QC control.

[0269] The purpose of performing Liquid QC with each new lot of test devices is to ensure that the devices, reagents, lot-specific calibration, and a specific Analyzer are performing together as designed. Occasionally Liquid QC will fail its performance specifications due to random error (imprecision), however: 1) Re-read and confirm that all procedures described in the Instructions for Use when running Liquid QC were performed exactly as described. 2) Re-test the Liquid QC for that specific HIGH RISK or LOW RISK QC vial that failed two more times (3 replicate tests total). 3) If the second and third test results (2 out of 3) pass requirements, the lot will pass performance requirements and will be unlocked for use. If 2 or 3 of the repeated tests again fail requirements, it may indicate a problem with the lot of test of devices and the specific lot will be locked out (blocked from use) on that specific Analyzer. The User may try to import and use another lot of devices.

[0270] VIII. Using the Analyzer to Run a Test1) The “Insert Ctrl QC” dialog will appear. Insert the EQC cassette into the opening in the front of the instrument.2) Once the EQC has passed, the Analyzer will prompt you to insert the test cassette.3) Dilution of specimen in preparation for testing a) Important Note: Specimen testing includes (1) Specimen dilution, (2) Insertion of testing cassette, (3) Scanning of specimen being tested, and (4) addition of diluted specimen to testing cassette. In order to avoid specimen mix-ups and identification errors during these steps, it is best practice to keep only the specimen being tested in the testing area in front of you where you perform dilution, specimen scanning, and testing. b) Obtain 1 bottle of Dilution BufferNote: each bottle is sufficient to test more than 40 devices. Buffer bottle is stored at RT (15°C-30°C). c) Prepare a calibrated pipette for pipetting sample by setting to 15pL d) Prepare a calibrated pipette for adding Dilution Buffer by setting to 80pL e) Pipette SOpil of Dilution Buffer into the dilution tube. f) Pipette 15 pl of the plasma sample into the dilution buffer. Visually confirm accurate pipetting of plasma with no bubbles.g) Mix sample and Dilution Buffer by pipetting up and down slowly 3-5 times with the 80pL pipette to mix the reagents, while avoiding foaming. Discard the tip. Note: This diluted sample can be used for up to 6 hours after preparation. h) Using a new pipette tip, transfer 80pL of diluted sample to the sample application port on the test cassette that is already inserted into the Analyzer. Important Note: Do not apply the diluted sample before the cassette is inserted.4) Tear open 1 device pouch containing test cassette. Confirm that the device has a blue line. Place the device into the Analyzer, following the arrow printed on the device to orient the direction of insertion, and push the device until there is a clear stop. The sample port of the device will be exposed and accessible.5) When prompted, use the barcode scanner to scan the specimen ID from the specimen vial. Add sample within 5 minutes of scanning the ID to prevent Analyzer from timing out.6) After scanning in the specimen ID, the Analyzer will prompt the User to add sample to the test device, but before adding sample the User must first prepare diluted specimen.7) After the sample has been applied, the Analyzer will automatically begin its 22 minute countdown.8) Do not remove the cassette while the test is running. If the cassette is removed, an error will appear, the run will not complete, and you will need to start testing over with a new diluted specimen and test cassette.9) Upon completion of the 22 minute countdown, the \ Analyzer will display the result.10) Select the “Print” option to print the test result on the paired Bluetooth printer, or select the “Exit” Option to return to the “Insert Cassette” dialogue screen and prepare to run the next test.

[0271] Testing specimens with manual identification and without barcode scanning:

[0272] If the specimen does not have a barcode label, or the label ID is not recognized by theAnalyzer, specimens may be tested manually without barcode scanning. If the barcode scanner is turned on, turn off its power using the switch on the bottom. If it is already off, leave the power off. If the Analyzer is on, exit to the menu and log off, then power down the Analyzer. Turn the Analyzer on without turning the barcode scanner. Log in and perform EQC. Insert a new cassette for testing. Instead of requesting that the specimen barcode be scanned, the instrument will ask for the sample to be added directly. Add the pre-diluted sample to start the test. Note that the test will be performed without any associated identifier entered into the Analyzer. When the results are ready, they may be printed out but the sample ID will simply read “00000”. The User should immediately transfer / write the specimen ID on the printed report.Example 10Exemplary Validation Study

[0273] PURPOSE / OBJECTIVE

[0274] The primary objective of this study was to demonstrate the clinical performance of the sFLT-1 lateral flow assay (LFA)Test as an aid in risk stratification for development of preeclampsia with severe features (sPE) within two (2) weeks of testing in pregnant women (23 0 / 7 weeks to 34 6 / 7 weeks gestation) hospitalized for hypertensive disorders of pregnancy in the PRAECIS study.

