WO2022026546A1 - Pregnancy detection tool and methods - Google Patents

Abstract

Disclosed are non-human pregnancy test methods, devices, and kits. These methods, devices and kits provide early and efficient pregnancy detection in bovines, sheep, and other ruminants or ungulates. There are numerous proteins that are expressed exclusively during pregnancy or are present at increased concentrations that can be measured within numerous bodily fluids and used to determine if an animal is pregnant or not-pregnant. The methods, devices, and kits may be used in the field at ambient temperatures without the necessity of laboratory equipment or analysis tools.

Description

PREGNANCY DETECTION TOOL AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application no. 63/057,565, filed 28 July 2020, which is hereby incorporated by reference in its entirety as though fully set forth herein.

BACKGROUND

[0002] In both beef and dairy cow operations, it is imperative that cows are bred within a narrow margin of time in order for the business to be profitable. Pregnant and non-pregnant (“open”) cows must be differentiated early in order to either attempt to get them pregnant again or remove them from the herd.

[0003] There are currently over nine million dairy cows in the United States alone.

The dairy industry has seen margins become thinner over the past decade as bulk milk prices have failed to keep up with cost of doing business. In order to remain viable, dairies have had to find new and innovative ways to make production more efficient. A rapid, on-farm pregnancy test that could be used by producers would be beneficial, as it would reduce the costs and logistics associated with current methods of pregnancy detection.

[0004] As of January 1, 2020, there were over 31 million beef cows in the United States. Based on USD A research, less than 25% of beef operations utilize rectal palpation or ultrasound for determination of pregnancy. Many producers do not perform pregnancy checks due to cost, difficulty, or lack of access to a veterinarian in rural areas. A simple, cost-effective method of pregnancy detection that does not require a veterinarian would be beneficial to this population of producers.

[0005] Given its significance to profitable production, various methods of pregnancy detection in the bovine have been investigated previously. Rectal palpation by a veterinarian is the oldest method of pregnancy detection and remains a mainstay today. With the advent of smaller, more affordable ultrasound units, pregnancy detection via trans-rectal ultrasound has become more common within beef and dairy herds. However, the necessity of a veterinarian adds cost and logistical difficulty in coordinating a time that is appropriate for producers and veterinarians alike. This becomes more apparent in rural areas where many beef producers are utilizing pregnancy detection at a similar time during the year and there may be a shortage of veterinarians able to perform the work. Some producers may not have any veterinarian available whatsoever. A simple means of pregnancy detection would be of benefit to this population.

[0006] For these and other reasons, the veterinary medical arts remain in need of simpler means of pregnancy detection.

SUMMARY OF THE INVENTION

[0007] In a general and overall sense, the invention relates to techniques for detecting pregnancy in an animal, such as a bovine, and field-ready kits, tools, and methods that may be used for determining if an animal is pregnant or not without an invasive veterinary examination of the animal.

[0008] In some embodiments, an early animal pregnancy detection method is provided.

[0009] In other embodiments, kits for detecting animal pregnancy are disclosed.

[0010] In yet other embodiments, a device or “tool”, for detecting pregnancy in an animal are disclosed.

[0011] The animals as described herein are of many species, including those of bovines and other production and non -production species of the infraorder of Pecora.

[0012] The detection methods and kits may be further characterized as suitable for use outside of a laboratory setting, such as in a field setting (i.e. in the barn, working system, etc.), without the need for centrifugation, refrigeration, or sophisticated analytical devices.

[0013] In particular applications, a biological sample of the animal to be tested is processed to determine the presence and/or level of a molecule associated with pregnancy, such as an antigen or certain hormones (see Table 1 in the “Detailed Description” section below). In some embodiments, the pregnancy-associated indicator molecule comprises an indicator molecule capable of demonstrating the presence of a hormone or other protein, peptide, or antigenic component thereof, that has been determined to be indicative of a state of pregnancy in that type of animal. The antigen may comprise one or more antigens, proteins, or peptides that are specific for non-human animal pregnancy, in an amount that is sufficiently increased to a detectable level that is higher than an animal that is not pregnant. Stated another way, the level of the chosen antigen, peptide, protein, or fragment thereof detected in the test animal, in a pregnant animal, is higher compared to homeostatic levels of the antigen or other component thereof, detected in a biological sample obtained from a non-pregnant animal. By way of example, the antigen may comprise a peptide hormone, such as progesterone.

[0014] By way of example, and not limitation, antigens that may be used for the present methods and kits include: Pregnancy specific protein B (PSPB), PAG- 1,4, 5, 6, 7, 9, PSP-60, Early Conception Factor (ECF), progesterone, and interferon-tau. These antigens are referred to herein as pregnancy antigens as used in the description of the present invention. As part of the methods or kits, any single antigen or combination of pregnancy antigens may be used.

[0015] A biological sample may comprise whole blood, serum, plasma, saliva, urine, milk, or exhaled breath.

[0016] As used herein, reference to “not” a value or parameter generally means and describes “other than” a value or parameter. For example, the method is not used for the detection or treatment of cancer or any other type of disease.

[0017] As used herein and in the appended claims, the singular forms “a,” “or,” and “the” include plural referents unless the context clearly dictates otherwise.

[0018] These and other aspects and advantages of the present invention will become apparent from the subsequent detailed description and the appended claims. It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention.

[0019] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. Other features of the disclosure are apparent from the following detailed description and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The following figures are included to illustrate certain aspects of the present invention and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, as will occur to those skilled in the art and having the benefit of this disclosure. [0021] Figure 1. Illustrates a horizontal representation of possible layers within a vertical flow assay device such as that described in Examples 3 and 4. For the purposes of this figure, the layers are placed next to one another. As part of a functional vertical flow assay device, ready for field use by a user, the layers will be stacked on top of one another to provide an assembly in a vertical orientation. The elements shown in Figure 1 are as follows:

1 - Sample application area. Acts to separate plasma from whole blood and direct flow to either end of device.

2 - Layer for control of direction and rate of sample flow through assay. This may also contain signal enhancers and/or washing buffers.

3 - Contains conjugated antibodies to one or more markers of bovine pregnancy (see Table 1).

4 - Layer for control of direction and rate of sample flow through assay. This may also contain signal enhancers and/or washing buffers.

5 - Nitrocellulose membrane.

6 - Sample addition site.

7 - Test spot with capture antibodies embedded in nitrocellulose membrane.

8 - Control spot(s) (may include positive and/or negative control spot).

[0022] Figure 2 Illustrates key components of various layers, placement of conjugated anti pregnancy antibodies, and placement of capture antibodies within a vertical flow assay device such as that described in Examples 3 and 4. Note that the layers have been drawn spatially separated. This is for illustration purposes only. In the actual device these layers would be stacked upon one another in the order shown. The elements shown in Figure 2 are as follows:

9 - Sample application area. Acts to separate plasma from whole blood and direct flow to either end of device.

10 - Layer for control of direction and rate of sample flow through assay. This may also contain signal enhancers and/or washing buffers.

11 - Conjugated antibodies within membrane. 12 - Layer for control of direction and rate of sample flow through assay. This may also contain signal enhancers and/or washing buffers.

