Lateral flow assay for detecting pathogens in the milk of cows with mastitis

Abstract

The present invention provides a lateral flow device for detecting lipoteichoic acid (LTA) as a Gram-positive bacterial identifier in an animal milk sample, the device comprising: a) a strip (formed of a material that allows capillary flow of fluid along a portion of the strip); b) a sample pad (located proximal to one end of the strip for receiving a milk sample); c) a conjugate pad (located within the strip, such that in operation the sample flows by capillary action through the strip from the sample pad to the conjugate pad, mobilizing a conjugate comprising an anti-LTA antibody bound to a detection agent, contained in the conjugate pad); and d) a test line (comprising anti-LTA antibody immobilized within the strip along a band disposed substantially perpendicular to the direction of sample flow along the strip, such that when a formed complex comprising the mobilized anti-LTA antibody conjugate and LTA in the sample comes into contact with the immobilized anti-LTA antibody in the test line, the presence of LTA in the sample is indicated by a visible color change).

Description

Technical Field

[0005] ,

[0006]

[0001] The present invention relates to a concentration-based lateral flow (LF) test for directly detecting target Gram-positive bacteria in milk samples collected from individual mammary quarters (compartments) of mastitis cows.

Background Art

[0002] Cows are usually milked at least twice a day. Most dairy farms have equipment sufficient to milk over 20 cows at a time. The milking machine causes milk secretion from the udder by creating a pulsating vacuum around the teat, mimicking the action of a calf.

[0003] Milk is usually placed in a milk storage vat or silo on the farm and stored at low temperature for 48 hours or less. After the milk is collected, the storage vat and stainless steel pipes are thoroughly cleaned, and then the farmer milks again.

[0004] Milk is collected from the farm every 24 or 48 hours by a tanker equipped with a highly insulated special stainless steel body to keep the milk cold during transportation to the processing factory. The driver of the milk tanker is a certified milk grader qualified to evaluate the milk prior to collection. The driver of the tanker grades and, if necessary, rejects the milk based on temperature, vision, and smell. Representative samples are collected from the pickup at each farm before transferring to the tanker by pump. After collection, the milk is transported to the factory site and stored in a refrigerated silo until processing.

[0005] Milk samples are collected from the farm vat before collection and from the bulk milk tanker upon arrival at the factory. Samples from the bulk milk tanker are tested for antibiotics and temperature before entering the factory's processing area. Farm milk samples are tested for milk fat, protein, somatic cell count, and bacterial count of the bulk milk. If the milk does not meet the quality standards, it is rejected. Most farmers receive payment based on the quality and composition of their milk.

[0006] Mastitis is inflammation of the mammary glands and mammary tissue due to microbial infection or physical trauma. It remains the most common and economically burdensome disease of dairy cows worldwide. Financial losses due to mastitis can occur in both latent and clinical cases. Clinical mastitis is readily apparent and easily detected by the appearance of abnormalities in milk or udder, or secondary clinical signs. Current diagnosis of latent mastitis is based on the results of indirect tests such as somatic cell count (SCC) as an indirect indicator of infection, the California Mastitis Test (CMT), or the electrical conductivity of milk. Typically, once mastitis is diagnosed, the cow's milk is extracted, and the infected udder is treated with intra-mammary infusion of antibiotics.

[0007] Early detection of mastitis improves treatment outcomes, and the ability to detect Gram-positive bacteria facilitates treatment decisions. All currently approved intramammary injection products are labeled as being for the treatment of Gram-positive bacterial mastitis pathogens (e.g., Staphylococcus and Streptococcus). Few antibiotics are labeled as being for the treatment of Gram-negative bacterial mastitis pathogens (e.g., Escherichia coli). Due to regulatory and environmental pressures, the use of broad-spectrum antibiotics and / or blanket therapy in the management of bovine mastitis continues to decline. Therefore, it is becoming increasingly important to accurately and rapidly diagnose the causative pathogens of mastitis, promote targeted use of narrow-spectrum antibiotics, and ensure treatment success and favorable outcomes. It is desirable to concentrate diagnostic efforts on the early diagnosis of mastitis (e.g., based on clinical signs, dairy records, SCC, etc.), then determine the causative pathogen, and initiate timely and appropriate antibiotic treatment.

[0008] Currently, the diagnosis of mastitis typically relies on (1) SCC as an indirect indicator of infection, and (2) in vitro milk culture (a laboratory-based pathogen identification method that usually requires the shipment of milk samples and typically takes several days to several weeks). Neither method meets the needs of dairy farmers and veterinarians seeking early detection and identification of mastitis pathogens. Therefore, it would be desirable to provide point-of-care testing to detect mastitis pathogens in milk to guide antibiotic selection. In particular, diagnostic instruments for Gram-positive bacterial identifiers would provide a more comprehensive, animal-specific customer solution to meet customer needs and complement treatment options on the market. If point-of-care testing is performed after the first milking and before the next milking, it would be desirable to ensure the quality and composition of milk collected from farms, reduce financial losses, and improve treatment outcomes. [Overview of the project]

[0009] Lipoteichoic acid (LTA) is a major pro-inflammatory structure present within the cell wall layer of almost all Gram-positive bacteria. It plays a crucial role in bacterial infection, inflammation, and the initiation and progression of septic shock. This invention provides a lateral flow device for detecting LTA expressed on the surface of Gram-positive bacteria as an identifier for mastitis Gram-positive bacteria in animal milk samples. The device comprises: a) a strip (formed of a material that allows capillary flow of fluid along a portion of the strip); b) a sample pad (located proximal to one end of the strip to receive a milk sample); c) a conjugate pad (located within the strip, where, during operation, the sample flows through the strip from the sample pad to the conjugate pad by capillary action, recruiting a conjugate containing an anti-LTA antibody bound to a detection agent, which is contained within the conjugate pad); and d) a test line (containing an immobilized anti-LTA antibody within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, so that when the formed complex containing the recruited anti-LTA antibody conjugate and LTA in the sample comes into contact with the immobilized anti-LTA antibody in the test line, the presence of LTA in the sample is indicated by a visible color change). In one embodiment, the strip is formed of nitrocellulose.