[0275] Secondary objectives of this study were:• To determine whether the SFLT-1 LFA test can be used to predict the short-term risk (within 2 weeks) of adverse maternal outcomes;• To determine whether the SFLT-1 LFA test can be used to predict the short-term risk of adverse fetal / neonatal outcomes;• To compare the performance of the sFLT-1 test alone to the performance of clinical and laboratory factors per American College of Obstetricians and Gynecologists (ACOG) Guidelines in predicting the maternal development of sPE, and to establish the performance of the sFLT-1 test in combination with the above factors in predicting the maternal development of sPE; and• To compare time to delivery in women with positive (HIGH RISK) vs. negative (LOW RISK) sFLT-1 test results.

[0276] SCOPE

[0277] The Preeclampsia Risk Assessment: Evaluation of Cut-offs to Improve Stratification (PRAECIS) study was a multicenter, prospective, non-interventional clinical evaluation of women with singleton pregnancies of 23 0 / 7 to 34 6 / 7 weeks’ gestation hospitalized with a hypertensive disorder of pregnancy. Study subjects provided serum and EDTA plasma specimens upon enrollment, and were followed up for two weeks or until delivery; incidence of progression to sPE, adverse maternal outcomes or adverse fetal / neonatal outcomes within the follow-up period were all captured as study endpoints. Frozen EDTA plasma specimens from the PRAECIS evaluation were eligible for inclusion in the current study.

[0278] ACCEPTANCE CRITERIA

[0279] The following null hypotheses were tested in this evaluation:Ho: Sensitivity < 65% vs. Hi: Sensitivity > 65%, ANDHo: Specificity < 65% vs. Hi: Specificity > 65%, where the sensitivity was defined as the proportion of subjects with an adjudicated diagnosis of sPE for whom the SFLT-1 LFA test result was positive ("High Risk"), and the specificity wasdefined as the proportion of subjects with an adjudicated diagnosis of "no sPE" for whom the SFLT- 1 LFA test result was negative ("Low Risk").

[0280] The primary study objective was considered to have been met if the lower limit of the two-sided 95% Wilson score confidence interval for both the sensitivity and the specificity were greater than 65%.

[0281] MATERIALS AND EQUIPMENT

[0282] Samples: The original PRAECIS sample population included 556 samples from enrolled subjects who met study eligibility criteria and from whom an adjudicated diagnosis of sPE or no SPE within 2 weeks of enrollment was obtained. A total of 13 samples were not included in this validation study due to insufficient sample volume required to run the Gravidas sFLT-1 Test. Therefore, a total of 543 samples were included in the current validation study, including 180 samples from subjects who developed sPE within 2 weeks of enrollment, and 363 samples from subjects who did not develop sPE within 2 weeks of enrollment.

[0283] SAMPLE SIZE

[0284] A total of 543 samples were included in the current validation study, including:• 180 samples from subjects who developed sPE within 2 weeks of enrollment (for the purpose of estimating SFLT-1 LFA test sensitivity), and• 363 samples from subjects who did not develop sPE within 2 weeks of enrollment (for the purpose of estimating SFLT-1 LFA test specificity).

[0285] Assuming that the true test sensitivity is 75% or higher, a minimum of 180 samples from subjects with sPE provided a minimum statistical power of 82.8% to reject the null hypothesis and conclude that the true sensitivity is significantly greater than 65%, at a significance level of a = 0.05.

[0286] Assuming that the true test specificity is 74% or higher, a minimum of 363 samples from subjects without sPE provided a minimum statistical power of 95.2% to reject the null hypothesis and conclude that the true specificity is significantly greater than 65%, at a significance level of a = 0.05.

[0287] DATA ANALYSIS PLAN

[0288] All hypotheses were tested at the 5% significance level unless otherwise specified, and analyses of study data will be performed using the statistical programming language R, version 4.1.1 (R Foundation for Statistical Computing, Vienna, Austria).

[0289] Primary endpoint analyses

[0290] Primary endpoint analyses were conducted using all available data from enrolled subjects. Subjects from whom valid SFLT-1 LFA test results were not obtained were not included inthe analysis population. All subjects included in the validation cohort were adjudicated independently for a diagnosis of sPE or no sPE within 2 weeks of enrollment.

[0291] Results were reported in the format of a 2x2 contingency table, showing test result (High Risk / Low Risk) versus independently adjudicated diagnosis (sPE / no sPE).

[0292] The sensitivity was computed as the proportion of subjects with an adjudicated diagnosis of sPE for whom the SFLT-1 LFA test result was positive (“High Risk”). The specificity was computed as the proportion of subjects with an adjudicated diagnosis of “no sPE” for whom the SFLT-1 LFA test result was negative (“Low Risk”). The positive predictive value (PPV) was computed as the proportion of subjects with a positive (“High Risk”) SFLT-1 LFA test result for whom an adjudicated diagnosis of sPE was made. The negative predictive value (NPV) was computed as the proportion of subjects with a negative (“Low Risk”) SFLT-1 LFA test result for whom an adjudicated diagnosis of “no sPE” was made. The accuracy was computed as the proportion of subjects for whom the SFLT-1 LFA test result was concordant with the adjudicated diagnosis (i.e. High Risk test result and sPE diagnosis, or Low Risk test result and “no sPE” diagnosis).