13 - Test spot

14 - Control spot(s) (may include positive and/or negative control spot)

[0023] Figure 3. Illustrates potential flow of sample within a vertical flow assay device such as described in Examples 3 and 4. Sample is applied to 15. This separates the plasma from whole blood. 16 comprises a membrane that acts to control rate and direction of sample flow. It may also have signal enhancing and/or washing reagents imbedded. 17 is comprised of a membrane with conjugated (gold, fluorescent dye, or other labeling agent) anti -pregnancy protein antibodies (monoclonal or polyclonal) that will bind to the desired pregnancy protein(s) (see Table 1) as the sample flows through the layer. The analyte and antibody are now bound and will flow through the remainder of the assay in this arrangement. The membrane will also serve to direct the flow of fluid. 18 serves a similar role to that of 16 (channel/adjust rate of fluid flow, add washing and/or signal enhancing reagents). 19 is comprised of a nitrocellulose membrane with anti -pregnancy antigen antibodies embedded that will bind to the target pregnancy protein(s). When bound this creates a sandwich where the capture antibody is bound to the nitrocellulose membrane via capture antibodies on one side and the pregnancy protein on the other. Attached to the pregnancy protein is the antibody with the detector molecule conjugated to it. The concentration of these bound indicator molecules results in a colorimetric or fluorescent change that is visible to the human eye or detectable through the use of an electronic device. There will also be a positive control indicator and/or a negative control indicator. 20 comprises an optional absorbent wicking pad that acts to collect biological sample and/or other reagents (for example, excess sample, reagents) at the end of the process. The elements shown in Figure 3 are as follows:

15 - Sample application

16 - Layer for control of direction and speed of flow as well as possible addition of signal enhancing and/or washing reagents.

17 - Membrane with conjugated antibodies embedded 18 - Layer for control of direction and speed of flow as well as possible addition of signal enhancing and/or washing reagents.

19 - Nitrocellulose membrane with capture antibodies embedded.

20 - Absorbent pad to collect excess sample at end of process.

[0024] Figure 3 A. Illustrates an alternative embodiment of potential flow of sample within a vertical flow assay device such as described in Examples 3 and 4. In this embodiment, the vertical flow assay device includes a top case portion A and a bottom case portion B. Each layer (four layers are shown in this embodiment) comprises a double-sided adhesive portion C and a transparency film portion D. Sample can be applied to the top case portion A or directly to the first layer. Subsequent layers can separate the sample. If the sample comprises whole blood, the subsequent layers can serve to separate out the plasma. Glass fiber channels 16A, 16B, and 16C are shown in association with the first, third, and fourth layers, respectively. The glass fiber channels 16A, 16B, and 16C act to control rate and direction of sample flow. They may also have signal enhancing and/or washing reagents imbedded. Conjugated antibodies can be embedded within one or more of the glass fiber channels 16A, 16B, and 16C. For example, the conjugated antibodies can include gold or fluorescent dyed anti -pregnancy protein antibodies (monoclonal or polyclonal) that will bind to the desired pregnancy protein(s) (see Table 1) as the sample flows through the channels. The analyte and antibody are now bound and will flow through the remainder of the assay in this arrangement. The channel will also serve to direct the flow of fluid. Nitrocellulose membrane 19A includes capture antibodies, such as anti -pregnancy antigen antibodies embedded that will bind to the target pregnancy protein(s). When bound this creates a sandwich where the capture antibody is bound to the nitrocellulose membrane via capture antibodies on one side and the pregnancy protein on the other. Attached to the pregnancy protein is the antibody with the detector molecule conjugated to it. The concentration of these bound indicator molecules results in a colorimetric or fluorescent change that is visible to the human eye or detectable through the use of an electronic device. There will also be a positive control indicator and/or a negative control indicator. An optional absorbent waste or wicking pad 20A can be included near the bottom case portion B. This pad that acts to collect biological sample and/or other reagents (for example, excess sample, reagents) at the end of the process. The elements shown in Figure 3 are as follows: A - Top case portion

B - Bottom case portion

C - Double-sided adhesive portion

D - Transparency film portion

16A, 16B, and 16C - Glass fiber channels

19A - Nitrocellulose membrane

20A - Waste pad

[0025] Figure 4. Illustrates components of a lateral flow assay device as described in Examples 1 and 2. 21 is the sample application spot. It is comprised of a material that is suitable for separating plasma from a whole blood sample. It also acts to channel separated sample to 22. 22 is a membrane that serves to control direction and/or rate of flow of sample. It may also include signal enhancing and/or washing reagents. 23 is a membrane with conjugated (gold, fluorescent dye, or other labeling agent) anti -pregnancy protein antibodies (monoclonal or polyclonal) that will bind to the desired pregnancy protein(s) (see Table 1) as the sample flows through. It can serve both to channel and/or adjust rate of fluid flow as well. 24 is a membrane that serves to control direction and/or rate of flow of sample and to add washing and/or enhancing reagents as needed. 25 is comprised of a nitrocellulose membrane with anti pregnancy protein antibodies embedded that will bind to the target pregnancy protein(s). When bound this creates a sandwich where the capture antibody is bound to the membrane on one side and the pregnancy protein on the other. Attached to the pregnancy protein is the antibody with the detector molecule conjugated to it. The concentration of these bound indicator molecules results in a colorimetric or fluorescent change that is either visible to the human eye or detectable through the use of an electronic device. 26 is a membrane that provides spatial separation of test line and control line to allow for easier distinction of both the test line and control line. This will contribute to speed and/or direction of sample flow. It may also contain signal enhancing and/or washing reagents. 27 is a control line (positive and/or negative). 28 is a membrane that provides spatial separation of test line and control line to allow for easier distinction of both the test line and control line. This will also contribute to speed and/or direction of sample flow. 29 is an absorbent pad to collect the sample at the end of the test process. The elements shown in Figure 4 are as follows:

21- Sample application site.

22- Membrane that can serve both to channel and adjust rate of fluid flow and to add washing and/or enhancing reagents as needed.

23- Membrane that contains conjugated antibodies to one or more markers of bovine pregnancy (see Table 1).

24- Membrane that can serve both to channel and adjust rate of fluid flow and to add washing and/or enhancing reagents as needed.

25- Nitrocellulose membrane with capture antibodies embedded.

26- Zone that provides spatial separation of test line and control line to allow for easier distinction of both the test line and control line.

27- Control line (negative and/or positive)

28- Zone that provides spatial separation of test line and control line to allow for easier distintion of both the test line and control line.

29- Absorbent pad.

30- Backing.

[0026] Figure 5. Illustrates flow of sample through a lateral flow assay device as described in Examples 1 and 2. Sample is applied to the sample application area as shown by 31. Sample flows laterally through capillary action through membranes. 32 demonstrates the conjugated antibodies. These antibodies are conjugated with fluorescent or gold nanoparticles that will create the colorimetric or fluorescent change that indicates the presence of analyte. 33 is the line of capture antibodies embedded in a nitrocellulose membrane. These will bind to the desired analyte. This results in a sandwich with the capture antibodies embedded in the nitrocellulose membrane; the analyte (protein indicating bovine pregnancy) forms the middle; the conjugated antibody forms the upper layer. This concentration of conjugated antibody is concentrated causing a detectable colorimetric or fluorescent change. 34 is a control line (positive and/or negative). 36 is a simplified rendering indicating that analyte has bound to the conjugated antibodies. 37 demonstrates the sandwich that is formed with capture antibodies, analyte, and conjugated antibodies. The elements shown in Figure 5 are as follows:

31- Sample

32- Conjugated anti -pregnancy antigen antibodies

33- Test line

34- Control line

35- Absorbent pad

36- Lateral flow assay with sample bound to conjugated antibodies.

37- Sample and conjugated antibodies bound to capture antibodies.

[0027] Figure 6. Illustrates possible case designs as described in Example 8.

Fig. 6A- Illustrates design of case made of two halves that are sealed during the manufacturing process. In this case it has sample application and result viewing areas that would work with a lateral flow assay as described in Examples 1 and 2.

Fig. 6B- Illustrates design of case made of a single piece of material with sample application and viewing areas appropriate for a lateral flow assay as described in Examples 1 and 2.

Fig. 6C- Illustrates the end-on view of a reusable case that incorporates a removable test insert (such as shown in Fig. 6B). This illustrates the opening that would allow for the insertion and removal of the test insert.

Fig. 6D- Illustrates design of case made of two halves that are sealed during the manufacturing process. In this case it has a sample application and result viewing area that would work with a vertical flow assay as described in Examples 3 and 4.

