Device for detecting misfolded proteins and method of using same - Patents.com

Abstract

A simple diagnostic device that can be used at the point of care to detect possible pre-eclampsia in pregnant mammals, particularly human females, providing early information as to whether a woman is at risk of or has pre-eclampsia and therefore should undergo immediate therapeutic intervention. The present invention relates to diagnostic devices for detecting proteins of interest associated with a disease or disorder in a mammal, as well as methods of using these devices. In particular, the proteins of interest can be misfolded proteins associated with a particular misfolded protein disorder in a mammal, including mammals suspected of or at risk of having such a disorder.

Description

[Background technology]

[0001] According to the American College of Obstetricians and Gynecologists, hypertensive disorders of pregnancy, including preeclampsia, complicate approximately 10% of pregnancies worldwide and are a leading cause of maternal and fetal morbidity and mortality [Reference: Hypertension in Pregnancy, Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy, Obstetrics and Gynecology 122 Vol. 122, No. 5, November 2013 (ACOG 2013 guidelines)]. Furthermore, these conditions are a leading cause of preterm birth and associated perinatal complications [Reference: Ananth CV, Vintzileos AM. J Matern Fetal Neonatal Med. 2006:19(12):773-82]. Hypertension during pregnancy can be classified as 1) preeclampsia-eclampsia, 2) chronic hypertension, 3) chronic hypertension with mixed preeclampsia, or 4) gestational hypertension.

[0002] Pre-eclampsia-Eclampsia is a poorly understood pregnancy-related condition that is a leading cause of maternal mortality, premature birth, and increased healthcare costs for childbirth. Globally, 76,000 maternal deaths are attributable to pre-eclampsia. Pre-eclampsia is responsible for one-fifth of pregnancy-related deaths in the United States, and the condition can lead to seizures, organ failure, and death. It most commonly occurs after about 20 weeks of pregnancy, and women are at risk throughout the postpartum period. The condition can cause seizures or convulsions, known as eclampsia. Pre-eclampsia can be classified as mild, severe, less severe, more severe, or as pre-eclampsia without severe features or with severe features. HELLP syndrome, a subtype of pre-eclampsia, is characterized as patients with symptoms of hemolysis, elevated liver enzymes, and low platelet counts. Preeclampsia that presents with an unusual constellation of symptoms is known as atypical preeclampsia.

[0003] The only known treatment is to deliver the baby, and as a result, preeclampsia is the leading cause of medically indicated preterm birth, estimated at 17% of all preterm births. The cost to the US healthcare system is estimated to be over $13 billion for preeclampsia delivery and nursing of the mother and infant. Currently, preeclampsia remains a challenge to diagnose, as it can only be characterized by its symptoms, most often high blood pressure and the presence of protein in the urine. Research aimed at improving the diagnosis of preeclampsia has generally explored known blood biomarkers that are up- or down-regulated, but few, if any, discoveries have led to globally useful diagnostic agents.

[0004] A study utilizing urine samples from women with severe preeclampsia who required medically indicated delivery (MIDPE) due to a diagnosis of preeclampsia and an unbiased mass spectrometry protein profiling approach found unique nonrandom cleavage products of SERPINA-1 and albumin. The finding that fragments of SERPINA-1 tend to misfold and form supramolecular aggregates led to the proposal that preeclampsia may be a misfolding disorder similar to Alzheimer's disease [see U.S. Pat. No. 8,263,342 and Buhimschi et al., Am J Obstet Gynecol. 2008 Nov;199(5):551.e1-551.16.doi:10.1016 / j.ajog.2008.07.006]. Furthermore, misfolded proteins were found in the urine of women with preeclampsia based on their binding to the Congo Red (CR) dye ("Congo red affinity"). These misfolded protein(s) or "supramolecular aggregates" bound to conformational state-dependent anti-amyloid aggregate antibodies were associated with a highly active amyloid precursor protein (APP) processing pathway and amyloid-like protein deposits in placentas from women with preeclampsia. [See Buhimschi et al., Sci. Transl. Med. 6, 245ra92 (2014)]. The percentage of CR retained after washing (attributable to binding to misfolded proteins) was measured by dot blot affinity assay (as a percentage of original CR) and the results were reported as Congo red retention (%) (CRR).

[0005] In a feasibility study of 80 women (40 with medically indicated delivery and 40 with healthy pregnancies as "controls"), the CRR (%) was significantly higher in those with severe preeclampsia (P<0.001), with sensitivity and specificity of 100%. In a validation study of 582 women (in cross-sectional and longitudinal cohorts), women with severe preeclampsia and preeclampsia complicated by pre-existing hypertension or proteinuria had a higher CRR (%) than all other clinical categories (P<0.001). Furthermore, 75% of women diagnosed with mild preeclampsia, 89% with severe preeclampsia, and 91% with mixed preeclampsia had a higher CRR than all other groups (P<0.05). Overall, CRR alone had a sensitivity of 85.9% and a specificity of 85.00, a positive likelihood ratio of 95%, and a negative likelihood ratio of 95% in the validation cohort in predicting preeclampsia requiring MIDPE. CRR was superior to currently used clinical screening methods for preeclampsia (P<0.001 compared with blood pressure or urinary protein dipstick; P=0.004 compared with blood pressure combined with urinary protein at the American College of Obstetricians and Gynecologists (ACOG) recommended cutoffs) (Buhimschi et al., Sci. Transl. Med. 6, 245ra92 (2014) and U.S. Patent No. 9,229,009).

[0006] A simple paper-based assay was created using the fact that Congo Red also binds to cellulose. When a normal urine-dye mixture is applied to the test paper, the dye bound to the cellulose in the paper is visualized as a tightly centered red dot. In contrast, in urine from women with Congo Red affinity urinary proteins, the dye is no longer bound to the cellulose because it is bound to the protein, but instead disperses diffusely and is visualized as a halo. (See US Patent Publication No. 20150293115.) In a clinical study of 346 women who presented to a labor and delivery triage center to confirm they did not have preeclampsia, the patients' urine was tested using a simple paper assay with CR (CRD). The CRD test has been demonstrated to have a sensitivity of 79%, a specificity of 89%, a negative predictive value of 91%, and a positive predictive value of 74% for the diagnosis of preeclampsia as defined by the ACOG 2013 guidelines [Ref: Rood et al. 2016 AJOG vol. 214, no. 1, supplement, pp. S24-S25]. The CRD test requires a step in which urine is mixed with a dye before being applied to the test strip, and the results can be difficult to read and interpret. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Pat. No. 8,263,342 [Patent Document 2] U.S. Pat. No. 9,229,009 [Patent Document 3] US Patent Application Publication No. 2015 / 0293115 [Non-patent literature]

[0008] [Non-Patent Document 1] Hypertension in Pregnancy, Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy, Obstetrics and Gynecology 122, Vol. 122, No. 5, November 2013 (ACOG 2013 Guidelines) [Non-Patent Document 2] Ananth CV, Vintzileos AM.J Matern Fetal Neonatal Med.2006:19(12):773-82 [Non-Patent Document 3] Buhimschi et al., Am J Obstet Gynecol. 2008 Nov;199(5):551.e1-551.16.doi:10.1016 / j.ajog.2008.07.006 [Non-Patent Document 4] Buhimschi et al., Sci. Transl. Med. 6, 245ra92 (2014) [Non-Patent Document 5] Rood et al. 2016 AJOG Vol. 214, No. 1, Special Issue, pp. S24-S25 Summary of the Invention [Problem to be solved by the invention]

[0009] In view of the above, there remains a very important unmet need for a simple diagnostic device that can be used at the point of care to detect possible pre-eclampsia in pregnant mammals, particularly human females. Such a device could potentially save the lives of thousands of pregnant women as well as their unborn children by providing early information on whether a woman is at risk for or has pre-eclampsia and therefore should undergo immediate therapeutic intervention.

[0010] All patents and publications mentioned herein are incorporated by reference in their entirety. [Means for solving the problem]

[0011] Summary of the Invention The present invention includes a diagnostic device for detecting at least one protein in a mammalian biological sample. The device includes a) a sample receiving material, b) a detection reagent, c) a trap, and d) a capillary bed, the sample receiving material being capable of receiving a biological sample; the detection reagent being reactive (i.e., binding) to at least one protein present in the biological sample; the trap being in contact with the sample receiving material and capable of separating the detection reagent bound to at least one protein in the biological sample from the detection reagent not bound to at least one protein in the biological sample, thereby allowing the detection reagent bound to at least one protein in the biological sample to flow through the trap and the detection reagent not bound to at least one protein in the biological sample to be captured by the trap; the capillary bed being in contact with the trap and configured to contain the biological sample after the biological sample has flowed through the trap. The capillary bed indicates the detection reagent being bound if at least one protein is detected in the biological sample. The sample receiving material, the trap, and the capillary bed are configured to be in contact in sequence. The sample receiving material of the device may, for example, include the detection reagent. Additionally, the detection reagent may be on or within the sample receiving material.