[0293] The 95% two-sided Wilson score confidence interval associated with each of the above estimates was computed.

[0294] Handling of Missing Data for Secondary Endpoints

[0295] The secondary endpoints of adverse maternal outcomes and adverse fetal / neonatal outcomes were composite endpoints; study subjects may have had missing data for a subset of clinical variables making up one or more of these endpoints. For these subjects, missing data was be imputed using multivariate imputation by chained equations, with relevant covariates such as admission systolic and diastolic blood pressure, serum creatinine, ALT, AST, maternal age, parity, body mass index, gestational age, and platelet count were included in the imputation model as predictors. Imputation procedures utilized predictive mean matching. A total of 50 sets of multiple imputations were generated for each secondary endpoint; analyses (as described below in section 9.3) were conducted for each set, and a pooled estimate of each metric will be computed, along with a 95% interval estimate. Results were pooled according to Rubin’s rules.

[0296] Secondary (Exploratory) Endpoint Analyses

[0297] Adverse Maternal Outcomes: Estimates of performance measures (Sensitivity, Specificity, PPV, NPV, and accuracy) were computed, along with the associated 95% interval estimates. Calculations will be performed with missing values imputed as defined in section 9.2 above.

[0298] Adverse Fetal / Neonatal Outcomes: Estimates of performance measures (Sensitivity, Specificity, PPV, NPV, and accuracy) were computed, along with the associated 95% interval estimates. Calculations will be performed with missing values imputed as defined in section 9.2 above.

[0299] Area under the Receiver Operating Characteristic Curve (ROC AUC): Using the quantitative TL value associated with each Gravidas s-FLT-1 Test result, a ROC curve was generated for the test as an aid in discrimination between subjects who developed sPE within 2 weeks of enrollment and those who did not develop sPE. Separate ROC curves were also be generated for the following ACOG defined clinical covariates:• Systolic blood pressure on admission;• Diastolic blood pressure on admission;• Alanine aminotransferase (ALT) level in U / L;• Aspartate aminotransferase (AST) level in U / L;• Serum creatinine level in mg / dL; and• Blood platelet count.

[0300] For each covariate, the ROC AUC was computed, along with the associated 95% confidence interval. Differences in ROC AUC values between covariates and associated 95% confidence intervals were computed using DeLong’s method.

[0301] A logistic regression model was constructed, relating the probability of observing sPE within 2 weeks as a function of the SFLT-1 LFA test result and all other ACOG defined clinical covariates as defined above. The ROC AUC and associated 95% confidence interval were computed; separate estimates were generated using the full model with all clinical covariates included, and with a reduced model including only those covariates which were statistically significant contributors to the model (at a significance level of a = 0.05).

[0302] The above analyses were conducted with respect to the subset of the analysis population for which complete case data was available for all relevant clinical covariates. The analyses were repeated with missing data imputed using multivariate imputation by chained equations; relevant covariates such as admission systolic and diastolic blood pressure, serum creatinine, ALT, AST, and platelet count were included in the imputation model as predictors. Imputation procedures utilized predictive mean matching. A total of 50 sets of multiple imputations were generated, and a pooled estimate of the ROC AUC was computed, along with a 95% interval estimate. Results were pooled according to Rubin’s rules.

[0303] Time to Delivery

[0304] Kaplan-Meier time-to-delivery curves were generated for patients with “High Risk” vs. “Low Risk” SFLT-1 LFA test results. A log-rank test was conducted at a significance level of a = 0.05 to test the hypothesis of lack of association between sFLT-1 Test result and time to delivery.

[0305] A multivariate proportional hazards Cox model for time to delivery was fitted as a function of sFLT-1 Test result and other ACOG defined clinical covariates (such as admission systolic and diastolic blood pressure, urinary protein / creatinine ratio, ALT and AST levels, serum creatininelevel, maternal age, parity, body mass index, and platelet count). An estimate of the hazard ratio and associated 95% confidence interval was generated, to assess the relationship between the qualitative sFLT-1 Test result and time to delivery after adjusting for all other ACOG defined clinical covariates. Continuous variables will be modeled using penalized splines. The proportional hazards assumption was checked using the Schoenfeld residuals test.

[0306] The above analyses were conducted with respect to the subset of the analysis population for which complete case data was available for all relevant clinical covariates. The analyses were repeated with missing data imputed using multivariate imputation by chained equations; relevant covariates such as admission systolic and diastolic blood pressure, urinary protein / creatinine ratio, ALT and AST levels, serum creatinine level, maternal age, parity, body mass index, and platelet count were included in the imputation model as predictors. Imputation procedures utilized predictive mean matching. A total of 50 sets of multiple imputations were generated, and a pooled estimate of the ROC AUC was computed, along with a 95% interval estimate. Results were pooled according to Rubin’s rules.