Fig. 6E- Illustrates design of case made of a single piece of material with a sample application and viewing area appropriate for a vertical flow assay as described in Examples 3 and 4. Fig. 6F- Illustrates the end-on view of a reusable case that incorporates a removable test insert (such as shown in Fig. 6E). This illustrates the opening that would allow for the insertion and removal of the test insert.

[0028] Figure 7. Illustrates one possible embodiment of a heating element within the case (44) of a lateral or vertical flow immunoassay. Sample would be applied to 45. Some sample would then be channeled out (46) the tubular structure (47) that houses the substance that, when exposed to fluid, would generate heat.

44- Case of lateral or vertical flow immunoassay as though the top has been removed and the inside is being shown.

45- Sample application area.

46- Channels to move fluid from sample application area to heating element.

47- Tubular heating element.

[0029] Figure 8. Illustrates a possible embodiment of compartments within the case of a lateral or vertical flow assay that materials may be added to in an effort to regulate humidity within the case. This figure is of the case opened and looking down at it.

48- Perimeter of the case.

49- Sample application area.

50- Represents possible configuration of compartments within the case that could have substances within them that help regulate humidity within the case.

[0030] Figure 9. Illustrates possible embodiment of method for mixing sample and reagent (conjugated antibodies, signal enhancing agents, buffers, etc.) in solution. Sample enters the compartment via a membrane that allows for one-way fluid flow (51). Dried reagent is within the compartment (53). It is activated when a fluid sample is added. 52 is a membrane that will dissolve to allow fluid to flow out after a pre-determined amount of time. It could also be a porous membrane coated in a dissolvable substance that dissolves after a pre-determined amount of time and allows fluid to pass through to the next step in assay. While this figure shows a cube with entire sides devoted to inflow and outflow, it could also be designed to be a different shape and have inflow and outflow regions of varying sizes and locations. 54 illustrates sample within the compartment with reagent and analyte joining in solution.

51- One-way membrane that allows for flow of sample into compartment.

52- Material that either dissolves after pre-determined amount of time or is coated in material that does the same to allow for fluid outflow.

53- Dried reagent within the chamber.

54- Fluid sample.

55- Demonstrate joined reagent and analyte.

[0031] Figure 10- Illustrates construction of a thin membrane that allows for one-way flow of a fluid sample. Two membranes have the appropriate incisions made before being joined.

The first membrane fluid encounters has circular holes made it in it through a process appropriate for the chosen material. The second membrane has crescent shaped incisions made in it of the same radius and layout as the first membrane. These two membranes are then joined slightly offset so that the crescent flap may be pushed in the direction of fluid flow but cannot go in the opposite direction because it encounters the first membrane.

Fig. 10A- Top view of first membrane encountered with circular holes.

Fig. 10B- Top view of second membrane encountered with crescent shaped incisions in the same layout as the first membrane.

Fig. IOC- Top view after the membranes are joined.

59- The solid circles represent the holes of the first membrane.

60- Dotted crescent lines represent the relative position of the second layer to the first. Demonstrates the offset that prevents the flap from opening in the opposite direction to fluid flow.

Fig. 10D- Side view of the two joined membranes.

62- Dotted lines represent holes extending full thickness through the first membrane. 63- Dotted lines represent the opening created by the crescent shaped incision in the second membrane. Demonstrate the offset from the first layer.

64- Illustrate the crescent flap side view with it in the open position to allow fluid flow in one direction.

DETATEED DESCRIPTION

Terms and Abbreviations

[0030] Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. All references herein are incorporated by reference. The following explanations of terms and abbreviations are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure.

[0031] All numbers expressing quantities of components, molecular weights, percentages, temperatures, times, length, and so forth, as used in the specification or claims are to be understood as being modified by the term "about" unless otherwise indicated.

[0032] As used herein, "comprising" means "including" and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise.

[0033] The term "sample" describes any type of sample suspected to contain a desired target protein to be assayed for detection of such target protein. In some embodiments a biological sample from a subject suspected of being pregnant, will be used, such as blood, plasma or serum, or other bodily fluids that may contain the target protein. These may include, for example, plasma, serum, spinal fluid, lymph fluid, secretions from the respiratory, gastrointestinal, or genitourinary systems including tears, saliva, milk, urine, semen, hepatocytes, and red or white blood cells or platelets. Samples may also be obtained from tissue cell culture, such as cultured hepatocytes or leukocytes, and constitute cells, including recombinant cells, or medium in which the target may be detected. In some cases, a tissue sample may be used in the assay or processed for use in the assay, for example, by a conventional method used to extract proteins from the sample. [0034] The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity.

[0035] An "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multi specific antibodies formed from antibody fragments.

[0036] An "antibody that binds to the same epitope" as a reference antibody refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50% or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50% or more.

[0037] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. [0038] "Native antibodies" refer to naturally occurring immunoglobulin molecules with varying structures. For example, native IgG antibodies are hetero-tetrameric glycoproteins of about 150,000 Daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CHI, CH2, and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also called a variable light domain or a light chain variable domain, followed by a constant light (CL) domain. The light chain of an antibody may be assigned to one of two types, called kappa (k) and lambda (l), based on the amino acid sequence of its constant domain.

[0039] "Radioimmunoassay" and "RIA" refers to in vitro assay techniques in which radioactive labelled antigen is mixed with unlabeled antigen (the test sample) and allowed to bind to an antibody or antigen binding fragment thereof. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine.

[0040] Enzyme-linked immunosorbent assays (ELISA) are standard in the art and can be found at, for example, Ausubel, F. M. et ak, (Current Protocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley & Sons, Inc., 1991). ELISA typically uses an enzymatic reaction to convert substrates into products having a detectable signal (e.g., fluorescence). Each enzyme in the conjugate can covert hundreds of substrates into products, thereby amplifying the detectable signal and enhancing the sensitivity of the assay. ELISA assays are understood to include derivative and related methods, such as sandwich ELISA and microfluidic ELISA.

[0041] The term "heterologous polypeptide" or "heterologous protein" refers to a polypeptide or protein that is derived from a different source, a native polypeptide in which modifications have been made to alter the native sequence, or a native polypeptide whose expression is quantitatively altered as a result of a manipulation of the gene encoding the polypeptide by recombinant DNA techniques. In the context of a fusion protein or peptide, a "heterologous polypeptide sequence" refers to a polypeptide sequence that comprises one or more subsequences that are not found in the same relationship to each other in nature.

[0042] Certain immunological markers are present only during pregnancy or are increased sufficiently during pregnancy to be considered markers of a positive pregnancy status.

Pregnancy associated glycoproteins (PAGs) have been known for some time. This class of proteins has since been refined to include several different proteins. Despite the discovery and prior knowledge of other pregnancy specific proteins, PAGs have been the most dependable class of protein found in blood or milk for recognition of bovine pregnancy. They are commonly used in commercially available tests. Commercial lab-based tests for veterinary pregnancy have been primarily enzyme-linked immunosorbent assays (US8541187, US20140024057). Idexx Laboratories offers a visual ELISA that relies on color changes that are indicative of pregnant v. not pregnant, without the need for special equipment.. This tool, however, requires many time- consuming steps and other complexities that render it not useful for the untrained user. These tests have been effectively used for diagnosis of pregnancy in sheep and bovine.

[0043] Pregnancy specific glycoproteins are proteins that are specific to pregnancy. These proteins are produced by binucleate and trophodectoderm cells of the bovine and ovine placentas and embryo. Within this class of protein there are numerous related proteins including: Pregnancy specific protein B (PSPB), PAG- 1,4, 5, 6, 7, 9, PSP-60. The serum concentration of PAGs has been effectively correlated to pregnancy. A PAG concentration of 0.33ng/ml in cows and 0.54ng/ml in heifers was 95% accurate at determining pregnancy status at day 30. PAGs can also be found in milk, which makes this an appropriate biological sample for detecting pregnancy.