[0010] In one embodiment, the milk sample is concentrated with bacterial cells. In another embodiment, the milk sample is from a mastitis-prone cow's milk. In a further embodiment, the milk sample is derived from an animal selected from the group consisting of dogs, cats, horses, goats, sheep, or animals of the Bovidae family. In a particular embodiment, the milk sample is derived from an animal of the Bovidae family.

[0011] In one embodiment, the device further includes a wicking pad for receiving and holding samples after they have passed through a test line and any control line. In another embodiment, the anti-LTA antibody in the conjugate and test line is a monoclonal antibody. In one embodiment, the detection agent conjugated to the anti-LTA antibody is selected from the following: metal nanoparticles or nanoshells, non-metal nanoparticles or nanoshells, enzymes, and fluorescent molecules. In a particular embodiment, the detection agent comprises metallic gold nanoparticles or nanoshells.

[0012] In one embodiment, the lateral flow device is a dipstick. In another embodiment, the sample pad portion of the dipstick is immersed in a milk sample. In one embodiment, the sample pad portion of the dipstick is immersed in a milk sample concentrated with bacterial cells. In a further embodiment of the device, the strip is housed in a cassette.

[0013] In one embodiment, the sample pad portion of the device includes a filter membrane for removing one or more components from a sample. In a particular embodiment, the one or more components removed from the sample by the filter membrane of the sample pad are cells, cellular material, fats, or particulate matter.

[0014] In one embodiment, the device further includes a control line positioned substantially perpendicular to the direction of sample flow along the strip. In another embodiment, the conjugate pad portion of the device further includes an antibody that is not specific to Gram-positive bacteria, the antibody being bound to a detection agent to form a second antibody conjugate, and during operation, the sample flows from the sample pad to the conjugate pad and recruits the second antibody conjugate. This passes through the test line without reaction and crosses the control line.

[0015] In one embodiment, the antibody deposited on the control line of the device is one that can bind to the mobilized second antibody conjugate as it crosses the control line, and such binding on the control line is indicated by a visible color change. In another embodiment, the antibody in the second antibody conjugate is derived from an animal species other than the species from which the milk sample was collected.

[0016] In one embodiment, the detection agent conjugated to the antibody in the second antibody conjugate is selected from the following: metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules. In a particular embodiment, the detection agent conjugated to the antibody in the second antibody conjugate includes metallic gold nanoparticles or nanoshells.

[0017] The present invention further provides a method for detecting lipoteichoic acid (LTA) as a mastitis Gram-positive bacterial identifier in animal milk samples, the method comprising using a device described according to any of the above embodiments. In one preferred embodiment, the milk sample is enriched with bacterial cells.

[0018] The present invention further provides a method for detecting lipoteichoic acid (LTA) as an identifier for mastitis Gram-positive bacteria. The method comprises contacting an animal milk sample with a conjugate containing an anti-LTA antibody conjugated to a detection agent, thereby forming an antibody-antigen complex between the anti-LTA conjugate and LTA present on Gram-positive bacteria in the sample; capturing the formed antibody-antigen complex with an anti-LTA antibody; and detecting the captured complex. In certain embodiments of the method, the milk sample is enriched with bacterial cells.

[0019] In another embodiment of this method, the anti-LTA antibody used in the conjugate and for capturing the antibody-antigen complex is a monoclonal antibody. In a further embodiment of this method, the detection agent conjugated to the anti-LTA antibody is selected from the following: metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules. In a particular embodiment, the detection agent comprises metallic gold nanoparticles or nanoshells.

[0020] In one embodiment, the method of the present invention is capable of detecting target Gram-positive bacteria with a concentration of ≥100 CFU / mL. [Brief explanation of the drawing]

[0021] [Figure 1] Figure 1 shows a schematic diagram of the lateral flow (LF) device of the present invention. [Figure 2] Figure 2 shows a schematic diagram of one aspect of the workflow of a concentration-based LF assay according to the present invention. [Figure 3] Figure 3 shows a schematic diagram comparing a conventional diagnostic method for identifying pathogens in mastitis cows with the point-of-care diagnostic method of the present invention.

[0022] Other objects, aspects, features, and advantages of the present invention will become apparent from the following description. However, the detailed description and specific examples, while indicating preferred embodiments of the present invention, will make various changes and modifications apparent to those skilled in the art within the spirit and scope of the present invention, and it should be understood that they are provided for the purpose of illustration only.

Modes for Carrying Out the Invention

[0023] Definitions Throughout this specification, unless the context requires otherwise, the word “comprise, comprises, comprising” is to be understood to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements.

[0024] The terms “lipoteichoic acid” and its abbreviation “LTA” may be used interchangeably herein. LTA is a major pro-inflammatory structure present within the cell wall layer of almost all Gram-positive bacteria. It plays an important role in the initiation and progression of bacterial infection, inflammation, and septic shock. LTA is a complex glycosyl-phosphate-containing polymer that is linked to the membrane of Gram-positive bacteria via a lipid anchor.

[0025] The term "antibody" (also referred to as "immunoglobulin") is a Y-shaped protein of the immune system that specifically identifies foreign substances or antigens, such as components of bacteria, yeast, parasites, and viruses. Since each tip of the antibody's 'Y' contains an antigen-binding site specific to a particular epitope on an antigen such as LTA present on gram-positive bacteria, these two structures can bind precisely. The production of a given antibody increases when exposed to an antigen (e.g., a microbial or viral antigen) that specifically interacts with that antibody. Thus, detecting antigen-specific antibodies in a subject's sample informs whether the subject is currently or has previously been exposed to a given microorganism such as a virus, bacterium, fungus, or parasite. "Antibodies" typically contain all or part of the Fc region and may also contain one or more antigen-binding sites, facilitating detection by antibody-specific binding agents such as antigens or antigen peptides. Antibodies can be, for example, antibodies of the IgG, IgE, IgD, IgM, or IgA type. In one aspect, the antibodies for use in the devices and methods herein are monoclonal antibodies (mAbs). In one aspect, the term "antibody" as used herein may contain all or part of the Fc region or may contain only the antigen-binding portion of the antibody, such as a Fab fragment.