[0307] As an exploratory analysis, multivariate logistic models were fitted to describe sensitivity and specificity as a function of hypertensive condition, gestational age and maternal age, with gestational age and maternal age modeled as categorical variables (as defined above). Factors shown to be significant predictors of sensitivity or specificity at a significance level of a = 0.05 were identified.

[0308] RESULTS

[0309] Demographic Characteristics

[0310] The original PRAECIS sample population included 556 samples from enrolled subjects who met study eligibility criteria and from whom an adjudicated diagnosis of sPE or no SPE within 2 weeks of enrollment was obtained. A total of 13 samples were not included in this validation study due to insufficient sample volume required to run the Gravidas sFLT-1 Test. Therefore, a total of 543 samples were included in the evaluable sample population, including 180 samples from subjects who developed sPE within 2 weeks of enrollment, and 363 samples from subjects who did not develop sPE within 2 weeks of enrollment.

[0311] Of the 543 study samples, 507 (93.4%) were provided from subjects upon their first enrollment in the PRAECIS study, while 36 (6.6%) were provided from subjects upon subsequent enrollments. The distribution of race among the 507 unique study subjects was:• White / Caucasian: 275 (54.2%);• Black / African American: 149 (29.4%);• Asian: 31 (6.1%);• Hawaii or Other Pacific Islander: 3 (0.6%);• American Indian or Alaskan Native: 2 (0.4%);• Other: 21 (4.1%);• Unknown or Undeclared: 26 (5.1%).

[0312] A total of 85 out of the 507 unique study subjects identified as Hispanic (16.8%).

[0313] Among the 543 enrollments from which study samples were obtained, the mean age at enrollment was 31.8 years, with a standard deviation of 5.8 years and a range of 18 to 50 years. A total of 184 enrollments (33.9%) occurred at a maternal age of 35 years or greater. The mean gestational age at enrollment was 30.4 weeks, with a standard deviation of 3.0 weeks and a range of 23 to 34 weeks. A total of 218 enrollments (40.1%) occurred at a gestational age of 30 weeks or less.

[0314] Primary Endpoint Analyses

[0315] Of the 543 enrollments, preeclampsia with severe features (sPE) was observed within2 weeks of enrollment in 180 cases (33.1%). Table 18 below displays a 2x2 contingency table, showing the frequencies of SFLT-1 LFA test results (High Risk / Low Risk) vs. adjudicated diagnosis (sPE / no sPE).Table 18: SFLT-1 LFA test result vs. Adjudicated Diagnosis (n = 543 enrollments)The lower limit of the 95% confidence interval for both sensitivity and specificity exceeded 65%; therefore, the acceptance criteria for the primary endpoint analyses were met.

[0316] SECONDARY ENDPOINT ANALYSES

[0317] Adverse Maternal Outcome: 19Of the 507 first enrollments, preeclampsia with severe features (sPE) was observed within 2 weeks of enrollment in 166 cases (32.7%). Table 2 below displays a 2x2 contingency table, showing the frequencies of SFLT-1 LFA test results (High Risk / Low Risk) vs. adjudicated diagnosis (sPE / no sPE) among the subset of first enrollments.Table 19: SFLT-1 LFA test result vs. Adjudicated Diagnosis (n = 507 first enrollments).The lower limit of the 95% confidence interval for both sensitivity and specificity exceeded 65% within this subset of first enrollments; therefore, the acceptance criteria for the primary endpoint analyses were met within this subset.

[0318] Secondary Endpoint Analyses

[0319] Adverse Maternal Outcomes: Of the 543 enrollments, the secondary endpoint of adverse maternal outcome within 2 weeks of enrollment was missing in 1 case. Of the 542 enrollments for whom outcome status was available, 49 (9.0%) resulted in an adverse maternal outcome within 2 weeks of enrollment. Table 20 below displays a 2x2 contingency table, showing the frequencies of SFLT-1 LFA test results (High Risk / LowRisk) vs. outcome status after 2 weeks (adverse outcome / no adverse outcome).Table 20: SFLT-1 LFA test result vs. Adverse Maternal Outcome Status.Accuracy = 56.0%; 95% confidence interval = (51.8%, 60.2%)

[0320] Adverse Fetal Outcomes

[0321] Of the 543 enrollments, the secondary endpoint of adverse fetal outcome was missing in 9 cases. Of the 534 enrollments for whom fetal outcome status was available, 282 (52.8%) resulted in an adverse fetal outcome. Table 21 below displays a 2x2 contingency table, showing the frequencies of SFLT-1 LFA test results (High Risk / Low Risk) vs. fetal outcome status (adverse outcome / no adverse outcome). Parameter estimates are shown, when excluding the missing values and also after imputation of the 9 missing values.Table 21 : SFLT-1 LFA test result vs. Adverse Fetal Outcome Status

[0322] Area under the Receiver Operating Characteristic Curve (ROC AUC)

[0323] Figure 13 displays ROC curves for the SFLT-1 LFA test (based on the internal continuous response) and other clinical covariates, as aids in the discrimination between subjects who developed sPE within 2 weeks of enrollment vs. those who did not. Table 22 below displays the ROC AUC estimate and associated 95% confidence interval for each covariate. (Note that missing covariate values were excluded from these individual ROC analyses.)Table 22. ROC AUC estimates for the SFLT-1 LFA test and for other clinical covariates.Differences between the ROC AUC for the Gravidas sFLT-1 internal continuous response and the ROC AUC for each of the other clinical covariates were computed, along with associated 95% confidence intervals; results are shown in Table 23 below. (Note that missing covariate values were excluded from these paired ROC analyses.)Table 23: Differences in ROC AUC between the SFLT-1 LFA test and other clinical covariates.