[0044] By way of example, Table 1 provides an exemplary list of proteins that may be used in the practice of the various embodiments of the present methods and testing devices: TABLE 1

[0045] PAG levels have also been associated with predicting embryo survival, with higher levels on day 28 of pregnancy correlating with increased embryo survival. Another study inversely linked milk production to circulating PAG levels. These findings of these studies suggest that a test that interprets not only the presence but also the concentration of PAGs may be beneficial in predicting embryonic loss.

[0046] Progesterone is a hormonal marker of pregnancy in cattle. While always present, it is greatly elevated during pregnancy. It has been known to be a possible tool in determining bovine pregnancy. Research has found that while a single sample may be used to suggest pregnant versus not-pregnant, serial samples are more accurate for diagnosing pregnancy as high concentrations may be influenced by a variety of factors. Progesterone has a fairly high sensitivity but low specificity. It is most useful as a negative predictor of pregnancy. To the best of the inventors knowledge this has not been combined in a single test with the aforementioned markers of pregnancy in order to enhance the overall sensitivity and specificity of the test. [0047] Early conception factor (ECF) can be detected in as little as 2-4 hours following fertilization and is measurable in early pregnancy. Embryonic loss is common within the first 28 days of pregnancy in cattle. Despite the fact that it can be found very early during pregnancy, ECF has less value as a marker of pregnancy due to high rates of embryonic loss during early pregnancy. Previously a commercial lateral flow assay was patented and produced for detection of ECF but was found to be a poor predictor of pregnancy and has since been removed from the market.

[0048] Estrone sulfate is a conjugated steroid of estrone. It has been shown to be detectable at day 60 of pregnancy. It becomes more reliable after day 100 of pregnancy. Circulating levels, however, are heavily influenced by genetics, weight, parity status, and environment. Due to wide variability in circulating levels of estrone sulfate it has not been widely studied in academia or commercial settings as a means of pregnancy diagnosis. When used in combination with other pregnancy markers it may aid in increasing specificity and sensitivity of a pregnancy test, particularly when used in animals that are past day 100 of pregnancy.

[0049] Interferon-tau has been widely studied and is considered critical in the bovine recognition of pregnancy pathway. It is produced by the conceptus starting between days 14-16. Interferon-tau stimulates numerous changes in gene expression in maternal cells. Circulating levels are below the detectable limits of point of care tests. The development of a diagnostic test that could rapidly detect interferon-tau or the changes it induces could make this a more useful marker of pregnancy.

[0050] The examples herein may include the use several specific amino acid and/or nucleic acid sequences, but it will be appreciated by one of ordinary skill in the art that other sequences are readily amenable for use in any and all modifications of the disclosed methods and kits.

[0051] The term "kit" refers to a composition of matter containing one or more ingredients necessary to practice the method of detecting pregnancy status according to the invention. In various embodiments one or more indicators of pregnancy via commercial antibodies (see Table 1)^.

[0052] A kit may also contain at least one biological sample preservative or additive for a sample, such as an agent that prevents degradation of proteins, a reaction buffer in which antibody and biological sample are mixed, a negative control sample, a positive control sample, one or more reaction containers, such as tubes or wells, a colorimetric chart, a pipette (disposable or disposable pipette tips to be used with a reusable pipette) to dispense the appropriate amount of sample, a packaging material, an instruction for use in detecting the same. Additional items that may be included within the kit are those required for sample collection including, but not limited to, blood tubes (EDTA, heparinzed, non-coated, etc.), needles, syringes, and vacutainers. In the event that the test is intended to be used with a smart device, a detection box may be included within or separately from the kit wherein the test is placed within the box in a predetermined spot and the smart device is placed within a holder to capture an image of the result. The box may or may not have artificial lighting within it. The box itself may have the electronics required to interpret the test without the user using their own smart device. This setup would allow for consistent interpretation of the results by the smart device by eliminating the lighting and background variables associated with photography outside of a controlled environment and thereby decrease the risk of incorrect and/or uninterpretable results.

[0053] The examples below are provided only for illustrative purposes and not to limit the scope of the present invention. Numerous embodiments within the scope of the claims will be apparent to those of ordinary skill in the art, thus the following non-limiting examples only describe particular embodiments of the invention. The present invention relates to colorimetric and fluorescent read-out systems capable of detecting a variety of biomolecules, including methods and kits relating thereto.

[0054] For all of the examples of diagnostic devices in this patent, they may be designed as qualitative, semi -quantitative, or quantitative designs. The exact method of how this is achieved may vary depending on if it is a lateral flow and vertical flow assay but will be clear to those skilled in the art.

[0055] To facilitate a better understanding of the present invention, the following examples of preferred or representative embodiments are given. In no way should the following examples be read to limit, or to define, the scope of the invention.

EXAMPLE 1 - LATERAL FLOW ASSAY WITH NITROCELLULOSE MEMBRANE

AND COLORIMETRIC INDICATOR

[0056] In one form of the invention a lateral flow assay device is provided according to the present invention, for the detection of pregnancy in an animal, such as a bovine animal. [0057] In some embodiments, a biological sample (blood, serum, plasma, saliva, urine, or milk; the “test” sample) from an animal is used to determine if the animal is pregnant or not pregnant. A blood sample can be applied to a membrane that separates plasma from whole blood. The sample then flows laterally through membranes that are coated in gold nanoparticle- conjugated (or other conjugation technique) anti -pregnancy antigen(s) detection antibodies (either mono-clonal or poly-clonal). The fluid continues to flow laterally until it reaches a membrane with capture antibodies embedded for detection and positive and/or negative controls. The resulting binding in the case of a pregnant animal causes a colorimetric change that is either distinct enough to be interpreted with the human eye or via a smart device (for example an iPhone, iPad, etc.) with an application designed to interpret and record the result.

[0058] Reagents (in dry or wet forms) may be placed on, embedded within, or otherwise incorporated into the device in order to extend the shelf life and/or stabilize the proteins incorporated within the device (i.e., antibodies) and/or provide a stable pH and/or act to prevent non-specific binding. These agents may include, but are not limited to, in various concentrations and combinations, trehalose, EDTA, bovine serum albumin, phosphate buffered saline, Tween- 20, borate, etc.

[0059] The assay device has multiple zones.

[0060] Each zone may or may not be the same material. Materials may include, but are not limited to, filter paper, nitrocellulose, etc.

[0061] Each zone may or may not have wax or other appropriate material applied (via printer, laser etching, paraffin impregnated material, etc.) that will create channels and pores to control fluid flow through the device.

[0062] A nitrocellulose membrane, if included, may be further optimized for use and channel printing techniques through combination with a transparent sheet and wax.

[0063] A plastic, metal, or other appropriate material can encase the entire test with openings for sample application and reading of results.

[0064] Zone 1 (e.g., including elements 21 and 31 in Figures 4 and 5, respectively) is the application site. Zone 1 may utilize either biochemical or mechanical filtration techniques. Filtration utilizing either technique enhances analyte detection and therefore improves both the specificity and sensitivity of the test by removing extraneous cells that could otherwise impede proper fluid flow rates and analyte binding. Biochemical filtration techniques rely on pretreatment of the sample pad to agglutinate red blood cells. Mechanical filtration relies on fibrous components to provide a porous matrix that is optimized in size to hold back red blood cells but allow plasma to flow through at the appropriate rate. Possible materials include, but are not limited to glass, micro-glass, cotton, or a blend of the aforementioned materials. The separation zone may be combined with parafilm, or other similar material, to further optimize fluid flow and particle size that can make it through. It may be combined through a heat press and then laser cut.