[0026] The term "protein" refers to polymers of amino acid residues and their variants, as well as synthetic and natural analogs. Thus, these terms apply to natural amino acid polymers and their natural chemical derivatives, as well as amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, e.g., chemical analogs of the corresponding natural amino acids.

[0027] The term "antigen" means a molecule having a distinctive surface feature or epitope that can stimulate a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to an antigen. Antigens can be proteins, carbohydrates, or lipids, but only protein antigens are classified as immunogens because carbohydrates and lipids cannot elicit an immune response on their own.

[0028] The "antigen-binding site" or "binding region" of an antibody refers to the portion of the immunoglobulin molecule involved in antigen binding. The antigen-binding site is formed by amino acid residues in the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly different (mismatched) stretches within the V regions of the heavy and light chains are called "hypervariable regions" and are sandwiched between more conservative adjacent stretches known as "framework regions" or "FRs." In the antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space, forming the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of the heavy and light chains are called "complementarity-determining regions" or "CDRs."

[0029] The term "nanoparticles" refers to uniform particles with a size of 1 to 200 nm. The term "nanoshell" refers to nanoparticles consisting of a core and a metal shell (usually gold). Lateral flow devices and kits Refer to the drawings. Figure 1 shows the LF device (1) of the present invention. The device (1) includes a sample pad (2) for preparing a target body fluid sample for capillary flow, a conjugate pad (3) containing a recruitable conjugate containing an anti-LTA antibody conjugated to a detection agent, a membrane (4), and a wicking pad (7) for receiving and holding the fluid that has moved from the sample pad (2) through the conjugate pad (3) and membrane (4) by capillary flow. A test line (5) containing an immobilized anti-LTA antibody is also shown. A band (6) is a positive control line. The device (1) may further include an adhesive band or cover tape (8) connecting the sample pad, the conjugate pad and nitrocellulose and a backing material (not shown).

[0030] When a fluid milk sample is placed on the sample pad (2), or when the sample pad (2) is immersed in a fluid milk sample, the fluid moves from the sample pad (2) to the conjugate pad (3) by capillary flow, where it comes into contact with an anti-LTA antibody conjugated to a detection agent (not shown). LTA, which is abundantly expressed on the surface of Gram-positive bacteria in the sample, reacts with the anti-LTA conjugate on the conjugate pad (3) to form a complex. The fluid then recruits the formed complex and transports it to the test line (5). Specifically, the anti-LTA antibody-LTA antigen-detection agent complex formed between some LTA antigen in the sample and the anti-LTA antibody conjugate on the conjugate pad (3) moves through the membrane (4) to the test line (5), where the complexed LTA is immobilized by the anti-LTA antibody deposited therein. If LTA is present in the sample, the accumulation of the detection agent on the test line (5) forms a visible signal, i.e., a color change, indicating a positive result. If LTA is not present in the sample, the conjugate will not be immobilized at the test line (5) and will continue to migrate to the wicking pad (7). The absence of a visible signal at the test line (5) means that the sample is negative for LTA, i.e., the sample is negative for Gram-positive bacteria, since LTA is expressed on the surface of Gram-positive bacteria.

[0031] In another embodiment of the device (1) in Figure 1, the conjugate pad (3) further contains an immunoglobulin that is not specific to Gram-positive bacteria. This immunoglobulin is bound to a detection agent (not shown) to form a second antibody conjugate, and during operation, the milk sample flows from the sample pad (2) to the conjugate pad (3) and recruits the second antibody conjugate. This passes through the test line (5) without reaction and crosses the control line (6). In one embodiment, what is deposited on the control line (6) is the antibody that the recruited second antibody conjugate can bind to as it crosses the control line (6), and binding on the control line (6) is indicated by a visible color change. In one embodiment, the immunoglobulin in the second antibody conjugate is derived from an animal species other than the species from which the milk sample was collected.

[0032] There is physical overlap and contact between the conjugate pad (3), sample pad (2), and membrane (4) to enable the formation of the LTA-gold conjugate complex and proper flow on the test strip. In one embodiment, the device is a dipstick, and the conjugate pad, sample pad, and membrane of the dipstick are covered with adhesive bands or cover tape and housed on a long backing card that provides a 15 mm long handle to the dipstick.

[0033] In one embodiment, a raw milk sample is first concentrated using a concentrating medium / broth (a suitable formulation thereof is shown in Figure 2 and disclosed in the Examples) according to the concentration method described below and in Figure 2. This is based on concentrating bacterial cells from the milk sample to a detectable level and then detecting them by a Gram-positive bacteria-specific LF test. In one embodiment, a milk sample from a mastitis-infected cow's milk plot is collected under sterile conditions before milking, in accordance with the guidelines of the National Mastitis Council (NMC), mixed with a concentrated broth, and incubated at 37°C for approximately 7 hours. In one embodiment, an aliquot of the concentrated milk sample is tested with an LF test according to the present invention before the next milking (8-12 hours) to enable diagnosis and appropriate antibiotic treatment.

[0034] As shown in Figure 2, in one embodiment, an aliquot of approximately 200-250 μL of concentrated milk sample is added to a test tube using a disposable pipette. Next, the sample pad portion (2) of the lateral flow dipstick is immersed in the concentrated milk sample in the test tube. Fluid flow is then initiated, causing the sample to move from the sample pad to the adjacent conjugate pad (3).