[0324] A logistic regression model was constructed, relating the probability of observing sPE within 2 weeks of enrollment as a function of the Gravidas sFLT-1 internal continuous response and all other clinical covariates. The model was first constructed using the subset of enrollments for whichcomplete data was available for all covariates (n = 491), and also after imputing missing covariate data according to the procedure outlined in section 9.3.3. Results are displayed in Table 24 below. In both The Gravidas sFLT-1 internal continuous response and the systolic blood pressure on admission were significant contributors to each model, at a significance level of a = 0.05.

[0325] A reduced logistic regression model was also constructed, relating the probability of observing sPE within 2 weeks of enrollment as a function of all significant covariates from the full model (i.e. the Gravidas sFLT-1 internal continuous response and the systolic blood pressure on admission). Both covariates remained significant in the reduced model (p < 0.0001 for each covariate). The ROC AUC associated with the predictive values from this reduced model was 0.903, with an associated 95% confidence interval of (0.877, 0.929).Table 24: Logistic regression model relating probability of sPE to the SFLT-1 LFA test and other clinical covariates.

[0326] Tests were performed to compare the discriminatory ability of the logistic regression models vs. the Gravidas sFLT-1 internal continuous response alone, in identifying subjects who developed sPE within 2 weeks of enrollment. Results are summarized in Table 25 below. Neither the full model (with all clinical co variates included) nor the reduced model (with Gravidas sFLT-1 and systolic blood pressure) resulted in a significantly higher discriminatory ability than the Gravidas sFLT-1 internal continuous response alone, when testing at a significance level of a = 0.05.Table 25: Differences in ROC AUC between fitted models and Gravidas sFLT-1 alone.

[0327] Time to Delivery

[0328] Figure 14 displays a Kaplan-Meier plot showing the probability of remaining undelivered as a function of number of days after enrollment, stratified by SFLT-1 LFA test result (High Risk vs. Low Risk). Among enrollments with known elapsed times to delivery, the median time to delivery was 8 days among subjects with a High Risk SFLT-1 LFA test result, and 29 days among subjects with a Low Risk test result. There was a significant difference in time to delivery between High Risk and Low Risk subjects (p < 0.0001, log-rank test).

[0329] A multivariate Cox proportional hazards model was constructed, relating the time to delivery as a function of the SFLT-1 LFA test result. The estimate of the hazard ratio as obtained from this model was 2.97, with an associated 95% confidence interval of (2.47, 3.56).

[0330] A second Cox model was constructed, relating the time to delivery as a function of the SFLT-1 LFA test result and all other relevant clinical covariates. Continuous variables were modeled using penalized splines. The full model was initially fitted using the subset of enrollments with no missing covariate data (n = 335 complete cases). The Schoenfeld residual test revealed that the proportional hazards assumption was appropriate (p = 0.380). The estimate of the sFLT-1 hazard ratio after adjusting for all other clinical covariates was 3.92, with an associated 95% confidence interval of (2.87, 5.36).

[0331] The full model was also fitted after imputing all missing covariate data according to the procedure outlined in section 9.3.4. The estimate of the sFLT-1 hazard ratio after adjusting for all other clinical covariates in the imputed model was 2.64, with an associated 95% confidence interval of (2.13, 3.28).

[0332] A summary of results obtained from the various Cox proportional hazards models in provided in Table 26 below.Table 26: sFLT-1 Hazard ratios as obtained from various Cox proportional hazards models.

[0333] A logistic regression model was constructed, relating the probability of obtaining a High Risk SFLT-1 LFA test result in subjects who progressed to sPE within 2 weeks of enrollment as a function of hypertensive condition, maternal age category and gestational age category (the sensitivity model). A separate logistic regression model was constructed, relating the probability of obtaining a Low Risk SFLT-1 LFA test result in subjects who did not progress to sPE within 2 weeks of enrollment as a function of hypertensive condition, maternal age category and gestational age category (the specificity model). The format of each model was:Logit(P) = Bo + Bi IPE + B2 ISUP.PE + B3 IGH + B4 IMAT.AGE + Bs IGEST.AGE , where• P = probability of High Risk test result (in sensitivity model) or• P = probability of Low Risk test result (in specificity model);• IPE = 1 if enrollment diagnosis = preeclampsia,= 0 otherwise;• ISUP>PE = 1 if enrollment diagnosis = superimposed preeclampsia,= 0 otherwise;• IGH = 1 if enrollment diagnosis = gestational hypertension,= 0 otherwise;• IMAT.AGE = 1 if maternal age at enrollment > 35 years,= 0 otherwise;• IGEST.AGE = 1 if gestational age at enrollment > 30 weeks,= 0 otherwise.