[0065] Zone 2 (e.g., including element 22 in Figure 4) is a membrane the utilizes microcapillary action to channel fluid to the next zone. The microcapillary design of this membrane can be altered to further optimize pore size and flow rate so that each incubation step is for the ideal amount of time and to reduce the number of extraneous cells and proteins that are able to pass through the membrane. Numerous materials may be used for this membrane including, but not limited to, nitrocellulose, plastic, or paper.

[0066] Zone 3 is a membrane between the sample pad (Zone 1) and the conjugate zone (Zone 4). This zone may or may not have dry washing and/or signal enhancing agents embedded within it that are activated once sample fluid passes through.

[0067] Zone 4 (e.g., including elements 24 and 32 in Figures 4 and 5, respectively) is a conjugate region, where gold-conjugated antibodies are embedded that can be of any combination of anti -pregnancy antigen antibodies (see Table 1). It is at this stage that these will bind to the analyte. The analyte-antibody combination will continue to flow as one through the remainder of the system.

[0068] Zone 5 is another space that can serve in the same capacity as zone 3.

[0069] Zone 6 (e.g., including elements 25 and 33 in Figures 4 and 5, respectively) is the positive test line. It has capture antibodies embedded in a nitrocellulose membrane.

[0070] Zone 7 (e.g., including elements 26 in Figure 4) offers a small space between the test and control lines to allow for clear differentiation of the two zones. [0071] Zone 8 (e.g., including elements 27 and 34 in Figures 4 and 5, respectively) is a control line (this may include a positive control and/or a negative control).

[0072] Zone 9 (e.g., including element 28 in Figure 4) is a blank area to allow for visual contrast to enhance viewing of control responses.

[0073] Zone 10 (e.g., including elements 29 and 35 in Figures 4 and 5, respectively) is an absorbent wicking pad to collect sample at the end of the system. It also acts as a fluid sink which aids in controlling flow of sample in one direction across the assay.

[0074] Zone 11 (e.g., including element 30 in Figure 4) is a backing material that supports the afore mentioned zones. It may be made of a paper product, plastic, or other appropriate material.

[0075] See Figures 4 and 5.

EXAMPLE 2 - LATERAL FLOW ASSAY WITH FLUOROMETRIC INDICATOR

[0076] In the present example, a lateral flow assay is utilized that uses fluorometric detection methods.

[0077] Blood or plasma is applied to the sample application spot which contains a filter to rid it of extraneous cells. The sample then flows laterally through membranes that, in one zone, contain a fluorescent dye conjugated (or other conjugation technique) anti -pregnancy protein(s) (see Table 1) detection antibodies (mono-clonal or poly-clonal). The fluid continues to flow laterally through a membrane with anti -pregnancy antigen antibodies embedded for detection and controls. The resulting binding in the case of a pregnant animal causes a fluorometric change that is detected via a smart device (for example an iPhone, iPad, etc.) or other electronic that has an application designed to interpret and record the result.

[0078] The assay device has multiple zones.

[0079] Reagents (in dry or wet forms) may be placed on, embedded within, or otherwise incorporated into the device in order to extend the shelf life and/or stabilize the proteins incorporated within the device (i.e., antibodies) and/or provide a stable pH and/or act to prevent non-specific binding. These agents may include, but are not limited to, in various concentrations and combinations, trehalose, EDTA, bovine serum albumin, phosphate buffered saline, Tween- 20, borate, etc.

[0080] Each zone may or may not be the same material. Materials may include, but are not limited to, filter paper, nitrocellulose, etc.

[0081] Each zone may or may not have wax or other appropriate material applied (via printer, laser etching, paraffin impregnated material, etc.) that will create channels and pores to control fluid flow through the device.

[0082] The nitrocellulose membrane, if included, may be further optimized for use and channel printing techniques through combination with a transparent sheet and wax.

[0083] A plastic, metal, or other appropriate material can encase the entire test with openings for sample application and reading of results.

[0084] Zone 1 (e.g., including elements 21 and 31 in Figures 4 and 5, respectively) is the application site. Zone 1 may utilize either biochemical or mechanical filtration techniques. Filtration utilizing either technique enhances analyte detection and therefore improves both the specificity and sensitivity of the test by removing extraneous cells that could otherwise impede proper fluid flow rates and analyte binding. Biochemical filtration techniques rely on pretreatment of the sample pad to agglutinate red blood cells. Mechanical filtration relies on fibrous components to provide a porous matrix that is optimized in size to hold back red blood cells but allow plasma to flow through at the appropriate rate. Possible materials include, but are not limited to glass, micro-glass, cotton, or a blend of the aforementioned materials. The separation zone may be combined with parafilm, or other similar material, to further optimize fluid flow and particle size that can make it through. It may be combined through a heat press and then laser cut.

[0085] Zone 2 (e.g., including element 22 in Figure 4) is a membrane the utilizes microcapillary action to channel fluid to the next zone. The microcapillary design of this membrane can be altered to further optimize pore size and flow rate so that each incubation step is for the ideal amount of time and reduces the number of extraneous cells and proteins that are able to pass through the membrane. Numerous materials may be used for this membrane including, but not limited to nitrocellulose, plastic, or paper. [0086] Zone 3 is a membrane between the sample pad (Zone 1) and the conjugate zone (Zone 4). This zone may or may not have dry washing and/or signal enhancing agents embedded within it that are activated once sample fluid passes through.

[0087] Zone 4 (e.g., including elements 24 and 32 in Figures 4 and 5, respectively) is a conjugate region, where fluorescent dye conjugated antibodies are embedded that can be of any combination of pregnancy antibodies (see Table 1). Common fluorescent dyes include: R-PE, Alexa Flour 532, Atto 465. It is at this stage that these conjugated antibodies will bind to the analyte. The analyte-antibody combination will continue to flow as one through the remainder of the system.

[0088] Zone 5 is another space that can serve in the same capacity as Zone 3.

[0089] Zone 6 (e.g., including elements 25 and 33 in Figures 4 and 5, respectively) is the positive test line. It has capture antibodies embedded within a nitrocellulose membrane.

[0090] Zone 7 (e.g., including elements 26 in Figure 4) offers a small space between the test and control lines to allow for clear differentiation of the two zones.

[0091] Zone 8 (e.g., including elements 27 and 34 in Figures 4 and 5, respectively) is the control line (this may include a positive control and/or a negative control line).

[0092] Zone 9 (e.g., including element 28 in Figure 4) is a blank area to allow for visual contrast to enhance viewing of control responses.

[0093] Zone 10 (e.g., including elements 29 and 35 in Figures 4 and 5, respectively) is an absorbent wicking pad to collect sample at the end of the system and acts as a fluid sink which aids in controlling flow of sample in one direction across the assay.

[0094] Zone 11 (e.g., including element 30 in Figure 4) is a backing material that supports the afore mentioned zones. It may be made of a paper product, plastic, or other appropriate material.

[0095] See Figures 4 and 5.

EXAMPLE 3 - VERTICAL FLOW ASSAY WITH COLORIMETRIC INDICATOR

[0096] In the present example, a vertical flow assay is illustrated. The spatial relationship between components may vary. [0097] The biological sample (blood, plasma, serum, saliva, urine, or milk) is applied to a sample application area.

[0098] The device is made of multiple layers.

[0099] Reagents (in dry or wet forms) may be placed on, embedded within, or otherwise incorporated into the device in order to extend the shelf life and/or stabilize the proteins incorporated within the device (i.e., antibodies) and/or provide a stable pH and/or act to prevent non-specific binding. These agents may include, but are not limited to, in various concentrations and combinations, trehalose, EDTA, bovine serum albumin, phosphate buffered saline, Tween- 20, borate, etc.

[0100] Each layer may or may not be the same material. Materials may include, but are not limited to, filter paper, nitrocellulose, etc. Pores/channels through each layer may be symmetrical or asymmetrical to optimize fluid flow through the device. Orientation of asymmetrical pores/channels may be altered to optimize fluid flow.