[0012] Additionally, the device may be housed within a housing or cassette, which may include a well (or other entity) for applying the biological sample and may further include a window for reading the results obtained. The device may be used at the point of care in a variety of clinical and non-clinical settings, or in a clinical laboratory.

[0013] As mentioned above, the detection reagent binds to at least one protein in the biological sample. The at least one protein may be, for example, a misfolded protein, a protein aggregate, a supramolecular protein aggregate, and mixtures thereof and fragments of each protein. The at least one protein may include a β-sheet structure. In addition, the at least one protein may be Congola red affinity. The misfolded protein may include, for example, alpha 1 antitrypsin (SerpinA1), ceruloplasmin, heavy chain IgG, light chain IgG, interferon-inducible protein 6-16 (IF16-6, G1P3), albumin, mixtures thereof or fragments thereof, or fragments of each protein. However, the misfolded protein is not limited to these proteins. For example, the misfolded protein may be any protein (or combination of proteins) that causes or is associated with a protein misfolding disorder.

[0014] The detection reagent may be, for example, an azo dye, thioflavin T, or an analogue of an azo dye. An example of an azo dye that may be utilized in connection with the present invention is Congo Red (i.e., 4-amino-3-[4-[4(1-amino-4-sulfonato-naphthalen-2-yl)diazenylphenyl]phenyl]diazenyl-naphthalene-1-sulfonic acid) disodium. (Analogs of Congo Red may also be utilized, e.g., the compound of formula C 32 H 22 N 6 Na 2 O 6 S 2 Secondary diazo dyes of the following formula may also be used as detection reagents as described herein in connection with the devices of the present invention.) Congo Red may be preloaded on the sample receiving material referred to above.

[0015] The sample receiving material of the device of the invention may comprise, for example, nitrocellulose, cellulose, glass fiber, cotton, woven mesh, non-woven material, porous plastic, polymer and / or polyester, which may be, for example, polyethylene.

[0016] The traps of the devices of the invention may comprise, for example, nitrocellulose, cellulose, fiberglass, cotton / fiberglass, woven mesh, non-woven materials, polymers, and / or polysulfone.

[0017] The capillary bed of a device of the invention may comprise materials such as, for example, nitrocellulose, chromatography paper, polysulfone and / or cellulose.

[0018] It should be noted that the device of the present invention produces test results within about 10 minutes, preferably within about 5 minutes, more preferably within about 3 minutes, and most preferably within 1 minute. Moreover, qualitative or semi-quantitative results can be obtained by visualization. Moreover, if desired, semi-quantitative or quantitative results can be obtained, such that the amount of at least one protein is measured.

[0019] The present invention also includes a diagnostic device as described above, which is utilized for detecting misfolded proteins in a biological sample, for detecting aggregated proteins in a biological sample, and / or for detecting supramolecularly aggregated proteins in a biological sample, wherein the sample is obtained from a mammal, such as a human, a primate, or a genetically engineered mammal. In some cases, the mammal may be pregnant. The device as described above may be used to detect pre-eclampsia, which may be diagnosed when the detection reagent is reactive (i.e., binds) to misfolded proteins, aggregated proteins, and / or supramolecularly aggregated proteins (i.e., proteins associated with pre-eclampsia in pregnant mammals) contained in the biological sample. The trap of the device may be configured to competitively bind to the detection reagent of the device. The device may be configured as a lateral flow device or strip, including a sample receiving material, a trap, and a capillary bed.

[0020] Additionally, the present invention encompasses a method for detecting at least one protein in a mammalian biological sample, comprising the steps of: a) applying the mammalian biological sample to a sample receiving material of a diagnostic device of the present invention for a time and under conditions sufficient to allow binding of at least one protein to a detection reagent; and b) detecting the presence of a detection reagent on the capillary bed, wherein the presence of the detection reagent on the capillary bed indicates the presence of at least one protein present in the biological sample.

[0021] The method may be used to detect at least one protein in a biological sample of a mammal having or at risk of having a protein misfolding disorder. The method includes the steps of (a) applying the biological sample of the mammal to a sample receiving material of the diagnostic device described above for a time and under conditions sufficient to bind at least one protein to the detection reagent, and (b) detecting the presence of the bound detection reagent on the capillary bed, the presence of the detection reagent on the capillary bed indicating the presence of at least one protein present in the biological sample and indicating that the mammal has a protein misfolding disorder. Again, the at least one protein may be, for example, a misfolded protein, an aggregated protein, a supramolecular aggregated protein, or a mixture thereof, or a protein having a beta-sheet structure, such as a Congophilic protein. The at least one protein may be Congophilic and / or have a beta-sheet structure. More specifically, the misfolded protein may be, for example, alpha 1 antitrypsin (SerpinA1), ceruloplasmin, heavy chain IgG, light chain IgG, interferon-inducible protein 6-16 (IF16-6, G1P3), albumin, mixtures or fragments thereof, or fragments of each protein. The protein misfolding disorder may be, for example, pre-eclampsia, Alzheimer's disease, prion disease, or Parkinson's disease. Misfolded proteins found in other diseases or conditions characterized as protein misfolding disorders may also be detected using the device of the present invention. With regard to the diagnosis of pre-eclampsia using the device of the present invention, different forms of pre-eclampsia may be diagnosed, including, for example, mild pre-eclampsia, severe pre-eclampsia, atypical pre-eclampsia, hemodialysis-elevated liver enzymes-decreased platelet count (HELLP) syndrome, and eclampsia. Furthermore, the patient may suffer from a hypertensive disorder of pregnancy.Thus, the method may be utilized for the differential diagnosis of certain hypertensive disorders of pregnancy, such as to differentiate pre-eclampsia from hypertensive conditions such as chronic hypertension or gestational hypertension or hypertension due to other causes, or to differentiate between the types of pre-eclampsia mentioned above.

[0022] The biological sample used in the above method and applied to the sample receiving pad may be, for example, urine (clean catch, i.e. naturally collected), blood, saliva, tissue, interstitial fluid, serum, plasma, cerebrospinal fluid, amniotic fluid, or extracts (e.g., nasal secretions, earwax, fecal matter, and tissue extracted). The method is utilized in connection with biological samples from mammals, such as humans, primates, and genetically engineered mammals. The mammal may be pregnant. In the case of humans, the method may be utilized in connection with pregnant women from approximately 8 to 42 weeks (i.e., gestational age) of gestation, preferably from about 18 to 41 weeks, more preferably from about 20 to 41 weeks or from 20 weeks to delivery. However, the method of the present invention may also be utilized in connection with postpartum mammals. It should be noted that the at least one protein detected by the method using the device may be detected by visualization to obtain a qualitative or semi-quantitative result, or by measurement to obtain a semi-quantitative or quantitative result. After visualization, the at least one protein may be measured to obtain a semi-quantitative or quantitative result.

[0023] Additionally, the present invention includes a kit comprising the above-mentioned device. The kit may also include instructions for using the device, as well as a standard or control reagent. The kit may also include a sample applicator. [Brief description of the drawings]

[0024] [Figure 1]Figure 1 shows a photograph of a test strip of a device of the present invention in a cassette or housing. The left panel of Figure 1 illustrates the device in the housing before contact with a biological sample, with the device having a sample well, a result window, and a control line. The right panel shows the results of the device testing urine samples that were found to be negative, weakly positive, and positive for misfolded proteins associated with pre-eclampsia. [Diagram 2] 1 shows an example of a test strip embodiment of a device of the present invention, the strip includes a control dye, an indicator strip (i.e., a capillary bed), a trap (e.g., three pieces of material), a second sample pad (i.e., a sample receiving material), a first sample pad (i.e., a sample receiving material) and a dye dried onto the sample pad. [Diagram 3]

[0023] Figures 3A and 3B show an embodiment of a diagnostic device of the present invention. Figure 3A is a top view with a sample pad, trap and indicator strip. Figure 3B shows the same embodiment as Figure 3A, from a side view. The sample pad, trap and indicator strip may be butted together. Figure 3C shows an embodiment in which the sample pad, trap and indicator strip overlap each other (side view). [Figure 4] 4A shows an alternative embodiment of the invention having two sample pads, a trap and an indicator strip (capillary bed) in sequence, with each part overlapping with its neighbors. FIG. 4B shows another alternative embodiment of the invention having two sample pads, a triple trap and an indicator strip in sequence, with each part overlapping with its neighbors. [Diagram 5]FIG. 1 shows an example of a dipstick configuration of a test strip of the present invention. This embodiment is configured with a cover tape on the top surface of the test strip. At the top of the test strip, the tape may be opaque and used to hold the strip. In the middle, the tape is transparent and provides a window for viewing the results. The tape may be placed so that the transparent window is, for example, approximately 10 mm above the trap. The tape at the window may be surrounded by a color (e.g., white) to allow easy viewing of the results. Below the trap, the tape is opaque to cover the trap. At the bottom of the strip, the tape may display an arrow indicating which end of the strip should be immersed in the test sample and may display a line indicating the maximum level to immerse the test strip in the sample. The bottom end of the test strip may be free of cover tape to facilitate wicking of the test sample when immersed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention is a device and a method that utilizes this device.More specifically, the device is a lateral flow chromatography rapid test that can be used in several clinical and non-clinical environments to detect proteins of interest in biological samples.The device can be used to detect protein misfolding disorders in mammals.Mammals may be suspected or at risk of having one or more such disorders.