[0035] In one non-restrictive embodiment, deposited on the conjugate pad (3) are various gold conjugates, namely 1) anti-LTA antibodies bound to gold particles, and 2) non-Gram-positive bacterial immunoglobulins, e.g., chicken IgY, bound to gold particles. The gold-bound anti-LTA antibodies form complexes with LTA antigens of Gram-positive bacteria present in the milk sample. The formed complexes migrate along the test strip and form complexes with striped anti-LTA antibodies on the test line (5) of the membrane (4). When the anti-LTA antibody-LTA antigen complexes are captured on the sensitized test line (5), their accumulation results in the formation of a clearly visible pink / red band. The pink / red band on the control line ensures that the test has been performed correctly. For the control, in one embodiment, chicken IgY-gold conjugate on conjugate pad (3) moves across an adjacent membrane, where it passes through test line (4) without reacting and crosses another line (control line 6) where donkey anti-chicken IgY is deposited. The chicken IgY-gold conjugate binds to this control line, and a visible red line is formed due to the accumulation of gold colloid particles.

[0036] Figure 3 shows a comparison between conventional diagnostic methods for detecting mastitis, namely the conventional method using cell culture, which is the gold standard for identifying pathogens, and the point-of-care diagnostic method of the present invention. As shown in the figure, the conventional method may require 1 to 5 days to identify the pathogen before initiating timely and appropriate antibiotic treatment. In contrast, the point-of-care lateral flow test of the present invention uses a milk sample concentration step, which takes about 6 to 7 hours in one embodiment and about 7 to 7.5 hours in a preferred embodiment (not shown), but the test itself can be performed in about 10 minutes. This allows dairy farmers to obtain results in a timely manner, for example, before the next milking. This can help ensure the quality and composition of milk collected from the farm and reduce financial losses. At the same time, it can also improve treatment outcomes by at least guiding antibiotic selection.

[0037] The present invention also provides a kit comprising one or more LF devices described herein and instructions for using the devices to detect LTA antigens as an indicator of Gram-positive bacteria in a test sample.

[0038] The lateral flow device of the present invention detects LTA, which is abundantly expressed on the surface of Gram-positive bacteria. On one side, the lateral flow device comprises: a) a strip (formed of a material that allows capillary flow of concentrated milk sample along a portion of the strip); b) a sample pad (located proximal to one end of the strip to receive the concentrated milk sample); c) a conjugate pad (located within the strip, where, during operation, the concentrated milk sample flows through the strip from the sample pad to the conjugate pad by capillary action, recruiting a conjugate containing an anti-LTA antibody bound to a detection agent, which is contained in the conjugate pad); d) a test line (containing an immobilized anti-LTA antibody within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, so that when the formed complex containing the recruited anti-LTA antibody conjugate and LTA in the concentrated milk sample comes into contact with the immobilized anti-LTA antibody in the test line, the presence of LTA in the concentrated milk sample is indicated by a visible color change); and e) a control line (positioned substantially perpendicular to the direction of flow of the concentrated milk sample along the strip, where the control area is located where the sample is loading into the sample entry area (loading When introduced into the region, it includes (a) a sample and fluid communication; and (f) a wicking pad (to receive and hold the sample after it has passed through the detection band).

[0039] The anti-LTA antibody used in this invention may be one produced in response to lipoteichoic acid derived from Gram-positive bacterial species. In one respect, the anti-LTA antibody can specifically react with lipoteichoic acid of Gram-positive bacteria in bacterial infection samples. Gram-positive bacteria are characterized by a blue-violet reaction in Gram staining. The color reaction is caused by the complexation of crystal violet, the main Gram staining dye, with the iodine mordant. Upon application of a destaining agent, slow dehydration of the crystal violet / iodine complex is observed by the closure of pores that penetrate the cell wall.

[0040] In one embodiment, anti-LTA antibodies are produced against lipoteichoic acid derived from Staphylococcus epidermidis. In another embodiment, anti-LTA antibodies are produced against lipoteichoic acid derived from Streptococcus pyogenes. In yet another embodiment, anti-LTA antibodies are produced against lipoteichoic acid derived from Bacillus subtilis.

[0041] Anti-LTA antibodies produced against lipoteichoic acid derived from Gram-positive bacteria are commercially available. For example, a mouse monoclonal anti-LTA antibody (class IgG1) is commercially available from QED Biosciences, Inc. in San Diego, California (catalog number: 15711). This antibody was produced against the Hay strain of Staphylococcus epidermidis (ATCC #55133) and reacts with lipoteichoic acid from other Staphylococcus epidermidis strains (types I, II, and III), Staphylococcus aureus strains 5 and 8, Streptococcus pyogenes, Streptococcus fecaelis, and Streptococcus mutans. In addition, a mouse monoclonal antibody [clone G43J] against a Gram-positive bacterium belonging to class IgG1 (ab267414) is commercially available from Abcam (Cambridge, UK). Abcam antibodies are thought to have been produced in response to lipoteichoic acid derived from Bacillus subtilis. The inventors have also succeeded in producing monoclonal antibodies against lipoteichoic acid derived from Streptococcus pyogenes using methods well known in the art. Furthermore, they have succeeded in cloning the antigen-binding region (Fab fragment) of antibodies against lipoteichoic acid derived from Streptococcus pyogenes using recombinant methods. Lipoteichoic acid derived from Streptococcus pyogenes can be purchased, for example, from Sigma Aldrich (catalog number L3140-5MG). These are all non-limiting examples of anti-LTA antibodies that can be used in the present invention.

[0042] Suitable methods for immobilizing capture entities, such as anti-LTA antibodies, onto a solid phase include ionic, hydrophobic, and covalent interactions. Regarding the immobilization of conjugates onto a conjugate pad, typically the conjugate is sprayed onto the conjugate pad using a specialized sprayer similar to an airbrush. The reagent is then dried on the conjugate pad. Similarly, test and control lines are drawn onto a test strip (e.g., nitrocellulose) using a precision dispensing machine. The protein binds to the nitrocellulose and is thus immobilized.