[0101] Each layer may or may not have wax or other appropriate material applied (via printer, laser etching, paraffin impregnated material, etc.) that will create channels and pores to control fluid flow through the device.

[0102] The nitrocellulose membrane, if included, may be further optimized for use and channel printing techniques through combination with a transparent sheet and wax.

[0103] A plastic, metal, or other appropriate material can encase the entire test with openings for sample application and reading of results.

[0104] Layer 1 (e.g., including elements 1/6, 9, and 15 in Figures 1, 2, and 3, respectively) is a membrane that accepts the sample. This layer may utilize either biochemical or mechanical filtration techniques. Filtration utilizing either technique enhances analyte detection and therefore improves both the specificity and sensitivity of the test by removing extraneous cells that could otherwise impede proper fluid flow rates and analyte binding. Biochemical filtration techniques rely on pretreatment of the sample pad to agglutinate red blood cells. Mechanical filtration relies on fibrous components to provide a porous matrix that is optimized in size to hold back red blood cells but allow plasma to flow through at the appropriate rate. Possible materials include, but are not limited to glass, micro-glass, cotton, or a blend of the aforementioned materials. The separation layer may be combined with parafilm, or other similar material, to further optimize fluid flow and particle size that can make it through. It may be combined through a heat press and then laser cut.

[0105] Layer 2 (e.g., including elements 2, 10, and 16 in Figures 1, 2, and 3, respectively) is a membrane the utilizes microcapillary action to channel fluid to the next layer. The microcapillary design of this membrane can be altered to further optimize pore size and flow rate so that each incubation step is for the ideal amount of time and to reduce the number of extraneous cells and proteins that are able to pass through the membrane. Numerous materials may be used for this membrane including, but not limited to nitrocellulose, plastic, or paper. It may also be used to add signal enhancers and/or washing buffer. These may be dry and become activated when sample passes through this layer.

[0106] Layer 3 (e.g., including elements 3, 11, and 17 in Figures 1, 2, and 3, respectively) contains gold particle conjugated (or other conjugation technique) anti -pregnancy protein(s) (see Table 1 for possible analytes) detection antibodies on a nitrocellulose or other membrane. This colloidal gold particle acts as the reporter entity.

[0107] Layer 4 (e.g., including elements 4, 12, and 18 in Figures 1, 2, and 3, respectively) is similar in form and function in the same or similar manner as the second layer.

[0108] Layer 5 (e.g., including elements 7/8, 13/14, and 19 in Figures 1, 2, and 3, respectively) is a membrane that has anti -pregnancy (see Table 1) capture antibodies embedded for detection as well positive and/or negative control spots.

[0109] Layer 6 (e.g., including element 20 in Figure 3) is an absorbent wicking pad to collect sample at the end of the process.

[0110] The resulting binding in the case of a pregnant animal causes a colorimetric change that is either distinct enough to be interpreted with the human eye or via a smart device (for example an iPhone, iPad, etc.) with an application designed to interpret and record the result.

[0111] See Figures 1, 2, and 3.

EXAMPLE 4 - VERTICAL FLOW ASSAY WITH FLUOROMETRIC INDICATOR

[0112] In the present example, a vertical flow assay is illustrated. The spatial relationship between components may vary. [0113] The biological sample (blood, plasma, serum, saliva, urine, or milk) is applied to a sample application area.

[0114] The device is made of multiple layers.

[0115] Reagents (in dry or wet forms) may be placed on, embedded within, or otherwise incorporated into the device in order to extend the shelf life and/or stabilize the proteins incorporated within the device (i.e., antibodies) and/or provide a stable pH and/or act to prevent non-specific binding. These agents may include, but are not limited to, in various concentrations and combinations, trehalose, EDTA, bovine serum albumin, phosphate buffered saline, Tween- 20, borate, etc.

[0116] Each layer may or may not be the same material. Materials may include, but are not limited to, filter paper, nitrocellulose, etc. Pores/channels through each layer may be symmetrical or asymmetrical to optimize fluid flow through the device. Orientation of asymmetrical pores/channels may be altered to optimize fluid flow.

[0117] Each layer may or may not have wax or other appropriate material applied (via printer, laser etching, paraffin impregnated material, etc.) that will create channels and pores to control fluid flow through the device.

[0118] The nitrocellulose membrane, if included, may be further optimized for use and channel printing techniques through combination with a transparent sheet and wax.

[0119] The separation layer may be combined with parafilm, or other similar material, to further optimize fluid flow and particle size that can make it through. It may be combined through a heat press and then laser cut.

[0120] Layer 1 (e.g., including elements 1/6, 9, and 15 in Figures 1, 2, and 3, respectively) is a membrane that accepts the sample. This layer may utilize either biochemical or mechanical filtration techniques. Filtration utilizing either technique enhances analyte detection and therefore improves both the specificity and sensitivity of the test by removing extraneous cells that could otherwise impede proper fluid flow rates and analyte binding. Biochemical filtration techniques rely on pretreatment of the sample pad to agglutinate red blood cells. Mechanical filtration relies on fibrous components to provide a porous matrix that is optimized in size to hold back red blood cells but allow plasma to flow through at the appropriate rate. Possible materials include, but are not limited to glass, micro-glass, cotton, or a blend of the aforementioned materials. The separation layer may be combined with parafilm, or other similar material, to further optimize fluid flow and particle size that can make it through. It may be combined through a heat press and then laser cut.

[0121] Layer 2 (e.g., including elements 2, 10, and 16 in Figures 1, 2, and 3, respectively) is a membrane the utilizes microcapillary action to channel fluid to the next layer. The microcapillary design of this membrane can be altered to further optimize pore size and flow rate so that each incubation step is for the ideal amount of time and to reduce the number of extraneous cells and proteins that are able to pass through the membrane. Numerous materials may be used for this membrane including, but not limited to nitrocellulose, plastic, or paper. It may also be used to add signal enhancers and/or washing buffer. These may be dry and become activated when sample passes through this layer.

[0122] Layer 3 (e.g., including elements 3, 11, and 17 in Figures 1, 2, and 3, respectively) is a conjugate region, where fluorescent dye antibodies are embedded that can be of any combination of anti -pregnancy antibodies (see Table 1). Common fluorescent dyes include: R- PE, Alexa Flour 532, Atto 465. It is at this stage that these conjugated antibodies will bind to the analyte. The analyte-antibody combination will continue to flow as one through the remainder of the system.

[0123] Layer 4 (e.g., including elements 4, 12, and 18 in Figures 1, 2, and 3, respectively) is a membrane the utilizes microcapillary action to channel fluid to the next layer. The microcapillary design of this membrane can be altered to further optimize pore size and flow rate so that each incubation step is for the ideal amount of time and to reduce the number of extraneous cells and proteins that are able to pass through the membrane. Numerous materials may be used for this membrane including, but not limited to nitrocellulose, plastic, or paper. It may also be used to add signal enhancers and/or washing buffer. These may be dry and become activated when sample passes through this layer.

[0124] Layer 5 (e.g., including elements 7/8, 13/14, and 19 in Figures 1, 2, and 3, respectively) is a nitrocellulose membrane that has pregnancy protein (see Table 1) capture antibodies embedded for detection as well as negative and/or positive control spots. [0125] Layer 6 (e.g., including element 20 in Figure 3) is an absorbent wicking pad to collect sample at the end of the process.

[0126] The resulting binding in the case of a pregnant animal causes a fluorometric change that is interpreted via a smart device (for example an iPhone, iPad, etc.) with an application designed to interpret and record the result.

[0127] See Figures 1, 2, and 3.

EXAMPLE 5 -MULTIPLE ANTIBODIES/TRIAD PRECIPITATE

[0128] In the present example, a test tube containing multiple antibodies is described for detection of a pregnancy related antigen in a biological sample.