[0334] Overall, the SFLT-1 LFA test displayed a sensitivity of 92.8%, a specificity of 70.5% and a negative predictive value of 95.2% in this sample population. Both sensitivity and specificity were significantly greater than 65%, thus meeting study acceptance criteria. The above analyses provide support to a claim for the SFLT-1 LFA test as an aid in risk stratification for development of preeclampsia with severe features (sPE) within two weeks of testing in pregnantwomen (23 0 / 7 weeks to 34 6 / 7 weeks gestation) hospitalized for hypertensive disorders of pregnancy.

[0335] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0336] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0337] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).

[0338] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

Claims

WHAT IS CLAIMED IS:

1. A lateral flow device for detection of an analyte comprising one or more circulating fms-like tyrosine kinase 1 (Flt-1) protein isoforms in a biological sample, wherein the lateral flow device comprises:(i) a sample receiving region comprising an about 9-11 mm in-length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising: phosphate-buffered saline (PBS), a non-ionic copolymer surfactant, a heterophilic antibody blocking reagent, mouse immunoglobulin G (IgG), and sucrose, optionally at a ratio of: about 0.5-1.5x phosphate-buffered saline (PBS), about 0.5-1.5% w / v non-ionic copolymer surfactant, about 1.67-3.67 mg / ml heterophilic blocking reagent (HBR), about 0.5-1.5 mg / ml mouse IgG, and about 0.25-0.75% w / v sucrose;(ii) a conjugate region comprising an about 11-13 mm in-length absorbent pad having been saturated with a biotinylated antibody solution in a first portion of the conjugate region, the biotinylated antibody solution comprising a biotinylated anti-sFLT- 1 antibody having an anti-sFLT-1 antibody content at about 30.75-36.75 pg / mL, about 5-15% sucrose, about 1-3% trehalose, a coloring agent at about 40-60 pg / mL, and a biotinylated antibody diluent, optionally the coloring agent comprising FD&C blue #1 Powder, and an anti-sFLT-1 / control final conjugate solution in a second portion of the conjugate region, the anti-sFLT-l / control final conjugate solution comprising a detectably labeled monoclonal anti-VEGFRl antibody having a content of the monoclonal anti-VEGFRl antibody at about 0.05-0.15% w / v, a detectably labeled chicken immunoglobulin Y (IgY) having the chicken IgY content at about 0.0125-0.0375%, about 8-12% w / v sucrose, about 1-3% w / v trehalose, and a latex storage buffer, wherein the detectable labels independently comprise a particulate label, a metal colloid label, or a fluorescent label, optionally the particulate label comprising a colored latex bead about 400 nm in diameter, andwherein the biotinylated anti-sFLT-1 antibody and the detectably labeled monoclonal anti-VEGFRl antibody bind to different epitopes within sFLT-1;(iii) a test region comprising an about 24-26 mm in-length nitrocellulose membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.3-0.5 mg / mL poly streptavidin, 0.5-1.5X PBS, and 4-6% w / v sucrose, and the control line having been saturated with about 0.125-0.375mg / mL donkey anti-chicken antibody, 0.5-1.5X PBS, 0.5-1.5% w / v sucrose, and the coloring agent at 40-60 pg / mL, wherein the poly streptavidin is immobilized in the test line and the donkey anti-chicken antibody is immobilized in the control line,(iv) a wick region comprising an about 20-22 mm in-length absorbent pad; and(v) a backing card having about 55-65 mm in length; wherein each region is in capillary contact with at least one other region thereby permitting a sample fluid to flow from the sample receiving region to the test region, wherein the sample receiving region overlaps with the conjugate region, the conjugate region overlaps with the test region and the test region overlaps with the wick region.