[0043] The sample pad not only receives the milk sample for testing, but also removes components from the sample that may obstruct the capillary flow of the fluid through the strip or negatively affect the detection of the formed LTA antigen-anti-LTA antibody complex on the test line. Milk components that can be removed by the sample pad include cells, cellular material, fats, and particulate matter. To detect the LTA antigen in the target milk sample, the sample pad also acts as a milk filtration pad, removing milk components such as cells and fats that may obstruct the flow of the sample along the strip.

[0044] Nitrocellulose has been found to be a suitable material for manufacturing the strips. Other materials may also be suitable if they allow for the desired capillary flow rate and adequate detection sensitivity. For example, PVDF membranes, polyethylene membranes, nylon membranes, or similar types of membranes.

[0045] In one embodiment, the milk sample is of animal origin, for example, but not limited to, animals of the Bovidae family. In a particular embodiment, the milk sample is of dairy cow origin. In one embodiment, the milk sample is of mastitis milk from a bovine.

[0046] A detection agent is any agent that provides a detectable change when accumulated on the test line or control line. Accumulation of a detection agent on the test line indicates the presence of LTA antigen, which is abundantly expressed on the surface of Gram-positive bacteria, in the milk sample. In fact, the detection agent binds directly or indirectly to an anti-LTA antibody to form a conjugate contained in the conjugate pad, which can bind to the LTA antigen in the sample. In some embodiments, the conjugate pad may also contain a second antibody that is nonspecific to Gram-positive bacteria. The second antibody is directly or indirectly bound to the detection agent, and the sample recruits the second antibody conjugate during operation. This passes through the detection band without reaction and crosses the control line. The control line has an antibody deposited that can bind to the antibody present in the recruited second antibody conjugate. When the second antibody conjugate crosses the control line and the antibody deposited on the control line binds to it, binding is indicated by a visible color change.

[0047] The accumulation of the detection agent causes a visible color change or observable fluorescence, or any other appropriate change, in the test line and control line. In various specifically conceivable embodiments of the present invention, the detection agent conjugated to the anti-LTA antibody on the conjugate pad is selected from metal nanoparticles or nanoshells, non-metal nanoparticles or nanoshells, enzymes, or fluorescent molecules. In a particular embodiment, the metal nanoparticles or metal nanoshells conjugated to the anti-LTA antibody are selected from gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, or silica-coated gold shells. In one desired embodiment, the detection agent conjugated to the anti-LTA antibody comprises metallic gold nanoparticles or nanoshells.

[0048] In other specifically conceivable embodiments of the present invention, the detection agent conjugated to a second antibody (not specific to Gram-positive bacteria) on a conjugate pad is selected from metal nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, or fluorescent molecules. In certain embodiments, the metal nanoparticles or metal nanoshells conjugated to the second antibody are selected from gold particles, silver particles, copper particles, platinum particles, cadmium particles, composite particles, gold hollow spheres, gold-coated silica nanoshells, and silica-coated gold shells.

[0049] In one embodiment, the detection agent bound to the anti-LTA antibody on the conjugate pad may be the same as or different from the detection agent bound to the second antibody on the conjugate pad. In one desired embodiment, both the detection agent bound to the anti-LTA antibody and the detection agent bound to the second antibody are gold nanoparticles to create a colloidal gold conjugate.

[0050] method The present invention further provides a method for detecting lipoteichoic acid (LTA) as a Gram-positive mastitis bacterium identifier in animal milk samples, the method comprising using a device described according to any of the above embodiments. In one desired embodiment, the milk sample is concentrated with bacterial cells. In one embodiment, the milk sample is derived from a dairy cow. In one special embodiment, the milk sample is derived from a mastitis-inducing milk sac of a bovine collected under sterile conditions in accordance with the guidelines of the National Mastitis Council (NMC). Preferably, the method is performed after the first milking and before the next milking. Typically, cows are milked two to three times a day.

[0051] The present invention further provides a method for detecting lipoteichoic acid (LTA) as a mastitis Gram-positive bacterial identifier. The method involves contacting a milk sample from an animal with a conjugate containing an anti-LTA antibody conjugated to a detection agent, where an antibody-antigen complex is formed between the anti-LTA conjugate and LTA present on Gram-positive bacteria in the sample; capturing the formed antibody-antigen complex with an anti-LTA antibody; and detecting the captured complex. In certain embodiments of the method, the milk sample is enriched with bacterial cells, for example, by using the enrichment method and enrichment broth shown in Figure 2 and described in the Examples section. Given the low bacterial load in many clinical milk samples (approximately 100 CFU / mL), it is desirable to include a milk sample preparation step in which the milk sample is first enriched with bacterial cells. The enrichment method of the milk sample allows for the detection of both clinical and latent cases of mastitis.

[0052] In one embodiment of this method, the anti-LTA antibody in the conjugate and the anti-LTA antibody used to capture the antibody-antigen complex are monoclonal antibodies. In a further embodiment of this method, the detection agent conjugated to the anti-LTA antibody is selected from the following: metal nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, and fluorescent molecules. In a particular embodiment, the detection agent comprises metallic gold nanoparticles or nanoshells.