[0026] The device has several different embodiments described herein. Basically, it comprises a test strip for detecting a protein(s) of interest in a biological sample (see, for example, FIG. 1). The detection is carried out with the aid of a series of reactions. The test strip has a length of lateral flow assay or chromatographic material with capillary action and a first end where the transport of the chromatographic solvent begins. It also has a second end where the transport of the chromatographic solvent ends. The strip comprises a number of zones or regions located between the first and second ends (see, for example, FIGS. 2 and 3). The zones include a first zone that is impregnated with a detection reagent, for example a dye. This detection reagent specifically binds to the protein(s) of interest in the biological sample. The first zone also receives the biological sample. In comparison, the second zone downstream of the first zone retains the detection reagent that is not bound to the protein(s) of interest in the biological sample while transporting the detection reagent that is bound to the protein(s) of interest in the biological sample to the third zone. The third zone of the test strip is located downstream of the second zone and receives the sample after it has passed through the second zone. The third zone indicates the detection reagent if the protein of interest is present in the sample. It also includes a means for detecting the detection reagent-bound protein as a measurement of the protein(s) in the biological sample. In one embodiment, the device allows the presence of misfolded proteins in a biological sample from a patient to determine whether the patient has a protein misfolding disorder. The presence of the protein(s) can be qualitatively or semi-quantitatively determined by visualization, or may be semi-quantified or quantified by using a measurement entity that may be present in the device.After a biological sample is applied to the first zone, the first zone releases the detection reagent into the sample, the second zone separates the detection reagent bound to the protein(s) from the unbound detection reagent and transports only the bound detection reagent to the third zone, which then displays the bound detection reagent for viewing or measurement. The first zone may be a sample receiving material. The second zone may be a trap. The third zone may be a capillary bed or an indicator strip.

[0027] Specific elements or components of the device, and the properties or characteristics of these elements, are described in detail as follows.

[0028] Materials used in device components The sample receiving material, trap, and capillary bed can be made of the same or different materials. Materials are generally known in the art of lateral flow devices and chromatography [see reference: EMD Millipore Rapid Lateral Flow Test Strips Considerations for Product Development, available from EMD Millipore, Billerica, MA]. Membranes are selected based on physical and chemical properties that affect capillary flow and therefore reagent deposition and assay performance. These materials include, but are not limited to, for example, nitrocellulose, filter paper, chromatography paper, cellulose, plastic polymers, asymmetric polysulfone membranes, cotton, linters and / or glass fibers, polyester, polyethylene, and polysulfone. Membranes may be made of polymers, including, for example, nitrocellulose, polyvinylidene fluoride, nylon, and polyethersulfone. Pad materials are often used as sample receiving materials to allow controlled and uniform sample reception and easy flow to the adjacent strip material of the device. Pad materials are porous and are often made of cellulose (i.e., filter paper), glass fiber, mesh woven and synthetic nonwoven materials, or polyester. Filter matrices may be used to receive the sample, especially if it is desired to separate and remove unrelated materials contained in the sample from the portion of the sample to be assayed (e.g., to separate and remove cellular material from the fluid). These filter matrices may be, for example, cellulose, asymmetric polysulfone membranes (including, but not limited to, Vivid™ plasma separation membranes and asymmetric submicron (BTS) polysulfone membranes). Absorbent pads, for example as wicks at the ends of the strips of the device, may be used to draw the sample through the lateral flow strip and increase the amount of sample assayed, thus increasing the sensitivity of the assay.These absorbent pads are often cellulose or cotton linters, and are best selected based on thickness, compressibility, and uniformity of total volume. The entire strip may be assembled on a backing card, which is often a card made of a plastic backing and adhesive. While these various materials are often considered for a specific purpose in the lateral flow device as described herein, each material can be considered for its properties suitable for the purposes of the receptive material, trap, and indicator strip of the present invention. See the examples for further discussion of materials.

[0029] Other materials utilized in the construction of test strips, particularly indicator strips, are known in the art of lateral flow technology and chromatography.

[0030] An element or component of a device Sample Receiving Material (Sample Pad) The first element of the device (hereafter referred to as the sample receptive material or sample pad) acts as a sponge, holding excess sample fluid to be tested. The receptive material not only absorbs the sample, but also allows it to flow or wick up to the next adjacent material. The receptive material is typically inert to, and therefore does not react with, the proteins of interest that may be present in the sample as well as the detection reagents (e.g., dyes), allowing the proteins and detection reagents to flow or wick up through it to the adjacent materials in the lateral flow device.

[0031] The sample receiving material may be immersed in a sample from a mammal or patient, or alternatively, the sample may be applied indirectly or directly to the sample receiving material. The sample may be applied to the sample receiving material by, for example, a dropper with a metering tip, a pipette, a volumetric pipette, or a pipette capable of repeatedly dispensing a patient sample. If the sample receiving material is configured to be immersed in a biological sample (see, for example, FIG. 5), the sample receiving material may be relatively long (for example, but not limited to, about 10 mm). If the sample receiving material is configured to receive a sample applied by, for example, a dropper or pipette, the sample receiving material may be relatively short (for example, but not limited to, between about 5 mm and about 10 mm). Typically, the width may be 2 mm to 10 mm, and most typically, 2.5 to 5 mm (±0.5 mm). Variations in the length and width of the sample receiving material are possible and depend on factors such as the size of the cassette or housing and the ability of the biological sample to mix sufficiently with the detection reagent.

[0032] In particular, the sample receiving pad serves as a capillary matrix in which the biological sample and the detection reagent (e.g., dye) can freely mix. The sample pad may also have a detection reagent in a dry form suitable for an optimized chemical reaction between the analyte (e.g., a protein of interest in the biological sample to be detected) and the detection reagent. The detection reagent may be preloaded on the sample receiving material. In one embodiment, the biological sample (e.g., urine) dissolves the detection reagent when added to the sample receiving pad, and the mixture of the sample and the detection reagent dye is then transported across the device to an adjacent material such as a trap by flowing through the sample pad.

[0033] Alternatively, the sample pad comprises a series of two or more sample pads (see, for example, FIG. 4). For example, a first pad may receive the sample and a second may contain a detection reagent, whereby the biological sample is transferred from the first element (pad) to the second element (pad) containing the detection reagent in a dry format suitable for an optimized chemical reaction between the analyte of interest and the detection reagent. If two or more sample receiving materials are utilized, any one may contain the detection reagent. Preferably, the first sample receiving material contains the detection reagent and the second sample receiving material does not.

[0034] In yet another embodiment, the first sample pad is designed to receive the biological sample and separate and remove or retain insoluble materials that may be present in the sample. The filtered sample then flows through the first sample pad or flows to a second pad, and a detection reagent is incorporated into either the first or second sample pad to allow proper mixing of the detection reagent with the sample. The second pad may have the same composition as the first pad or a different composition.

[0035] Additionally, in another embodiment, the first sample pad receives the sample and also contains a detection reagent, and the second pad provides additional time for the detection reagent to mix with the analyte of interest before entering the next adjacent material in the strip, e.g., the trap.

[0036] In additional embodiments, the sample receiving material includes a substrate for a detection reagent, which is maintained in a dry state. The detection reagent may be on or within the sample receiving material. When a patient sample is added, the detection reagent is released. The substrate does not react with or absorb the patient sample, and when applied, the patient sample migrates through the matrix onto an adjacent material, such as a trap.

[0037] It should be noted that the sample may be applied or placed into the cassette or housing, for example, through a sample well or other entity for receiving the sample of the device. The sample well or entity may be located above the sample receiving material (e.g., a test strip) when it is assembled or housed in the cassette or housing (see FIG. 1).

[0038] Materials useful as sample receiving material or sample pad are generally known in the technical field of lateral flow devices and chromatography [see EMD Millipore Rapid Lateral Flow Test Strips Considerations for Product Development, available from EMD Millipore, Billerica, MA] and are selected based on the physical and chemical properties that affect sample reception, controlled and uniform capillary flow, and sample filtration.In addition, if the sample receiving material also contains detection reagent, ideally, the material is a suitable matrix for holding detection reagent and optimally releasing it when test sample is added.Pad materials are porous and are often made of cellulose (e.g., filter paper), glass fiber, mesh fabric, synthetic non-woven material, or porous plastic, such as polyester. Other materials that can be used as sample receiving materials include, for example, polysulfone asymmetric membranes, cotton / glass fiber materials such as Ahlstrom® 8950, plastic polymer membranes such as polyethylene (e.g., high density polyethylene), polytetrafluoroethylene, and porous glass fiber membranes (see, for example, Porex, Fairburn, GA). For further description of materials, see the Examples (i.e., Examples 2 and 6).