2. The lateral flow device of claim 1, wherein(i) the sample receiving region comprises an about 10 mm length absorbent pad, the absorbent pad having been saturated with a sample pad block solution, the sample pad block solution comprising about lx PBS, about 1% w / v non-ionic copolymer surfactant HO(C2H4O)a(- C3H6O)b(C2H4O)aH wherein a is 99-101 and b is 55-57, about 2.67 mg / ml heterophilic blocking reagent (HBR) (HBR Plus), about 1 mg / ml mouse IgG, and about 0.5% w / v sucrose;(ii) the conjugate region comprises an about 12 mm absorbent pad having with the biotinylated antibody solution in the first portion of the conjugate region, the biotinylated antibody solution comprising the biotinylated anti- sFLT-1 antibody having the anti-sFLT-1 antibody content at about 33.75 pg / mL, about 10% w / v sucrose, about 2% w / v trehalose, the coloring agent at about 50 pg / mL, and the biotinylated antibody diluent, andthe anti-sFLT-l / control final conjugate solution in the second portion of the conjugate region, the second portion of the conjugate region being different from the first portion of the conjugate region, and the anti-sFLT- 1 / control final conjugate solution comprising the anti-sFLT-l / control final conjugate solution comprising the latex bead-conjugated monoclonal anti- VEGF R1 antibody having the monoclonal anti-VEGF R1 antibody content at about 0.1% w / v, the latex bead-conjugated chicken IgY having the chicken IgY content at about 0.025% w / v, about 10% w / v sucrose, about 2% w / v trehalose, and the latex storage buffer;(iii) the test region comprises an about 25 mm in-length nitrocellulose CN95 membrane comprising a test line and a control line, wherein the test line having been saturated with about 0.4 mg / mL poly streptavidin, about IX PBS, about 5% w / v sucrose, and the control line having been saturated with about 0.25 mg / mL donkey anti-chicken antibody, about IX PBS, about 1% sucrose, about 50 pg / mL FD&C Blue powder,(iv) the wick region comprises an about 21 mm in-length absorbent pad; and(v) the backing carding having about 60 mm in length and optionally having cuts located 9, 16 and 46mm from a proximal end of the backing card, said proximal end being at or near the sample receiving region.

3. The lateral flow device of claim 1, wherein the biotinylated antibody diluent comprises about 0.5-1.5% w / v bovine serum albumin and 0.5-1.5X PBS, and / or wherein the latex storage buffer comprises about 0.5- 1.5% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 25-75 mM boric acid.

4. The lateral flow device of claim 1, wherein the biotinylated antibody diluent comprises about 1% w / v bovine serum albumin and IX PBS, and wherein the latex storage buffer comprises about 1% w / v casein in an alkaline borate buffer, the alkaline borate buffer containing about 50 mM boric acid.

5. The lateral flow device of claim 1, wherein the latex bead-conjugated mAb anti-VEGF R1 comprises Human VEGFRl / Flt-1 Antibody (Clone #49560) and about 400nm-in-diameter Red carboxylated polystyrene latex particles conjugated at a weight ratio of from 100:1 to 10: 1.

6. The lateral flow device of claim 1, wherein the latex bead-conjugated mAb anti-VEGF R1 comprises about Human VEGFRl / Flt-1 Antibody (Clone #49560) and 400nm red carboxylated PS latex particles conjugated at a weight ratio of about 40: 1.

7. The lateral flow device of claim 1, wherein the latex bead-conjugated Chicken IgY comprises Chicken IgY and about 400 nm Red Carboxylated Latex particles conjugated at a weight ratio of from 100: 1 to 10:1.

8. The lateral flow device of claim 1, wherein the latex bead-conjugated Chicken IgY comprises Chicken IgY and red carboxylated latex particles about 400 nm in size conjugated at a weight ratio of about 40:1.

9. The lateral flow device of claim 1, wherein the latex storage buffer comprises about 1% w / v of Casein in an alkaline borate buffer, the alkaline borate buffer containing about 50 mM of boric acid and pH adjusted to 8.6.

10. A kit, comprising: a lateral flow device of any one of claims 1-9; and a chase buffer.

11. The kit of claim 10, wherein the chase buffer comprises the non -ionic copolymer surfactant, potassium chloride, PBS, and a preservative biocide, optionally the preservative biocide comprising 2-Methyl-4-isothiazolin-3-one.

12. The kit of claim 10, further comprising a low external control, a high external control, or both, wherein the low external control comprises sFLT-1 at a first predetermined concentration, and the high external control comprises sFLT-1 at a second predetermined concentration, the first predetermined concentration being lower than the second predetermined concentration, and optionally the sFLT-1 comprises a polypeptide having an amino acid sequence of Ser27-His687 of SEQ ID NO: 1.

13. The kit of claim 12, wherein the low external control comprises about 1.5-2.5 ng / mL of sFLT- 1 and the high external control comprises about 3.5 -5.0 ng / mL sFLT-1.

14. The kit of claim 12, wherein the low external control comprises about 2.4 ng / mL of sFLT-1 and the high external control comprises about 3.6 ng / mL sFLT-1.

15. The kit of claim 10, further comprising a quality control diluent.

16. The kit of claim 15, comprising the quality control diluent, wherein the quality control diluent comprises 0.5-1.5X PBS, 0.09-1.15% w / v 2-Methyl-4-isothiazolin-3-one, about 4-6% w / v Sucrose, about 0.5 -1.5% w / v Trehalose, about 40-50 mg / mL bovine serum albumin (BSA), about 90-110 mM ethylenediaminetetraacetic acid (EDTA), and about 18-22 USP / mL Lithium Heparin salt.