[0053] The present invention also includes the following embodiments. Embodiment 1 A lateral flow device for detecting lipoteichoic acid (LTA) expressed on the surface of Gram-positive bacteria as an identifier for mastitis Gram-positive bacteria in an animal milk sample, the device comprising: a) a strip (formed of a material that allows for the capillary flow of fluid along a portion of the strip); b) a sample pad (located proximal to one end of the strip to receive a milk sample); c) a conjugate pad (located within the strip, and during operation, the sample flows through the strip from the sample pad to the conjugate pad by capillary action, recruiting a conjugate containing an anti-LTA antibody bound to a detection agent, which is contained in the conjugate pad); and d) a test line (containing an anti-LTA antibody immobilized within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, so that when a complex formed with the recruited anti-LTA antibody conjugate and LTA in the sample comes into contact with the immobilized anti-LTA antibody in the test line, the presence of LTA in the sample is indicated by a visible color change). Embodiment 2: The device according to Embodiment 1, wherein a milk sample is concentrated for bacterial cells. Embodiment 3: The device according to Embodiment 1 or Embodiment 2, wherein the milk sample is from a mastitis-inducing milk section of a bovine. Embodiment 4 The device according to any one of Embodiments 1 to 3, further comprising a wicking pad for receiving and holding a sample after it has passed through a test line. Embodiment 5: The device according to any one of Embodiments 1 to 4, wherein the anti-LTA antibody in the conjugate and test line is a monoclonal antibody. Embodiment 6 The device according to any one of Embodiments 1 to 5, wherein the detection agent conjugated to the anti-LTA antibody is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules. Embodiment 7 The device according to Embodiment 6, wherein the detection agent comprises metallic gold nanoparticles or nanoshells. Embodiment 8: The device according to any one of Embodiments 1 to 7, wherein the lateral flow device is a dipstick. Embodiment 9 The device according to Embodiment 8, wherein the sample pad portion of the dipstick is immersed in a milk sample. Embodiment 10: The device according to any one of Embodiments 1 to 7, wherein the strip is housed in a cassette. Embodiment 11 The device according to any one of Embodiments 1 to 10, wherein the sample pad includes a filter membrane for removing one or more components from a sample. Embodiment 12 The device according to Embodiment 11, wherein one or more components removed from the sample by the filter membrane of the sample pad are cells, cellular material, fat, or particulate matter. Embodiment 13 The device according to any one of embodiments 1 to 12, further comprising a reference line positioned substantially perpendicular to the direction of sample flow along the strip. Embodiment 14 The device according to Embodiment 13, wherein the conjugate pad further contains an antibody that is not specific to Gram-positive bacteria, the antibody being bound to a detection agent to form a second antibody conjugate, and during operation, a sample flows from the sample pad to the conjugate pad, mobilizing the second antibody conjugate, the second antibody conjugate passes through the test line without reacting, and crosses the control line. Embodiment 15 The device according to Embodiment 14, wherein the control line is deposited with an antibody that can bind to a mobilized second antibody conjugate as it crosses the control line, and the binding at the control line is indicated by a visible color change. Embodiment 16 The device according to Embodiment 14 or Embodiment 15, wherein the antibody in the second antibody conjugate is derived from an animal species other than the species from which the milk sample was collected. Embodiment 17 The device according to any one of Embodiments 14 to 16, wherein the detection agent bound to the antibody in the second antibody conjugate is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules. Embodiment 18 The device according to Embodiment 17, wherein the detection agent bound to the antibody in the second antibody conjugate comprises metallic gold nanoparticles or nanoshells. Embodiment 19 The device according to any one of Embodiments 1 to 18, wherein the strip is formed from nitrocellulose. Embodiment 20 The device according to any one of Embodiments 1 to 19, wherein the milk sample is derived from an animal selected from the group consisting of dogs, cats, horses, goats, sheep, or animals of the Bovidae family. Embodiment 21 The device according to any one of Embodiments 1 to 20, wherein the milk sample is derived from an animal of the Bovidae family. Embodiment 22 A method for detecting lipoteichoic acid (LTA) as a mastitis gram-positive bacterium identifier in an animal milk sample, comprising using the device described in any of Embodiments 1 to 21. Embodiment 23 A method for detecting lipoteichoic acid (LTA) as an identifier for mastitis gram-positive bacteria, the method comprising contacting a milk sample from an animal with a conjugate containing an anti-LTA antibody conjugated to a detection agent, thereby forming an antibody-antigen complex between the anti-LTA conjugate and LTA present on gram-positive bacteria in the sample; capturing the formed antibody-antigen complex with an anti-LTA antibody; and detecting the captured complex. Embodiment 24 The method according to Embodiment 23, wherein the milk sample is concentrated for bacterial cells. Embodiment 25 The method according to Embodiment 23 or Embodiment 24, wherein the anti-LTA antibody in the conjugate and the anti-LTA antibody used to capture the antibody-antigen complex are monoclonal antibodies. Embodiment 26 The method according to any one of Embodiments 23 to 25, wherein the detection agent conjugated to the anti-LTA antibody is selected from the group comprising metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules. Embodiment 27 The method according to any one of Embodiments 23 to 26, wherein the detection agent comprises metallic gold nanoparticles or nanoshells. The present invention will be further described with reference to the following embodiments. It will be understood that the claimed invention is not intended to be limited in any way by these embodiments. [Examples]

[0054] Example 1 - Milk Sample Concentration Method Typically, lateral flow testing alone is insufficient to detect the low bacterial load of approximately 100 CFU / mL associated with some mastitis milk samples. Given this situation, a milk sample preparation method that is suitable for dairy farm work and the daily activities of staff is desirable.

[0055] This embodiment describes the development of a concentration method. This method is based on concentrating bacterial cells from milk samples to a detectable level and then detecting them with a Gram-positive specific LF test. Milk samples from mastitis-prone cows are expected to be collected before milking according to NMC guidelines, mixed with concentrated broth, and incubated at 37°C for 7 hours. Finally, aliquots of the concentrated milk samples are tested with an LF test before the next milking (8-12 hours) to enable diagnosis and appropriate antibiotic treatment.