[0039] It should be noted that the sample receiving material used in the device of the present invention may be cellulose when the detection reagent is a dye with affinity to cellulose, provided that there is sufficient detection reagent present to bind to the denatured protein(s) of interest in the biological sample. More specifically, it is unacceptable for the cellulose to "outcompete" the detection reagent for binding to the protein(s) of interest, e.g., misfolded protein(s) in the biological sample. Instead, cellulose may be used for the sample receiving material, provided that the sample receiving material is in a matrix that is less reactive with the detection reagent than the denatured protein(s) of interest. (Cellulose is, for purposes herein, a cellulose having the formula (C 6 H 10 O 5 ) n It is defined as an organic compound having a structure of β(1->4) linked D-glucose units in a linear chain of hundreds to thousands of units, specifically a polysaccharide.

[0040] trap The fluid (e.g., the sample, or the sample mixed with the detection reagent) then flows from the sample receiving material or sample pad through a filter (hereinafter referred to as a "trap") designed to retain any unbound detection reagent. In particular, the trap serves in a lateral flow device to separate free detection reagent (e.g., dye) from protein-bound detection reagent. Specifically, the trap material allows the sample to flow through it to the next adjacent material, but retains, retards the flow of, or binds to the unbound detection reagent if the protein(s) of interest are not present in the biological sample.

[0041] The trap borders, but preferably overlaps, a sample pad or a series of sample pad elements that contain a detection reagent (e.g., dye) (see, e.g., FIG. 3). The trap functions to separate detection reagent that is bound to the protein(s) of interest of the test sample (e.g., misfolded protein(s)) from detection reagent that is not bound to the protein(s) of the test sample, thus retaining unbound detection reagent while allowing bound detection reagent to flow through. Alternatively, the trap may be a series of one or more filters of the same or different materials.

[0042] Without being bound by theory, the trapping material may contain a substrate for the detection reagent so that unbound detection reagent binds to the trapping material and does not flow to the next material. Detection reagent already bound to the protein does not bind to the substrate in the trap and flows to the next material in the strip. The substrate may be the trapping material (e.g., cellulose) or it may be a chemical modification or addition to the trapping material. Alternatively, structural features of the trapping material composition may retain unbound detection reagent.

[0043] The trap may also be constructed from multiple overlapping or contiguous pieces of material to optimize retention of unbound detection reagent (see FIG. 4). The multiple pieces may be made from the same or different materials. Filter matrices, such as thermoplastic polymers, such as cellulose, asymmetric polysulfone membranes (including, but not limited to, Vivid™ plasma separation membranes, and asymmetric submicron (BTS) polysulfone membranes), may be used in the trap. For further description of trap materials, see the Examples (e.g., Examples 2, 4, 5, 8, 13, 14, and 16).

[0044] It was unexpected and quite surprising that there were so many nitrocellulose materials that actually performed well and allowed urine and dye-bound proteins to flow through the device. For example, Whatman® AE99 nitrocellulose membranes performed extremely well. (See Table 1). Some cellulose materials performed reasonably well (e.g., Ahlstrom® 601, 319, 247, Whatman® CF1, CF3, CF4; EMI11513, 5475, 5493), while others did not perform well (e.g., Ahlstrom® 270). Surprisingly, the materials that performed well in terms of retaining unbound dye while allowing protein-bound CR dye to flow were Vivid™ Plasma Separation Material (Pall Corporation) and Asymmetric Submicron (BTS) Polysulfone Membrane (Pall Corporation). (See Tables 1 and 2).

[0045] When the detection reagent is a dye such as Congo Red, the trap material may be, for example, filter paper, cellulosic, and / or materials such as EMI 11513, EMI 5475, EMI 5493, 1281, 642, Standard 17, C048, LF1, LF1, VF2, CF1, CF3, Ahlstrom 319. The trap may be, for example, about 5-10 mm long, or may be a series of pieces each 5-10 mm long.

[0046] In one embodiment, the trap retains free detection reagent (e.g., dye) but allows protein to flow past the bound dye, hi a particular embodiment, the trap is composed of cellulose and the detection reagent is Congo Red.

[0047] Detection Reagents The detection reagent is a substance that is reactive to the protein(s) of interest in the sample. For example, the detection reagent may be a substance that is reactive to or has binding affinity with misfolded protein(s) (e.g., Congophilic protein), aggregated protein, and / or supramolecular aggregated protein present in a biological sample from a mammal, such as a patient sample. The detection reagent may be preloaded on the reagent pad (e.g., coated on the reagent pad or the reagent pad is immersed in the detection reagent or dye). The reagent pad may be a sample receiving material or a sample receiving pad.

[0048] In one aspect, the detection reagent is a dye that stains the subset of proteins of interest in the biological sample if present. In one embodiment, the detection reagent can react with misfolded proteins. For example, the dye can be an azo dye, such as Congo Red (CR) or its analogues, buffered or unbuffered. Alternatively, other dyes can be used as detection reagents, but only if they have affinity (and can bind or react with) the misfolded protein, aggregated protein, and / or supramolecular protein(s) of interest in the biological sample or patient sample. Examples of such dyes include, but are not limited to, Congo red analogues such as those described in Sellarajah S et al., Synthesis of analogues of Congo red and evaluation of their anti-prion activity, J Med Chem. 2004 Oct. 21; 47(22):5515-34; and Helene Rudyk et al., Screening Congo red and its analogues for their ability to prevent the formation of PrP-res in scrapie-infected cells, Journal of General Virology (2000), 81, 1155-1164. The detection reagent for detecting misfolded protein(s) may also be, for example, Thioflavin T.

[0049] Moreover, in one aspect of the present invention, the detection reagent is not only present in the device in a dry form, but may also be present in other forms. The form of the detection reagent is suitable for optimal mixing with the sample when applied, and for binding to the protein of interest. The form of the detection reagent is suitable for the long-term stability or shelf life of the device. In one embodiment, the dried detection reagent is a dye. Furthermore, the dye may be Congo Red, and may be present in the device in an amount of, for example, 0.1ug to 800ug, more preferably 0.2ug to 480ug, even more preferably 1ug to 400ug, even more preferably 2.5 to 120ug.

[0050] Congo Red (CR) (e.g., buffered or unbuffered) may be pre-applied to the sample receiving material. For example, a CR solution may be applied to the material during kit manufacture and allowed to dry prior to assembly and packaging (see FIG. 2 and Example 6).

[0051] A detection agent is detectable, ie, visible to the naked eye or otherwise detectable, for example, by visual examination and / or a machine or electronic reader(s).

[0052] The present invention provides a detection reagent that is incorporated into the test device, for example during the manufacture or assembly of the device. This is an improvement over previous devices for detecting misfolded proteins, which required mixing of a dye with a biological sample before adding to the test. In a preferred embodiment, the test strip contains a sample receiving material that contains a detection reagent. When a biological sample is added to the sample receiving material of the test device, it is mixed with the detection reagent in the sample receiving material, and the biological sample-detection reagent mixture flows through the device.

[0053] Capillary bed ("indication strip") The sample (e.g., urine), with or without the detection reagent bound to the protein, passes through the trap and onto the capillary bed (hereafter referred to as the "indicator strip") where it accumulates. Thus, the indicator strip draws up the sample and flows it, indicating the presence or absence of the detection reagent. In particular, the indicator strip draws up the sample and flows it, indicating the presence or absence of the analyte bound to the detection reagent. The detection reagent or the bound reagent can then be visualized by human or mechanical means (to obtain a qualitative result) and / or can then be measured (i.e., semi-quantitatively or quantitatively). The indicator strip optimally provides a uniform flow of the sample throughout, and a relatively uniform indication of the detection reagent when the protein of interest is present. In certain embodiments, the intensity or concentration of the detection reagent on the indicator strip can correspond to the amount of the protein of interest in the sample. In another embodiment, the distance the detection reagent flows up the indicator strip can indicate the amount of the protein of interest in the biological sample. Furthermore, both the intensity of the detection reagent and the distance it moves up the indicator strip can indicate the concentration of the protein in the sample. Furthermore, when detecting misfolded protein aggregates or supramolecular aggregates, both the intensity of the detection reagent and the distance it climbs the indicator strip can indicate the size of protein aggregates present in the sample. Suitable materials for the capillary bed include, for example, nitrocellulose or chromatography paper. Polysulfone asymmetric membranes also provide suitable indicator strips. Other suitable materials include CytoSep® membranes, such as CytoSep® 1660 and MN-260. The capillary bed of the lateral flow strip is aligned under the result viewing window of the lateral flow strip cassette or housing (described below). See the examples for further description of materials (e.g., Examples 2, 3, and 9). If protein-bound detection reagent is present, the detection reagent is visualized in the window (see FIG. 1).

[0054] Wick Optionally, the device contains a wick. The wick may be located after the third zone or indicator strip in the strip or device of the invention. In use, a biological sample (e.g., fluid) applied to the device continues to migrate from the indicator strip into the final porous absorbent material, or "wick". The wick can act as a sample reservoir and also function to draw the sample through the strip. Absorbent pads, for example as wicks at the end of the strip of the device, can be used to draw the sample through the lateral flow strip, increasing the amount of sample assayed and increasing the sensitivity of the assay. These absorbent pads are often cellulose or cotton linters and are optimally selected based on thickness, compressibility, and uniformity of total volume.