[0129] Two groups of antibodies are used in the present assay format. The first group of antibodies bind to pregnancy proteins. The second group of antibodies are designed to bind both the pregnancy protein antibody(s) and the pregnancy protein(s). The antibodies are conjugated (or other form of conjugation) to a metal element, such as gold.

[0130] The first group and the second group of antibodies will bind to antigen present in the biological sample upon the binding, and a triad structure is formed. The biological sample may be a bovine urine sample or other biological sample.

[0131] As more of these “triads” form in the reagent mixture, a mass is formed that can be precipitated out of the solution. This mass may be separated through gravity or centrifugation. The resulting precipitate with colorimetric change indicates a positive or negative test for pregnancy.

EXAMPLE 6 - TEST STRIP WITH MICROCAPILLARIES

[0132] In the present example, a biological sample is applied to the end of a test strip containing microcapillaries and pregnancy protein specific antibodies within those microcapillaries.

[0133] Gold-conjugated anti-pregnancy antibodies are bound to the surface of the microcapillary. [0134] In operation, when a biological sample, such as a blood sample of a bovine or other animal, flows over the bound antibodies, pregnancy proteins contained within the biological sample will bind to the antibodies.

[0135] The antibodies bind more tightly to the pregnancy protein than they do to the substrate and therefore are not carried in the fluid. Infrared light at a frequency most absorbed by the gold nanoparticles is used to determine the concentration of gold conjugated antibodies in the sample (much like a pulse oximeter determines hemoglobin oxygen saturation).

EXAMPLE 7 - METHOD FOR ASSESSING BOVINE PREGNANCY - TOOL

IDENTIFICATION CODE

[0136] Any form of the above described embodiments may be used within a system of diagnosing an animal as pregnant or not pregnant. For the methods described herein, any one of the described assay platform designs may be used as a platform, or tool, for conducting the pregnancy tests. As used in the description of the present invention, the term “tool” is used to refer to a particular assay platform design.

[0137] In one method the animal is restrained (head catch, squeeze chute, trailer, breeding box, halter, etc.) and blood is drawn from the jugular vein or caudal vein (“tail vein”) or via small prick in the dermis, or saliva is collected from the mouth, or milk is collected from the udder, or urine is collected via catherization or caught when the animal is urinating. Each tool, or assay platform design device, will be given an identification number. The tool identification code will be recorded, such as by taking a photograph of the identification number (e.g., with a smart device). Where the photograph of the tool is obtained with a smart device, the smart device will be configured to possess a connection to a radio frequency identifying device tag (RFID) that is unique to a specific animal. An electronic application (“app”) within the device will connect the identifier of the animal to that of the “tool”. The sample is applied to the “tool”. After the “tool” has run the test, another picture is taken of the “tool”. The application interprets the result of the test and correlates it with the animal identification to create a spread sheet for the user.

[0138] In a particular embodiment, a smart device is used to take pictures of both the animal identification device (e.g., ear tag) and the presence or absence of a pregnancy indicating identifier (such as those listed in Table 1) being employed by the particular “tool” configuration being used, in an electronic application (the “app”). The application records the animal identification number or combination of letters and matches this with the particular pregnancy indicating identifier result produced by the tool, after a biological sample of the subject animal is contacted with the tool. The biological sample may be collected using any one of the methods described above. The test result on the tool is imaged upon completion and the application, provides an interpretation of the results to provide a result of pregnant or not pregnant or how many days pregnant or what range of days pregnant the animal is. It then matches this result with the animal identification number from the animal from which the biological sample was obtained.

[0139] In another embodiment, the sample may be obtained by any of the means described above, with the user recording the animal identifier from which the biological sample was obtained, on the “tool”. The biological sample is then applied to the tool, such as to provide contact between the biological sample and the indicator molecule. Upon permitting the components in the biological sample to interact with the indicator molecule on the tool, a result is recorded, and the result is interpreted through a software program on a smart device (an “app”). The animal test results may be recorded on paper, in a software programmed report format, or any number of alternative formats of the user’s choosing.

[0140] In another embodiment, the sample is gathered through any of the means described above, a user writes the animal identifier from which the biological sample was obtained, on the “tool” for record keeping purposes, and the presence/absence of a pregnancy associated protein, peptide or fragment thereof is determined by a colorimetric indicator means. When the test is complete, a distinct color change will be indicative of a positive pregnancy result, while the absence of a colorimetric change will be indicative of a negative pregnancy result. The user may then record the result in the form of their choosing.

[0141] In another embodiment, the sample is gathered through any of the means described above, a smart device is used to either take a picture of the animal ID tag or recognizes the RFID tag, the sample is applied to the “tool” assay format of choice, and a change in fluorescence is detected. The fluorescent color change is read by the smart device that is equipped with a special adapter on the device camera which can interpret the fluorescent change. When the test is complete, a distinct fluorescent color change will be indicative of a positive pregnancy result, while the absence of a colorimetric change will be indicative of a negative pregnancy result. The user may then record the result in the form of their choosing.

EXAMPLE 8 - CASE DESIGN FOR TOOL

[0142] The tools described in example 1, 2, 3, 4, and 6 may be used with the following embodiments for design of the case that holds tool. For each of these embodiments the appropriate application opening and result viewing windows for each test may be created (through cutting, drilling, plastic molding, etc.). The case may be made of any appropriate material such as, but not limited to, plastic, a paper product, glass, metal, etc.

[0143] For all embodiments the case may or may not have some form of groove or channel that allows it to lock into place in a device that allows for the sample to be read by an electronic device as discussed in example 7.

[0144] In one form the case is comprised of two halves. The tool is placed appropriately within the pieces and they are permanently joined. This occurs during the manufacturing process.

[0145] In another embodiment, the case is reusable. The case is produced as a solid piece or two joined halves. On one side of the case there is an opening that allows the tool to be inserted into the case for use. Once sample is applied and the test is complete the inserted tool may be removed and disposed of.

[0146] See Figure 6.

EXAMPLE 9 - HEATING ELEMENT INCORPORATED WITHIN THE TESTING

SYSTEM

[0147] This example provides for the various embodiments of a method to regulate the temperature of an immunoassay (such as those described in examples 1-4). The temperature at which an assay is stored and/or operated can contribute greatly to the sensitivity of the test. This is especially critical in the setting of bovine pregnancy recognition as this often occurs in the natural elements which can have ambient temperatures that vary widely.

[0148] In one embodiment of the heating element a part of the sample (some form of fluid) is channeled (through gravity, microcapillary action, etc.) from the sample application area into a tube (may be nanometers to millimeters in radius) that contains a chemical agent that is activated by exposure to fluid and will then cause an exothermic reaction that releases energy as heat.

This heat will warm the interior of the case allowing for a more favorable environment for the reaction kinetics of the assay. The tube running throughout the case may be filled with substances favorable to an oxidation-reduction reaction such as, but not limited to, magnesium or iron. This process may be further expedited by adding sodium chloride or another accelerant to the environment.

[0149] In another embodiment of the heating element there are panels that may be removed from the casing exposing substances contained within the plastic casing that create an exothermic reaction (thereby releasing heat) when combined with oxygen. These may include but are not limited to cellulose, iron, activated carbon, vermiculite, and/or salt.

[0150] In another embodiment of the heating element there is a thin printed film that can be connected to a commercially available battery (for example a AA battery). The film may be incorporated within the casing with an adapter that allows for the battery to be connected via a reusable connecting cord.

[0151] In another embodiment a device is made that attaches to a smart device (for example the iPhone, iPad, etc.). This device has a slot that the test (including case) is inserted into after sample is applied. The device has an electric heating element incorporated that can be turned on or off depending on ambient temperature. It may receive energy from either a battery incorporated into the device or it may plug into the smart device. The device will align the test with the camera of the smart device and allow for a precise image to be obtained for processing by an application. This could be incorporated into certain embodiments such as those described in Example 7.

[0152] See Figure 7.