17. The kit of claim 15, comprising the quality control diluent, wherein the quality control diluent comprises IX PBS, 0.105% w / v 2-Methyl-4-isothiazolin-3-one, about 5% w / v Sucrose, about1% w / v Trehalose, about 45 mg / mL BSA, about 100 mM EDTA, and about 20 USP / mL Li Heparin salt.

18. An analyzer for analyzing a lateral flow device, comprising: an opening to receive a lateral flow device of any one of claims 1-9; a barcode scanner; a light source optionally being an illumination light-emitting diode (LED); a camera, lens, fdter assembly to record the image of a lateral flow strip in the lateral flow device; a battery or electric power cord; a display on the outside of the analyzer; and a computer readable medium comprising computer executable instructions to process the image, integrate the test line (TL) signal, perform background subtraction, calculate background-subtracted TL signal, convert the TL signal into sFLT plasma or serum concentrations.

19. The analyzer of claim 18, further comprising one or more of a pSD card port; a battery port; a USB port; and a printer.

20. A system, comprising a kit of any one of claims 10-17; and an analyzer of any one of claims 18-19, configured to measure the intensity of the test line, control line, or both on the lateral flow device.

21. The system of claim 20, further comprising an analyzer control cassette configured to test the analyzer’s electronics and photometric detection system.

22. A method of detecting a level of soluble fms-like tyrosine kinase 1 (sFlt-1) in a plasma or serum sample with a lateral flow device of any of one of claims 1-9, the method comprising: applying (a) a mixture containing or consisting of a plasma or serum sample and a chase buffer, or (b) the plasma or serum sample and the chase buffer concurrently or sequentially, to the sample receiving region of the lateral flow device; measuring the level of the latex beads at the first location in the test region; and calculating the level of sFlt-1 based on the measured level of the latex beads.

23. The method of claim 22, wherein measuring the level of the latex beads is performed at about 20-30 minutes after applying the mixture or the plasma or serum sample to the sample receiving region.

24. The method of claim 22, wherein measuring the level of the latex beads is performed at about 25 minutes after applying the mixture or the plasma or serum sample to the sample receiving region.

25. The method of claim 22, wherein the plasma or serum sample is from a pregnant woman.

26. The method of claim 22, wherein the plasma or serum sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant.

27. The method of claim 22, wherein measuring the level of the latex beads at the first location in the test region, or calculating the level of sFlt-1 based on the measured level of the latex beads, or both is performed with an analyzer of any one of claims 18-19.

28. A method of prognosticating a woman’s risk of having preeclampsia with severe features (sPE), comprising assaying a plasma or serum sample obtained from the women to detect a level of soluble fms-like tyrosine kinase 1 (sFlt-1) with a lateral flow device of any of one of claims 1- 9, the method comprising: pre-mixing the plasma or serum sample with a chase buffer to obtain a mixture sample and applying the mixture sample to the sample receiving region of the lateral flow device; measuring the level of the red latex beads at the first location in the test region; calculating the level of sFlt-1 based on the measured level of the red latex beads; and prognosticating the women’s risk of having sPE as high when the level of sFlt-1 is higher than a reference level or prognosticating the women’s risk of having sPE as low when the level of sFlt-1 is less than the reference level, wherein the reference level is 2.0-6.0 ng / mL, wherein a high risk of having sPE is having sPE within three weeks from having her plasma or serum sample assayed.

29. The method of claim 28, wherein measuring the level of the red latex beads is performed at about 20-30 minutes after applying the mixture sample to the sample receiving region.

30. The method of claim 28, wherein measuring the level of the red latex beads is performed at about 25 minutes after applying the mixture sample to the sample receiving region.

31. The method of claim 28, wherein the plasma or serum sample is from a pregnant woman who is between 23 0 / 7 weeks to 34 6 / 7 weeks pregnant.

32. The method of claim 28, wherein measuring the level of the red latex beads at the first location in the test region, or calculating the level of sFlt-1 based on the measured level of the red latex beads, or both is performed with an analyzer of any one of claims 18-19.

33. The method of claim 28, wherein the reference level is 3.0 ng / mL.

34. The method of claim 28, wherein a high risk of having sPE is having sPE within two weeks from having her plasma or serum sample assayed.

35. The method of claim 28, wherein a high risk of having sPE is having sPE within one weeks from having her plasma or serum sample assayed.

36. A method of treating or managing preeclampsia or eclampsia for a patient in need thereof, comprising: prognosticating the women’s risk of having sPE by the method of any one of claims 28-35, and initiating one or more evaluation or preparedness for the woman whose risk of having sPE is prognosticated as being high, or providing standard of care expectant management to the woman whose risk of having sPE is prognosticated as being low.

37. The method of claim 36, wherein one or more evaluation or preparedness comprises step-up care for the woman, providing neonatal intensive care unit (NICU) consult, increase frequency of tests to assess severity of the preeclampsia or eclampsia, increase frequency of fetal assessments, planning for steroids or magnesium treatment.

38. The method of claim 36, further comprising administering steroids or magnesium to the women when the risk of having sPE is prognosticated as being high.

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