[0055] Backing card The device may have a backing card. The entire strip may be assembled onto a backing card (such as those available from Lohmann, Orange, VA), which is often a card made of a plastic backing and adhesive.

[0056] Case / Cassette In one embodiment of the present invention, the diagnostic device is housed, contained, or enclosed in a housing or cassette. The device may further include a housing or cassette, including but not limited to a cartridge, plastic device, or extruded plastic part configured for this purpose, that houses the device. Several generic cassette housings are commercially available (e.g., from Kanani Biologicals, Gujarat, India or EASE-Medtrend Biotech LTD, Shanghai, China) or may be custom manufactured for this purpose (see, e.g., U.S. Design Patent Application No. 29 / 533,647). The device may be configured in a housing or cassette having a sample well for receiving a sample, where the sample well is located above the sample receiving material of the strip when the strip is assembled or housed in the cassette. Additionally, the device may be configured such that the display strip or capillary bed of the strip is located below the result viewing window of the housing when the device is erected in the housing or cassette.

[0057] In one embodiment, the device of the invention also includes an electronic reader capable of quantifying the results, such as the intensity of the detection reagent (e.g., dye) on the capillary bed (i.e., the result window), and may further include a display screen (e.g., an LED screen) for displaying the results. The reader may be part of the housing or an element integrally assembled with the housing, such as an element within the result window. Such readers are described, for example, in Venkatraman, Biosensors and Bioelectronics Vol. 74, Dec. 15, 2015, pp. 150-155, PCT Application WO2013083686A1, PCT Application WO2004010143A2, and PCT Application WO2006010072A2.

[0058] Cover tape The device, with or without a housing, may further include a cover, such as a protective adhesive tape (e.g., available from Lohmann, Orange, VA) or other material that protects the device from damage and allows the test results to be properly read. For example, the device may be configured to be used as a dipstick, such as a urine dipstick. In this embodiment, the lateral flow strip may be covered with a protective adhesive tape (see FIG. 5 and Example 11).

[0059] Control Reagents In a preferred embodiment, a control reagent may be present on the capillary bed, visible in the viewing window, before the device is used. (See Figures 1 and 2, Example 12.) As the biological sample (e.g., fluid) flows through the capillary bed, the control reagent dissolves, wicks up into the indicator strip, diffusing the line of control reagent and / or carrying the control away, so that the control reagent disappears or fades in the result window, or some other difference may be visualized or measured. A change in the control reagent in the viewing window indicates that the test sample (e.g., fluid) has been added and properly flowed through the device, and serves, for example, as a trial control (see Example 12). The control reagent may be visually detectable, e.g., by the naked eye, or otherwise detected or measured, e.g., by mechanical testing and / or electronic reader. In one embodiment, the control reagent is tartrazine. Other dyes that may be used as trial control reagents include FD&C Blue No. 1 - Brilliant Blue FCF, E133 (blue), FD&C Blue No. 2 - Indigotine, E132 (indigo), FD&C Green No. 3 - Fast Green FCF, E143 (turquoise), FD&C Red No. 3 - Erythrosine, E127 (pink), FD&C Red No. 40 - Allura Red AC, E129 (red), FD&C Yellow No. 5 - Tartrazine, E102 (yellow), FD&C Yellow No. 6 - Sunset Yellow FCF, E110 (orange).

[0060] Protein of interest to be detected As mentioned above, the present invention is directed to a device for detecting a protein of interest, more specifically, misfolded proteins, aggregated proteins, and / or supramolecular protein aggregates, and a method utilizing the device. It is known that α-helix is ​​an important structural motif of functional proteins in their native conformation. In contrast, protein conformational changes can lead to β-sheet structural motifs (i.e., β-sheet structures) or misfolded proteins, which in turn tend to cause protein aggregation and toxicity. Thus, misfolded proteins can be in the form of protein aggregates or supramolecular aggregates, and can be associated with misfolded protein disorders such as preeclampsia, Alzheimer's disease, prion disease, and Parkinson's disease.

[0061] In particular, the misfolded proteins, protein aggregates, and / or supramolecular aggregates associated with preeclampsia detected by the device and method of the present invention may include, but are not limited to, alpha 1 antitrypsin (serpin A1), ceruloplasmin, heavy chain IgG, light chain IgG, interferon-inducible protein 6-16 (IF16-6, G1P3), albumin, and fragments of each protein, mixtures thereof, and fragments of such mixtures. These proteins have binding affinity to the detection reagents (described below) utilized in the device of the present invention. For example, these misfolded proteins are Congo red-affinity, and have affinity for a dye called Congo red.

[0062] The device of the present invention may also be used to detect protein misfolding disorders other than preeclampsia. For example, the device may be used to detect Alzheimer's disease, cerebral beta amyloid angiopathy, degeneration of retinal ganglion cells in glaucoma, prion diseases, Parkinson's disease and other synucleinopathies, tauopathies, frontotemporal lobar degeneration (FTLD), FLTD-FUS, amyotrophic lateral sclerosis (ALS), Huntington's disease and other triplet repeat diseases, dementia (familial British and Danish types), hereditary cerebral hemorrhage with amyloidosis, CADASIL, Alexander's disease, various amyloid-related disorders, and other disorders that may be associated with glaucoma. The present invention may be utilized to detect misfolded proteins in misfolded protein disorders or conditions such as glaucoma, serinopathies, type II diabetes, inclusion body myositis / myopathy, cataracts, retinitis pigmentosa with rhodopsin mutations, medullary thyroid carcinoma, prolactin-producing pituitary adenomas, hereditary lattice corneal dystrophy, Mallory bodies, pulmonary alveolar proteinosis, amyloidogenic odontogenic tumors, cystic fibrosis, sickle cell disease, and critical illness myopathy.

[0063] Biological samples The protein(s) of interest to be detected can be found in a biological sample from a mammal. The biological sample can be, for example, urine obtained from a clean catch or natural urine collection, cerebrospinal fluid, amniotic fluid, or any bodily fluid sample that may contain the protein(s) of interest (e.g., blood, saliva, amniotic fluid, cerebrospinal fluid, plasma, or serum). The sample can also be an extract of excreta from a patient, for example, an extract from nasal secretions, fecal matter, or ear wax, or an analyte of tissue that is extracted with an appropriate solution and applied to the device. The protein of interest can be found in a biological sample from a mammal, for example, during pregnancy or after delivery.

[0064] patient The patient can be a mammal.Furthermore, the mammal can be pregnant, for example, a pregnant woman, a pregnant primate, or a genetically engineered animal model designed to have the physical symptoms and signs of pre-eclampsia (e.g., high blood pressure and urinary protein), such as those used in laboratory research.Preferably, in the case of diagnosing pre-eclampsia, the patient can be any pregnant woman.These pregnant women may be suspected of having pre-eclampsia or at risk of having pre-eclampsia. For example, this suspicion may be based on: 1) the presence of signs and symptoms of preeclampsia, e.g., as described in the American College of Obstetrics and Gynecology guidelines (ACOG) (Hypertension in Pregnancy, Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy, Obstetrics and Gynecology 122 Vol. 122, No. 5, November 2013), specifically, e.g., Table E-1 ("ACOG 2013 Guidelines"); and / or 2) the presence of one or more risk factors for preeclampsia (e.g., women with a previous pregnancy involving preeclampsia, women carrying multiple fetuses, women with cardiovascular or renal abnormalities, or women with an autoimmune disease such as lupus).

[0065] kit The present invention also includes a kit for detecting a protein of interest in a sample. In one embodiment, the kit includes a device for detecting misfolded proteins associated with pre-eclampsia present in a sample from a pregnant mammal. The kit includes the device of the present invention as described above, in any of the alternative embodiments described above. The kit may also include a means for applying a patient sample to a sample receiving material, such as a pipette (e.g., a fine-tip volumetric pipette available from Genesee Scientific, San Diego, CA) or a dropper, a control, and instructions for using the device. Thus, the device can be utilized as a standalone entity, but also in the form of a kit, which may be more advantageous in some clinical or non-clinical environments. The kit may be packaged in a foil or Mylar pouch. The kit pouch may be further packaged singly or in multiples, for example, 2, 5, 10, 15, 25, 50, or 100 kits per package.

[0066] Environment for using the device and method of utilizing the device The devices of the invention can be used, for example, in clinical laboratories (either in a hospital environment or outside of a hospital environment), rapid-care settings, physicians' personal exam rooms, emergency departments (e.g., in hospitals), or as patient-side tests or point-of-care devices by medical personnel, non-medical professionals, or even by the patient themselves when the patient is human. Additionally, the device can be used in combination with other diagnostic assays (e.g., immunoassays such as immunoassays that detect other proteins (i.e., biomarkers) associated with pre-eclampsia, such as sFlt-1, PlGF, PP-A, PP13, pentraxin, inhibin-A, and soluble endoglin), and / or other methods or findings commonly utilized in the diagnosis of pre-eclampsia (e.g., blood pressure measurements, laboratory tests used to diagnose pre-eclampsia, including platelet count, serum creatinine concentration, serum ALT (alanine aminotransferase) and AST (aspartate aminotransferase), and other signs or symptoms such as weight gain, dizziness, headache, blurred vision, etc.).