[0056] Five growth media were evaluated: (1) Todd-Hewitt broth (THB), (2) THB with colistin and nalidixic acid (LIM broth), (3) Brain Heart Infusion (BHI) broth, (4) Nutrient broth, and (5) Tryptic Soy Broth (TSB). The milk concentration method was tested by incubating different ratios of milk samples with different growth media at 37°C. After 7 hours of incubation, a mixture of 1.0 mL of milk sample and 1.0 mL of THB supported higher bacterial growth but also produced undesirable high nonspecific binding (NSB) on the LF dipstick. This high NSB was also observed in the other four media, but they supported only relatively low bacterial growth. Removal of the beef heart infusion component from THB significantly eliminated NSB, and bacterial growth was shown to be comparable to that observed with THB.

[0057] Based on the above findings, a concentrated broth specifically for testing was prepared to reduce NSB. This broth contains Millipore Sigma peptone special (20.0 g / L), dextrose (2.0 g / L), sodium chloride (2.0 g / L), disodium phosphate (0.4 g / L), dibasic sodium phosphate (0.4 g / L), sodium nalidixate (0.03 g / L), and sodium carbonate (2.5 g / L). All culture medium components are readily available from commercial sources.

[0058] Example 2 - Selection of Key Reagents The key reagents selected for the development of this assay are preferably those that detect a wide range of target Gram-positive bacteria or related antigens in milk samples. Three different bacterial biomolecules were selected to generate monoclonal antibodies (mAbs). Based on their reactivity with target Gram-positive bacteria, a commercially available anti-lipoteichoic acid (LTA) mAb (QED Bioscience Inc., California) was selected for the development of the LF assay (Table 1).

[0059] [Table 1]

[0060] For the development of the LF test, we selected a monoclonal anti-LTA antibody (class IgG1) (catalog number: 15711) commercially available from QED Bioscience Inc. (San Diego, California). This antibody was produced against Staphylococcus epidermidis Hay strain (ATCC #55133). The host animal species was mouse. In addition to Staphylococcus epidermidis Hay strain, the anti-LTA antibody reacts with clinical strains of Staphylococcus epidermidis (types I, II, and III), Staphylococcus aureus strains 5 and 8, Streptococcus pyogenes, Streptococcus fecal, and lipoteichoic acid of Streptococcus mutans. It does not react with Staphylococcus aureus peptidoglycan or peptidoglycan-rhamnose, nor with pneumococcal polysaccharides. This antibody does not cross-react with Escherichia coli (E. coli) or Haemophilus influenzae type B.

[0061] Example 3 - Development of a preliminary lateral flow dipstick This example describes one aspect of the device and method of the present invention. A lateral flow-based sandwich-type immunoassay format was selected. The test consists of nitrocellulose membranes laminated on an adhesive backing card. Both ends of the nitrocellulose membranes overlap with adjacent conjugate pads and adjacent absorbent pads. The sample pad overlaps with a conjugate pad. After all reagents have been deposited on each membrane, the card is cut into strips approximately 5 mm wide.

[0062] The test strip consists of the components disclosed in Table 2. Immunoassays were performed on clinical milk samples enriched with bacterial cells according to the method described in Example 1 and a preferred enrichment medium, using the test protocol described in Example 4.

[0063] [Table 2]

[0064] The conjugate can be prepared using a standard antibody conjugation method for colloidal gold. Mix the anti-LTA antibody with a buffer of the desired pH. Add the colloidal gold (nanoComposix, San Diego, California) to the antibody and mix for 5-10 minutes. Add a second basic buffer to the conjugate to raise the pH, and block the conjugate by adding BSA.

[0065] To prepare the control conjugate, adjust colloidal gold to the desired pH. Add a saturated amount of protein (e.g., chicken IgY) at 20-100 μg / ml to the gold and incubate for 10 minutes. Next, add a BSA blocker to the gold and incubate for another 10 minutes. Add a stabilizer buffer containing BSA and sucrose to the conjugate.

[0066] The conjugate is mixed with a conjugate diluent consisting of a surfactant, buffer, sucrose, and BSA at an optimized critical OD. The conjugate is sprayed onto a conjugate pad using an air jet sprayer.

[0067] The test line and control line reagents, anti-LTA antibody, and donkey-anti-chicken IgY are each diluted to optimized critical concentrations in a deposition buffer containing stabilizing sugars. The reagents are deposited onto nitrocellulose using a high-precision fluid handler capable of spraying microvolumes. The cards are stored at relative humidity <30%.

[0068] Example 4 - Test Protocol Add 1.0 mL of breast milk sample to a graduated test tube containing 1.0 mL of concentrated broth. • Incubate a test tube containing a mixture of milk and concentrated broth at 37°C for 7 hours in a standard bacteriology incubator (no special milk sample preparation equipment is required). After incubation, test the concentrated milk sample (200-250 μL) by placing the LF dipstick into the concentrated milk sample. • LTA antigen, which is abundantly expressed on the surface of Gram-positive bacteria in the concentrated milk sample, migrates to the conjugate pad and reacts with anti-LTA antibody conjugated to colloidal gold. A complex is formed between the anti-LTA antibody conjugate and the LTA in the sample. The formed complex migrates across the nitrocellulose, and the complexed LTA is immobilized by the anti-LTA antibody deposited on the test line. As colloidal gold particles accumulate on the test line, a visible red line is formed if LTA antigen is present, indicating a positive result. If LTA antigen is not present in the sample, the gold conjugate does not immobilize on the test line and continues to migrate to the absorption pad. The absence of a red line on the test line means that the sample is negative for LTA and does not contain Gram-positive bacteria. In one embodiment, a second conjugate (control conjugate) deposited on the conjugate pad consists of chicken IgY conjugated to colloidal gold. The control conjugate migrates across the nitrocellulose and is immobilized by anti-chicken IgY antibody on the second reaction line (control line). The accumulation of colloidal gold control conjugate particles forms a red control line. Since the control line is a procedural control, it indicates that the test was performed and flowed correctly. • Visually read the test strip after 10 minutes.