EXAMPLE 10 - METHOD TO CONTROL HUMIDITY WITHIN TEST CASE

[0153] Immunoassays are sensitive to the environment in which they are operated. The sensitivity of a test can be negatively affected by fluctuations in humidity as well levels that are consistently too high or too low. This example provides for a method to decrease the amount of humidity within the casing holding an immunoassay (such as those described in examples 1-4). [0154] The immunoassay is held within a case (plastic, metal, or other appropriate material). Storage spaces may be made within the case to house a substance or substances that can store moisture when humidity levels are above optimal levels and/or release moisture when humidity levels are below optimal levels. Such substances may be one or more of but are not limited to the following: Silica gel, activated carbon, graphene, plaster, calcium oxide, clay, zeolite.

[0155] See Figure 8.

EXAMPLE 11- METHOD TO HAVE ANALYTE AND REAGENT MIX IN

SUSPENSION

[0156] Reaction kinetics and efficiency can be improved by allowing conjugated antibody and analyte to mix in solution as opposed to on a surface as normally occurs in a vertical or lateral flow assay. Commonly, for these components to mix in solution multiple steps and added complexity are added to the test by requiring the user to carefully mix both in a separate setting and then apply to the assay. This example provides for a method to have analyte and conjugated antibody (or other reagent such as buffer or signal enhancing reagents) meet in solution while still maintaining the simplicity of the vertical or lateral flow immunoassay such as those described in examples 1-4. The method may be incorporated at one or more times in both vertical and lateral flow assays.

[0157] In this example a one-way semi-porous membrane forms one side of a container a holding container. The container contains detection antibodies (or other reagent) in a dry form.

[0158] The sample flows through the assay where it is then directed to the side of the cube that is comprised of the one-way semi-permeable membrane. It passes through this membrane and into the container.

[0159] Once in the container the sample containing the desired analyte and detection antibodies (or other reagent) mix.

[0160] In another region of the container a different membrane is a barrier that is or is coated in a substance that will dissolve after the desired amount of time being exposed to fluid (the sample that was applied). This dissolution allows the fluid to move to the next step in the assay.

[0161] In addition to allowing for analyte to mix with detection antibodies, the same principles may be used for improved results with signal enhancing agents, washing buffers, etc. [0162] See Figure 9.

EXAMPLE 12 - METHOD TO CONTROL ONE-WAY FLOW OF A FLUID THROUGH

A MEMBRANE

[0163] One-way flow of fluid through a porous membrane is advantageous in many settings including in diagnostic testing. This example provides for one method to allow flow of a sample fluid through a semipermeable membrane in one direction only.

[0164] This method comprises of two sheets of thin a non -porous membrane (of some suitable substance such as a plastic or cellophane). The outer sheet (on the side that sample fluid is coming from) has holes made in it (size commiserate with the desired flow rate and type of sample). The second layer (on the side that the fluid sample is going towards) there are crescent shape cuts in the membrane in the same layout as the aforementioned holes. This membrane is then offset slightly from the other membrane so that the crescent shaped flaps can only be deflected in the direction of fluid flow and not in the opposite direction. The two membranes are joined by means appropriate for the selected material.

[0165] See Figure 10.

Claims

CLAIMS What is claimed is:

1. A vertical flow assay device for detecting pregnancy, the device comprising a plurality of layers including: a first layer configured to separate or direct flow of an assay sample; a second layer embedded with an unbound conjugated antibody configured to bind to a pregnancy antigen during flow of the assay sample; and a third layer embedded with a bound capture antibody configured to bind to the pregnancy antigen during flow of the assay sample; wherein a threshold concentration of the pregnancy antigen is detectable in the third layer via a colorimetric or fluorescent change under a substantially ambient temperature; and wherein the pregnancy antigen comprises at least one of the following: pregnancy specific protein (PSP) B; pregnancy associated glycoprotein (PAG)-1, 4, 5, 6, 7, and 9; bovine interferon tau; and early conception factor (ECF).

2. The vertical flow assay device of claim 1, wherein the first, second, and third layers comprise a membrane or a filter.

3. The vertical flow assay device of claim 2, wherein first, second, and third layers further comprise at least one of the following reagents: trehalose, EDTA, bovine serum albumin, phosphate buffered saline, Tween-20, and borate.

4. The vertical flow assay device of claim 1, wherein the assay sample comprises at least one of the following: blood, plasma, serum, saliva, urine, and milk.

5. The vertical flow assay device of claim 4, wherein the assay sample comprises a bovine sample.

6. The vertical flow assay device of claim 1, wherein the unbound conjugated antibody comprises at least one of a gold particle conjugated antibody or a fluorescent dye conjugated antibody.

7. The vertical flow assay device of claim 1, wherein the colorimetric or fluorescent change is detectable via a smart device or an application.

8. The vertical flow assay device of claim 7, wherein the smart device or the application is configured to correlate the colorimetric or fluorescent change with a source of the assay sample.

9. The vertical flow assay device of claim 1, further comprising a heating element configured to maintain the assay sample at the substantially ambient temperature.

10. The vertical flow assay device of claim 1, further comprising a storage case configured to hold the first, second, and third layers; wherein the storage case comprises a plurality of storage spaces configured to house a moisture-retaining material.

11. A kit for detecting a selected animal pregnancy marker in a biological sample, said kit comprising: a first solution including an indicator molecule specific for a selected animal pregnancy marker, wherein the indicator molecule is configured to produce a visually detectable change under a substantially constant temperature and a specified reaction time; a vertical flow design assay device, said device comprising at least a first layer, a second layer, and a third layer, each layer comprising a surface, wherein: a surface of the first layer comprises a biological sample application layer configured to direct and control flow of the biological sample or to separate the biological sample; a surface of the second layer comprises a plurality of pores configured to channel flow of the biological sample or to store the first solution; a surface of the third layer comprises a biomolecular recognition element configured to bind to the selected animal pregnancy marker; and a control sample configured to generate a visually detectable change in the indicator molecule, the visually detectable change comprising a threshold intensity; wherein a positive reading is produced when the biological sample generates a visually detectable change with an intensity greater than the threshold intensity; and wherein a negative reading is produced when the biological sample does not generate a visually detectable change or generates a visually detectable change with an intensity less than the threshold intensity.

12. The kit of claim 11, wherein the selected animal protein marker includes at least one of the following: pregnancy specific protein (PSP) B; pregnancy associated glycoprotein (PAG)-1, 4, 5, 6, 7, and 9; bovine interferon tau; and early conception factor (ECF).

13. The kit of claim 11, wherein the indicator molecule is fluorometric or colorimetric.

14. The kit of claim 11, further comprising a storage case configured to house the vertical flow design assay device.

15. The kit of claim 14, wherein the storage case comprises a heating element configured to maintain the biological sample at the substantially constant temperature.

16. The kit of claim 14, wherein the storage case comprises a plurality of storage spaces configured to house a moisture-retaining material.

17. An animal pregnancy detection method comprising: measuring a level of an animal pregnancy -associated antigen in a test animal comprising contacting a biological sample of the test animal with a detecting reagent and obtaining a test animal antigen measurement; comparing the test animal antigen measurement to a measurement of the animal pregnancy-associated antigen in a biological specimen of a control animal; and detecting pregnancy in the test animal where the test animal biological specimen contains a greater concentration of the animal pregnancy-associated antigen compared to the level of the pregnancy-associated antigen in the biological specimen of the control animal.

18. The method of claim 17, wherein the animal pregnancy-associated antigen comprises at least one of the following: pregnancy specific protein (PSP) B; pregnancy associated glycoprotein (PAG)-1, 4, 5, 6, 7, and 9; bovine interferon tau; and early conception factor (ECF).

19. The method of claim 17, wherein the detecting reagent comprises a colorimetric detecting reagent or a fluorometric detecting reagent.

20. The method of claim 17, further comprising controlling a temperature or a humidity level of the biological sample of the test animal.