[0067] For example, the present invention includes a method of diagnosing or differentially diagnosing pre-eclampsia in a patient suffering from a hypertensive disorder of pregnancy or a patient who may be suspected of having pre-eclampsia, the method comprising the steps of a) determining the blood pressure of a pregnant patient (wherein a blood pressure higher than 140 / 90 mm / Hg in a pregnant patient may be indicative of pre-eclampsia in the pregnant patient), and b) applying a biological sample from the patient to a device of the present invention, whereby detection of at least one protein of interest in the patient's biological sample provides or assists in the diagnosis of pre-eclampsia in the patient, or serves as a differential diagnosis.

[0068] Furthermore, the present invention also encompasses a method of treating a pregnant mammal suspected of suffering from pre-eclampsia, comprising: a) applying a biological sample from the mammal to the device of the present invention to determine the presence of at least one protein that indicates that the pregnant mammal has pre-eclampsia; and b) allowing the pregnant mammal to deliver to treat pre-eclampsia. Furthermore, the device may also be used after delivery to determine whether the patient expresses misfolded protein(s) in the biological sample. If misfolded protein(s) are present, further patient management or therapeutic intervention (e.g., administration of magnesium sulfate or other antihypertensive agents) may be required to treat pre-eclampsia, or the patient may be monitored using the device of the present invention to indicate when the protein is no longer detectable in the biological sample. In addition, the device of the present invention may be utilized after therapeutic intervention to determine whether the treatment was successful or to measure changes (e.g., reduction, absence, or increase) in the amount or presence of the protein of interest present in the biological sample. Thus, the devices of the present invention may be used before and after treatment of a patient to determine whether further therapeutic intervention is necessary to resolve pre-eclampsia or other protein misfolding disorders, to determine whether therapeutic intervention has been effective and / or to administer alternative forms of therapeutic intervention or increased doses of a therapeutic agent.

[0069] Benefits of the device and how to use it The device of the present invention is advantageous over existing devices for at least the following reasons: 1) it reduces steps in the testing procedure, simplifying the testing procedure; 2) it can be optimally manufactured using standardized test materials; 3) it improves the stability of detection reagents in the test kit, resulting in a longer shelf life; 4) the results are simpler and easier to read overall, while 5) maintaining relatively low cost; and 6) it provides rapid results suitable for use at the point of care or in a clinical laboratory environment.

[0070] In particular, the provided devices and methods are far superior to known paper kits for detecting potential pre-eclampsia (see, e.g., U.S. Patent Application Publication No. 20150293115) in that they require fewer steps for the user, are easier to read results, and are more stable than paper kits, thereby providing a long shelf life of at least 6 months, preferably 1 year, even more preferably 2 years, even more preferably 3 years, even more preferably 4 years, and even more preferably 5 years. The devices of the present invention also produce rapid results suitable for minimally trained point-of-care users (i.e., within 3 minutes, preferably within 2 minutes, and more preferably within 1 minute of applying a biological sample (e.g., urine) to the device).

[0071] The invention can be illustrated by use of the following non-limiting examples. EXAMPLES

[0072] [ Example 1 Sample Device Figure 1 shows a photograph of a test strip of a device of the invention in a cassette or housing. The left panel of Figure 1 illustrates the device in the housing prior to contact with a biological sample. The right panel shows the results of the device testing urine samples known to be negative, weakly positive, and positive for misfolded proteins associated with pre-eclampsia.

[0073] 2 shows an example of a test strip embodiment of a device of the present invention. The strip includes a control dye, an indicator strip, a trap (e.g., three materials), a second sample pad, a first sample pad, and a dye dried onto the sample pad.

[0074] Figure 3A shows one embodiment of the diagnostic device of the present invention. Figure 3A shows a top view. Figure B shows the same embodiment as in Figure 3A, but from the side. The sample pad, trap, and indicator strip may be butted together. Figure 3C shows an embodiment in which the sample pad, trap, and indicator strip overlap each other (side view).

[0075] Figure 4 illustrates different material configurations within a test strip. Figure 4A illustrates an alternative embodiment of the invention having two sample pads, a trap, and an indicator strip (capillary bed), in sequence, with each part overlapping with its neighbor. Figure 4B illustrates another alternative embodiment of the invention having two sample pads, a triple trap, and an indicator strip, in sequence, with each part overlapping with its neighbor.

[0076] FIG. 5 shows an example of a dipstick configuration of the test strip of the present invention. This embodiment is configured with a cover tape on the top surface of the test strip. At the top of the test strip, the tape may be opaque and used to hold the strip. In the middle, the tape is transparent and provides a window for viewing the results. The tape may be placed so that the transparent window is, for example, approximately 10 mm above the trap. The tape at the window may be surrounded by a color (e.g., white) to allow easy viewing of the results. Below the trap, the tape is opaque to cover the trap. At the bottom of the strip, the tape may display an arrow regarding which end of the strip should be immersed in the test sample and may display a line indicating the maximum level to immerse the test strip in the sample. The bottom end of the test strip may be free of cover tape to facilitate wicking of the test sample when immersed.

[0077] [ Example 2 Materials Testing Various paper-like materials were tested for their ability to differentiate urine samples from pregnant women with or without preeclampsia. Congo Red (CR) dye (Sigma, St. Louis, MO) was added to the urine sample and a drop was added to the material to assess its suitable properties for receiving, trapping, and displaying strips. The resulting spots were visualized after about 3 minutes and evaluated for visual differences between urine from preeclamptic patients and urine from control normal pregnancies (Table 1). A result of "Excellent" indicates that the material was suitable for providing a visually observable difference between CR urine from preeclamptic patients and CR urine from control pregnant patients, and thus may be useful in diagnostic test devices. A result of "Poor" indicates that the material was not suitable for providing a visually observable difference between CR preeclampsia positive and CR control urine. On a scale of results from "poor" being the least suitable material, "subtle" being slightly better but not ideal, "acceptable" being better, "good" being even better, "better" being even better, and "excellent" being the most suitable material. A polysulfone asymmetric material was found to produce the best results. Certain cotton materials such as CF3 and CF4 performed well while other cottons such as Ahlstrom 270 did not perform well. Nitrocellulose and fiberglass materials generally did not perform well for this purpose.

[0078] [Table 1] TIFF0007648188000002.tif230163

[0079] Example 3 Test Strips Because BTS and Vivid™ demonstrated superior results in producing visually observable differences between CR urine from preeclamptic patients and control pregnant patients, test strips were assembled using Ahlstrom® 8950 (cotton / glass fiber composition) ("8950") sample pads and BTS or Vivid™ trial strips or capillary beds. Congo red was added to the urine sample, the sample was vortexed, and the sample was then applied to the 8950 end of the test strip. Results were observed on BTS or Vivid™ membrane trial strips. Urine from women with preeclampsia applied to strips containing Vivid™ membrane produced a very clear signal (pink / red Congo red staining) and negative urine showed no signal. The signal was volume dependent, with decreasing volume resulting in decreased signal. BTS strips showed no difference between positive and negative urine, with higher sample volumes both staining positive. When decreasing amounts of sample were applied, the signals of positive and negative urine decreased equally.

[0080] [Table 2]

[0081] These results indicate that the Vivid™ material was suitable for the diagnostic test device of the present invention, whereas the BTS did not perform well.

[0082] Example 4 Trap material evaluation Vivid™ and BTS membranes were evaluated as trapping materials by placing them between the sample pad and the trial strip (i.e., capillary bed or indicator strip) and allowing CR bound to pre-eclampsia positive urine to flow through to the indicator strip while assisting in the retention of CR dye when negative urine (i.e., urine from women without pre-eclampsia that does not contain the protein of interest) is applied. Test strips were assembled to include the 8950 as the test pad and either the Vivid™ membrane or the BTS membrane followed by a CytoSep® 1660 trial strip. CR spiked urine was applied to the 8950 and the results were observed on the CytoSep® 1660 trial strip.

[0083] Positive urine (i.e., urine containing the protein of interest from women with pre-eclampsia) - When Congo red dye was applied, CR staining was seen on all strips. Negative urine - When Congo red was applied, the strips made with the Vivid™ membrane trap were negative, but the strips made with the BST membrane trap showed some dye staining. The Vivid™ material was an effective trap at retaining the CR dye when negative urine was tested while allowing the CR dye to pass when positive urine was tested. BTS was a less effective trap.

[0084] Example 5 Evaluation of Materials for Use with Preloaded Detection Reagents in Test Strips Congo red dye was applied to the sample pad material and allowed to dry. A positive urine was applied and the results observed for dye released from the sample pad.