[0069] Example 5 - Sample Collection The sample set consisted of the following, which were received from different dairy farms after being stored on ice overnight. • Clinical milk samples (n=108) collected from infected cow's milk plots containing ≥100 CFU / mL of target Gram-positive bacteria. • Clean, culture-negative milk samples (n=103) collected from cows that did not develop mastitis. The mastitis-causing pathogens present in each clinical milk sample were counted and identified by direct culture and subsequent MALDI-ToF analysis.

[0070] Example 6 - Test Results - Limit of Detection (LoD) of Concentrated Lateral Flow Assay The limit of detection (LoD) of the alpha prototype test was estimated using milk samples spiked with various concentrations (CFU / mL) of each target Gram-positive bacterium. Each concentrated sample was tested 10 times, and the concentration (CFU / mL) at which the assay was visually positive in 9 out of 10 trials was considered the LoD specific to each target bacterium.

[0071] [Table 3]

[0072] Example 7 - Estimation of preliminary diagnostic ability After concentration at 37°C for 7 hours, aliquots of each concentrated milk sample were tested in three different lots of LF dipsticks. Visual results were recorded after 10 minutes. Culture results were used as reference. • Test strips from lots 1 and 2 detected the target Gram-positive bacteria in 105 / 108 clinical breast milk samples. Estimated diagnostic sensitivity and specificity were 97.2% (95% CI: 92.1–99.4%) and 95.1% (95% CI: 89.0–98.4%), respectively (Table 4). • Test strips from lot number 3 yielded 104 / 108 positive results. The estimated sensitivity and specificity of the diagnosis were 96.3% (95% CI: 90.8–99.0%) and 96.1% (95% CI: 90.4–98.9%), respectively (Table 4).

[0073] Lots 1 and 2 showed three false negatives and five false positives, while lot 3 showed four false negatives and four false positives.

[0074] [Table 4]

[0075] Example 8 - Equipment In one embodiment, the interpretation of the LF test results is based on visual evaluation by a human technician. It is designed for easy setup, short execution time, and easy readout. While the kit does not require a complex lateral flow reader or associated software, the LF test can also be based on evaluation of test results using a lateral flow reader, if desired. However, the milk sample concentration process requires a simple bacteriological incubator (e.g., a heat block) capable of maintaining a temperature of 37°C. Dairy farms practicing cultivation on-site have these incubators in their dairy office or dairy laboratory. In one embodiment, a portable incubator can also be provided to dairy farms that do not have one. [Explanation of Symbols]

[0076] (1) Lateral flow device (2) Sample pad (3) Conjugate pad (4) Membrane (5) Test line (6) Reference line (7) Wicking pad (8) Adhesive bands / cover tapes

Claims

1. A method for diagnosing a Gram-positive case of mastitis, comprising: providing a milk sample from an animal; concentrating the milk sample with respect to bacterial cells by combining the milk sample with a growth medium; then growing the bacterial cells in the sample to a detectable level by culturing the sample under appropriate culture conditions; contacting the concentrated milk sample with a conjugate containing an anti-lipoteichoic acid (LTA) antibody conjugated to a detection agent, thereby forming an antibody-antigen complex between the anti-LTA conjugate and the LTA present on the Gram-positive bacteria in the sample; capturing the formed antibody-antigen complex with an anti-LTA antibody; and detecting the captured complex.

2. The method according to claim 1, wherein the method can detect Gram-positive bacteria of 100 CFU / mL or more.

3. The method according to claim 1 or claim 2, wherein the anti-LTA antibody in the conjugate and the anti-LTA antibody used to capture the antibody-antigen complex are monoclonal antibodies.

4. The method according to any one of claims 1 to 3, wherein the detection agent conjugated to the anti-LTA antibody is selected from the group consisting of metal nanoparticles or nanoshells, nonmetal nanoparticles or nanoshells, enzymes, and fluorescent molecules.

5. The method according to any one of claims 1 to 4, wherein the detection agent comprises metallic gold nanoparticles or nanoshells.

6. The method according to any one of claims 1 to 5, wherein the milk sample is derived from an animal selected from the group consisting of dogs, cats, horses, goats, sheep, or animals of the Bovidae family.

7. The method according to claim 6, wherein the milk sample is derived from an animal of the Bovidae family.

8. The method according to claim 7, wherein the animal of the Bovidae family is a cow.

9. The method according to claim 8, wherein the milk sample is collected from the mastitis-inducing milk area of ​​a bovine under sterile conditions prior to milking.

10. The method according to claim 1, wherein the concentration step includes mixing a milk sample with a growth medium, and then culturing the milk sample at 37°C for 6-7.5 hours to produce a concentrated milk sample.

11. The method according to claim 10, wherein an equal volume of milk sample is mixed with a growth medium before incubating the milk sample.

12. The method according to any one of claims 9 to 11, wherein an aliquot of a concentrated milk sample is tested before the cow's next milking (8 to 12 hours).

13. The method according to any one of claims 1 to 12, wherein the method is performed using a lateral flow device.

14. The method according to claim 13, wherein the lateral flow device comprises: a) a strip, wherein the strip is formed of a material that allows capillary flow of fluid along a portion of the strip; b) a sample pad, wherein the sample pad is positioned proximal to one end of the strip to receive a milk sample; c) a conjugate pad, wherein the conjugate pad is positioned within the strip, and during operation, the sample flows through the strip from the sample pad to the conjugate pad by capillary action, recruiting a conjugate containing an anti-LTA antibody conjugate bound to a detection agent, which is contained within the conjugate pad; and d) a test line, wherein the test line contains an anti-LTA antibody immobilized within the strip along a band positioned substantially perpendicular to the direction of sample flow along the strip, so that when a complex formed comprising the recruited anti-LTA antibody conjugate and LTA in the sample comes into contact with the immobilized anti-LTA antibody in the test line, the presence of LTA in the sample is indicated by a visible color change.

15. The method according to claim 14, wherein the lateral flow device is a dipstick.

16. The method according to claim 15, comprising immersing the sample pad portion of a dipstick in a concentrated milk sample.

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