[0085] [Table 3]

[0086] The detection reagent (i.e., in this case, the dye) is ideally released from the sample pad material upon application of the biological sample, i.e., urine. Some materials, including POREX® 4894 and Porex® X-4897, release the dye well and are therefore very suitable for diagnostic test devices. In addition, some materials, such as Fusion material (glass microfiber) and Whatman® 33, also release the dye and are suitable.

[0087] Example 6 Evaluation of sample pads with dyes incorporated into test strips Sample pad materials including Porex® X4897 and POREX® 4894 infused with Congo Red dye were incorporated into test strips having a Vivid™ x trap and an MN-260 trial or indicator strip. Urine was applied to the sample pad of each strip and results were viewed on the MN-260 indicator strip.

[0088] Results: The sample pad material released dye when urine was added. Addition of pre-eclampsia positive urine resulted in positive staining of the MN260 material. In most cases, addition of pre-eclampsia negative urine did not stain the MN260; however, in some cases there was some staining at the beginning of the MN260 strip. These results indicate that the composition of the Porex®, Vivid™, and MN260 materials provides a means to differentiate pre-eclampsia positive from pre-eclampsia negative urine, thereby providing a useful diagnostic test device.

[0089] Example 7 Evaluation of test strips in lateral flow assay cassettes The strip was assembled from a dye-infused POR-4899 sample pad placed on top of a Fusion 5, followed by a Vivid X membrane to act as a trap, and then an indicating strip of MN260. The strip was placed into a universal cassette with the sample pad directly under the cassette's sample port and the MN260 visible through the cassette's result window. [75ul] Seventy-five microliters of urine was added into the sample port and the result was viewed in the result window. Red staining was visible in the result window when positive urine was added, whereas no staining was observed when negative urine was added. This configuration provided a useful diagnostic device to differentiate pre-eclampsia positive urine from pre-eclampsia negative urine.

[0090] Example 8 Trap Improvements Negative urines tested on the strips sometimes had some dye streaks that wicked up into the result strip, especially over time. Additional trap configurations were evaluated to see if they could better separate positive samples from negative samples and eliminate this streaking associated with negative urine. Strips were assembled using a dye-infused POR-4899 sample pad mounted on a Fusion 5, two offset but overlapping layers of Vivid™X traps, and a MN260 result strip. When tested by applying negative urine, the dual layer trap prevented dye leakage and streaking, resulting in an improved diagnostic test device for distinguishing pre-eclampsia positive urine from pre-eclampsia negative urine.

[0091] Example 9 Device Strip Configuration POR-4899 was lined with Congo Red dye solution dispensed at 6 μl / cm using a Kinematic dispenser. POR-4588 was lined at 4 μl / cm. The pads were dried at 37° C. for 1 hour and stored in a drying cabinet. Test strips were assembled using the lined POR material, one layer of Vivid™X trap, and MN260. The POR-4899 strips performed well when testing urine, but the strips containing POR-4588 caused dye leakage onto the indicator strip when testing negative urine. Further evaluation of POR-4588 lined at 4 μl / cm resulted in a clean flow of negative urine. POR-4588 was then evaluated lined at 6 or 8 μl / cm and assembled with dual Vivid™ traps. This strip assembly with a 6 μl / cm line of dye gave the best results when tested with positive urine and the least amount of dye leakage or streaking when tested with negative urine. Additionally, strips with 3 layers of Vivid™ traps and POR-4588 lined at 8 μl / cm gave good results and a stronger positive signal when evaluated.

[0092] [Table 4]

[0093] A device constructed with POR-4599 with Congo red applied at 8 μl / cm and dual vivid traps produced clean negative results when testing pre-eclampsia negative urine and strong positive results when testing pre-eclampsia positive urine and is the preferred diagnostic test device configuration.

[0094] Example 10 Evaluation of dipstick test designs. Test strips were assembled using POR-4588 and 3-layer Vivid™ traps lined at 6, 8, or 10 μl / cm, and MN260. The strips were dipped into 60 μl of urine. The urine was successfully drawn up into the strip, with the 8 μl / cm lined version producing the best positive test results along with clean negative test results. Thus, an optimal dipstick format for a diagnostic test device can be constructed in this way, which is useful for distinguishing pre-eclampsia positive urine from pre-eclampsia negative urine for diagnostic purposes.

[0095] Example 11 Further evaluation of diagnostic test device configurations Test strips made with two Porex® sample pads (one lined with dye), a three-layer Vivid™ trap, and MN260 were tested in cassettes / housings and as urine test dipsticks. The cassette housings improved results, likely as a result of controlled flow of the sample onto a central spot on the sample pad, better dye uptake, flow wicking to the center of the strip, and more uniform staining of the trial strip results. All five negative urines tested were negative, and of the five positive urines, two were weakly positive and three were positive. As a dipstick, the test strips were placed in 100 μl of urine in a test tube. At 3 minutes, both the positive and negative urine results were all negative. However, after more time, by 5 minutes, the positive urine results were two weakly positive and three positive, while the negative urines remained negative. Thus, these test configurations are suitable for a preeclampsia diagnostic test device.

[0096] Example 12 Evaluation of trial controls The purpose of this experiment was to investigate a control, or "trial control," that would indicate when the sample had been successfully wicked up and run off the strip. If no staining was observed on the indicator or result strip when a negative biological sample was tested, it would be difficult to tell whether this was a negative result or whether the test had not been performed correctly (i.e., no sample was actually added, insufficient sample was added, or insufficient sample wicking off the strip occurred). A line of water-soluble dye was sprayed across the trial strip material and positioned so that it would be visible at the top of the cassette's result window when assembled into the housing. A urine sample was added to the sample pad through the sample port and flow was observed through the result window. When the sample reached the control dye line, the line dissolved and ran with the sample. The disappearance of the trial control line was an easily visible indication that the sample had been successfully run off the strip. The control did not interfere with the reading of the urine test result. An example of a control line dye is tartrazine. Other dyes that may be used as trial controls include FD&C Blue No. 1 - Brilliant Blue FCF, E133 (blue), FD&C Blue No. 2 - Indigotine, E132 (indigo), FD&C Green No. 3 - Fast Green FCF, E143 (turquoise), FD&C Red No. 3 - Erythrosine, E127 (pink), FD&C Red No. 40 - Allura Red AC, E129 (red), FD&C Yellow No. 5 - Tartrazine, E102 (yellow), FD&C Yellow No. 6 - Sunset Yellow FCF, E110 (orange).

[0097] Example 13 Investigating alternative materials for traps Test strips were assembled using two Porex® sample pads (the first sample pad was marked with a line of Congo Red (CR) dye), various alternative trap materials as listed in Table 5, and a trial strip of MN260. Negative urine samples were tested on the assembled test strips to evaluate the ability of each trap to 1) retain CR dye in the absence of the protein of interest in the sample, and 2) not retain proteins bound to CR.

[0098] [Table 5]

[0099] RESULTS: Most of these trap materials showed promise in differentiating Congophilic protein-positive biological samples (Congophilic protein-positive urine) from Congophilic protein-negative biological samples (Congophilic protein-negative urine).

[0100] To further improve retention of detection reagent (CR) when testing negative samples, test strips were then fabricated using three layers of the selected trapping materials described in Table 6.

[0101] [Table 6]

[0102] Results: The triple layer trap was effective in keeping unbound CR detection reagent from entering the display strip. However, the triple trap configuration made with EMI 11513 and C048 also reduced the strength of the signal results when positive samples were tested. Of the traps tested, the triple trap made with 642 produced the best results for both positive and negative samples.

[0103] Example 14 Further investigation of trap materials To further investigate materials suitable for use as traps in the diagnostic test device of the present invention, strips were assembled having two Porex® sample pads (the first sample pad was lined with CR dye (8 ul / cm)), a trap as described in Table 7, and a trial strip of MN260. Strips of each configuration were tested with a Congophilic negative urine sample, two weakly positive Congophilic protein samples, and two strongly positive Congophilic protein samples.

[0104] [Table 7]

[0105] Surprisingly, the results for 5493 were weaker than those for 5475, and 5493 was approximately four times more absorbent and twice as fast in capillary rise than 5475. These results suggest that lower absorbency and slower capillary rise are favorable for a stronger signal. Similarly, CF3 was weaker than CF1 and twice as absorbent. Material 238 had a weaker signal and twice as fast in wicking as 237, and 319 had the strongest color results and slowest in wicking of the lines on this paper. MN640, a faster flowing material than MN615, had a weaker signal than MN615.

[0106] The results indicate that materials that are less absorbent and have slower capillary rise or wicking rates may be the preferred materials for a more robust signal.

[0107] Example 15 Diagnostic Test Device Evaluation A complete test strip was assembled including a dye-infused Porex® sample pad, Vivid™ membrane strip trap and MN260 capillary bed membrane mounted on top of Fusion 5 (a proprietary single layer matrix membrane made by GE). The strip was housed in a universal LFA cassette case. The urine sample was added to the circular sample well and the results were viewed in the rectangular window at 3 minutes.

[0108] Results: No dye was observed in the window for negative urine, a light pink color was observed for weakly positive urine, and a strong pink color was observed for strongly positive urine samples. These results indicate that this is a suitable configuration for a diagnostic test device to detect Congophilic protein in a sample to diagnose pre-eclampsia.

[0109] Example 16 Further evaluation of optimal trapping materials A complete test strip was assembled containing a dye-infused Porex® sample pad, a second Porex® pad (without dye), a single or double layer trap of 11513, 5475, 319, 247, or CF1, and a capillary bed of MN260 membrane with a Whatman 470 wick. The MN260 contained a line of tartrazine dye as a test control. The test strip was housed in an LFA-like cassette optimized for the addition of the test sample onto the dye-infused Porex® pad and optimized so that the viewing window was centered over the MN260 membrane so that the tartrazine dye could be observed before the test run but not after the test run. The urine sample was added to the circular sample well and the results were viewed in the rectangular window at 3 minutes.

[0110] result At the 3 minute mark, no dye was observed in the window for negative urine, a faint pink color was observed for weakly positive urine, and a strong pink / red color was observed for strongly positive urine samples. Additionally, a line of yellow tartrazine dye, which was clearly visible before sample addition, could be observed to move as the sample was drawn up, and was barely visible or completely gone at the 3 minute mark. These results indicate that this is a suitable configuration for a diagnostic test device for detecting Congolabinophilic protein in a sample to diagnose preeclampsia.

[0111] Example 17 Results from the device of the present invention vs. CRD test results The test device of the present invention was evaluated by testing 105 clinical urine samples from a study of Congo Red dot (CRD) testing in women suspected of having pre-eclampsia (Rood et al., 2016 AJOG, 214(1)s24-s25). Samples were selected to have 27 strongly positive, 28 negative and 50 weakly positive samples. The negative / positive status of the samples was determined by CRR (%) (Irina A. Buhimschi et al., Sci. Transl. Med. 6, 245ra92 (2014)). Samples were added to the test device and the results were visually scored as either negative (- or 0), weakly positive (+ or 1) or strongly positive (++ or 2) at 3 minutes.

[0112] Results showed that the test device results were concordant with the CRD results in 103 / 105 (98%) of the samples. Test results of the device of the present invention showed a sensitivity of 91%, specificity of 64%, accuracy of 82% with a positive predictive value of 83% and a negative predictive value of 79% when compared to a declared diagnosis of pre-eclampsia (based on ACOG guidance: Hypertension in Pregnancy Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy, 2013 Obstetrics & Gynecology 122(5):1122-1131).

[0113] When the test sample was added to the test sample well, a liquid front was observed moving up the strip by 30 seconds. The front met the yellow dye control line at approximately 1 minute and moved to the edge of the window by approximately 1.5 minutes. Once the liquid front moved into the result window, a strong positive result was clearly visible within 30 seconds and was visible thereafter. The entire result window was colored within 1.5 minutes. Weak positive results take longer to determine than strong positives because the liquid front generally appears negative and color appears in the result window approximately 1-3 minutes after sample addition. Example results are shown in Figure 1.

[0114] Example 18 Device evaluation using urine sample controls A test strip of the device was assembled containing a MN260 membrane capillary bed with a dye-infused Porex® 4588 sample pad of 120ug / device, a second Porex® pad (without dye), a double layer trap of Ahlstrom 319, and a wick of Whatman 470. The MN260 contained a line of tartrazine dye as a test control. The test strip was housed in an LFA-like cassette optimized for the test sample to be added onto the dye-infused Porex® pad and for the viewing window to be centered above the MN260 membrane so that the tartrazine dye could be observed before the test run but not after the test run.

[0115] Urine analytes from five patients with confirmed pre-eclampsia were pooled to create a positive assay control. The presence of Congo red affinity proteins was confirmed by Congo red dot blot test (Buhimschi 2014). Phosphate buffered saline (10 mM phosphate, pH 7.4, 150 mM NaCl) was used as a negative control and to create a dilution series of the positive assay control. The following dilutions were prepared (ratio of sample to total volume of buffer): no dilution, 1:4, 1:8, 1:15, 1:40. Each urine sample in this dilution series was tested in duplicate on the device.

[0116] Results: Each urine sample replicate at a particular dilution on the device exhibits the same staining density. The results of the test are shown in the table below.

[0117] [Table 8]

[0118] These data demonstrate that the diagnostic device is capable of detecting Congophilic proteins and that this effect is titratable. They also show that the device exhibits a progressive visual scale of red color that corresponds to increasing concentrations of Congophilic proteins.

Claims

1. 1. A method for determining whether a postpartum mammal suspected of having pre-eclampsia requires therapeutic intervention, comprising the steps of: a) applying a biological sample from said postpartum mammal to a sample receiving material of a diagnostic device for a time and under conditions sufficient to bind at least one misfolded protein of said biological sample to a detection reagent of said device, said detection reagent being selected from the group consisting of an azo dye and thioflavin T; and b) detecting the presence of said detection reagent on the capillary bed of said device, wherein the presence of said detection reagent on the capillary bed indicates the presence of said at least one misfolded protein present in the biological sample and further indicates that the postpartum mammal has pre-eclampsia and is in need of therapeutic intervention, and wherein the absence of said detection reagent on the capillary bed indicates that the postpartum mammal does not have pre-eclampsia, said capillary bed being in contact with a trap of said device, said trap being in contact with said sample receiving material, said sample receiving material, said trap and said capillary bed being configured to be in contact in sequence, said trap separating detection reagent bound to said at least one misfolded protein in the biological sample from unbound detection reagent, allowing flow of detection reagent bound to said at least one misfolded protein in the biological sample through said trap, and retaining, retarding the flow of or binding to unbound detection reagent.

2. 2. A pharmaceutical composition comprising magnesium sulfate for administration as said therapeutic intervention to a postpartum mammal when said postpartum mammal is indicated to have pre-eclampsia in step b) of claim 1 and when said postpartum mammal is in need of an anti-seizure agent.

3. If the postpartum mammal is indicated to have pre-eclampsia in step b) of claim 1, further comprising the steps of: c) applying an additional biological sample from the mammal that has undergone the therapeutic intervention to a sample receiving material of the diagnostic device for a time and under conditions sufficient to bind at least one protein of the additional biological sample to a detection reagent of the device to confirm that the therapeutic intervention has effectively resolved pre-eclampsia in the postpartum mammal; and d) detecting the presence of said detection reagent on the capillary bed of said device.

2. The method of claim 1, wherein the presence of the detection reagent on the capillary bed indicates the presence of the at least one protein present in the additional biological sample and indicates that the postpartum mammal still has pre-eclampsia and may require further therapeutic intervention, and the absence of the detection reagent on the sample receiving material of the device indicates that the pre-eclampsia has been effectively resolved.

4. If the postpartum mammal is shown to be free of pre-eclampsia in step b) of claim 1, further comprising the steps of: c) after a time interval, applying another biological sample from said mammal to a sample receiving material of a diagnostic device for a time and under conditions sufficient to bind at least one protein of said another biological sample to a detection reagent of said device; and d) detecting the presence of said detection reagent on the capillary bed of said device. wherein the presence of the detection reagent on the capillary bed indicates the presence of the at least one protein present in the other biological sample and indicates that the postpartum mammal has pre-eclampsia and may be in need of therapeutic intervention.

5. 5. A pharmaceutical composition comprising magnesium sulfate for administration to a postpartum mammal as said therapeutic intervention when said postpartum mammal is indicated to have pre-eclampsia in step d) of claim 4 and when said postpartum mammal requires administration of an anti-seizure agent.

6. If the postpartum mammal is shown to have pre-eclampsia in step d) of claim 4, the method further comprises the steps of: e) applying an additional biological sample from the mammal that has undergone a therapeutic intervention to a sample receiving material of the diagnostic device for a time and under conditions sufficient to bind at least one protein of the additional biological sample to a detection reagent of the device to confirm that the therapeutic intervention has effectively resolved pre-eclampsia in the postpartum mammal; and f) detecting the presence of said detection reagent on the capillary bed of said device.

5. The method of claim 4, wherein the presence of the detection reagent on the capillary bed indicates the presence of the at least one protein present in the additional biological sample and indicates that the postpartum mammal still has pre-eclampsia and may require further therapeutic intervention, and the absence of the detection reagent on the sample receiving material of the device indicates that the pre-eclampsia has been effectively resolved.

7. The method of claim 1 , wherein the azo dye is Congo Red.

8. 2. The method of claim 1, wherein the method is used in combination with at least one method selected from the group consisting of obtaining one or more blood pressure measurements from the postpartum mammal, obtaining one or more platelet counts from the postpartum mammal, measuring one or more serum creatinine concentration levels from the postpartum mammal, measuring one or more serum alanine aminotransferase (ALT) concentration levels from the postpartum mammal, and measuring one or more aspartate aminotransferase (AST) concentration levels from the postpartum mammal.

9. 9. The method of claim 8, wherein the method is used in combination with monitoring at least one symptom of pre-eclampsia selected from the group consisting of weight gain, dizziness, headache and blurred vision.

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