WO2019216775A1 - Device for detection of antibodies to a pathogen - Google Patents

Device for detection of antibodies to a pathogen Download PDF

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Publication number
WO2019216775A1
WO2019216775A1 PCT/NZ2019/050051 NZ2019050051W WO2019216775A1 WO 2019216775 A1 WO2019216775 A1 WO 2019216775A1 NZ 2019050051 W NZ2019050051 W NZ 2019050051W WO 2019216775 A1 WO2019216775 A1 WO 2019216775A1
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Prior art keywords
sample
antibodies
pathogen
detection
conjugate
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PCT/NZ2019/050051
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French (fr)
Inventor
Anton Pernthaner
Michael Patrick SEAWRIGHT
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Koru Diagnostics Limited
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Priority to PCT/NZ2019/050051 priority Critical patent/WO2019216775A1/en
Publication of WO2019216775A1 publication Critical patent/WO2019216775A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56938—Staphylococcus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56944—Streptococcus
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N2021/7756—Sensor type
    • G01N2021/7759—Dipstick; Test strip
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles

Definitions

  • the invention relates to a device for the detection of antibodies to a pathogen in a bodily fluid of an animal.
  • the invention relates to a lateral flow device that can be used for the rapid determination of infection in an animal, for example mastitis in a bovine cow.
  • LF tests are membrane-based immunoassay tests.
  • LF devices have a test strip consisting of a membrane, such as porous paper or a sintered polymer, which enables capillary flow of a sample and detection reagents.
  • the membrane also holds substances that form test and control lines.
  • One end of the membrane is fitted with a sample pad that contacts another pad used to store the detection conjugate.
  • the other end of the membrane has a wicking pad that holds fluids and reagents that have migrated via capillary action through the membrane.
  • LF devices exist for a wide range of targets including the detection of infectious agents, metabolic molecules, antibodies, toxins and drugs for use in human and veterinary applications. Products for detecting targets that are present in the environment or in agriculture are also in use.
  • LF tests are typically modelled on existing immunoassay formats and can be sandwich assays or competitive assays. Many assay variations are possible, but a positive signal is usually achieved by the specific accumulation of a detection complex at the test line.
  • LF devices usually need to be specifically developed in an iterative process for the desired application.
  • Each component of the test strip of a LF device requires specific evaluation followed by testing to determine the optimal combination of components.
  • the identification of the most suitable membrane is an important step in the test strip development as this determines the capillary flow properties that need to be compatible with the type of sample to be tested.
  • Samples therefore may need to be pre-treated in order to achieve the required capillary flow properties. For instance, viscous or highly concentrated samples, such as serum, may need to be diluted in a suitable buffer, corpuscular bodies such as cells and aggregates may need to be removed by centrifugation or fat globules may need to be removed by flotation for the sample to be amenable to capillary flow.
  • Sample pre-treatment can be a time-consuming process and often requires processing in a laboratory. This substantially extends the time from sampling to obtaining results which delays important treatment decisions. The delay in commencing treatment may be detrimental to the outcome of a disease. It would therefore be an advantage if some sample pre-treatment steps could be directly integrated in the assay.
  • Mastitis is a world-wide problem in every dairy industry. Approximately 15% of cows in New Zealand develop clinical mastitis. Current diagnostic procedures typically consist of aseptic sampling of milk in a sterile vessel, transportation to a specialised laboratory, followed by bacteriological examination. The entire process takes a minimum of 48 hours, but more often 72 hours or more are required before culture results are available for determining the most suitable treatment option. This delay in identifying the causal agent can mean the course of treatment is inappropriate or ineffective, the infection is prolonged, and farmers suffer higher economic losses than they would have otherwise. A rapid point-of-care test would be advantageous because informed treatment and management decisions could be made immediately after detection of the causal agent.
  • milk samples in LF assays are inherently difficult because milk contains cells and fat that may reduce capillary flow and therefore the effectiveness of the assay.
  • mastitic milk has highly elevated somatic cell counts (SCC), and may contain debris and bacterial conglomerates that prevent effective capillary flow.
  • the applicant has now developed a rapid and effective LF device for the testing of bovine milk samples in order to determine whether the cow that produced the sample has a specific mastitis infection.
  • the test is based on the use of Immunoglobulin A (IgA) antibodies for the detection of bacterial pathogens known to cause mastitis. It will be appreciated that the principle of using IgA antibodies to detect the presence of a pathogen in a sample may also be employed for the detection of other animal and human diseases.
  • IgA Immunoglobulin A
  • the invention provides a device for the detection of antibodies to a bacterial pathogen in a bodily fluid of an animal, the device comprising :
  • a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
  • conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti-IgG, anti-M or anti-IgA antibodies specific to the animal, which antibodies are bound to a detection agent;
  • a detection band comprising the pathogen, or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal;
  • wicking pad for receiving and retaining sample after passing through the detection band.
  • the antibodies are Immunoglobulin A (IgA), Immunoglobulin G (IgG), or Immunoglobulin M (IgM) antibodies.
  • the antibodies are IgA antibodies.
  • the antibodies are IgG antibodies.
  • the anti-Ig, anti-IgG, anti-M or anti-IgA antibodies comprising the conjugate are partially or fully substituted for one or more immunoglobulin binding proteins, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
  • the conjugate comprises Protein G or a Protein A/G fusion protein.
  • the device comprises
  • a strip formed of a material enabling capillary flow of fluid along a portion of the strip
  • sample pad located proximal to one end of the strip for receiving a sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
  • a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti-IgG, anti-M or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins, or a combination thereof, which antibodies or immunoglobulin-binding proteins are bound to a detection agent; d) a detection band comprising the pathogen, or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
  • the one or more components removed from the sample by filter membrane of the sample pad are cellular material, fats, or particulate matter.
  • the sample pad is a CytoSepTM sample pad.
  • the sample pad enables the sample to be applied to the strip without prior processing of the sample.
  • the strip may be formed from any suitable material that allows capillary transport of a fluid.
  • the strip is formed from a nitrocellulose material.
  • the animal is a human or is a bovine, ovine, caprine, equine, canine, feline, porcine, cervine, camelid or galline animal.
  • the animal is a human.
  • the animal is a bovine animal.
  • the bovine animal is a lactating bovine cow.
  • the bodily fluid is selected from the group comprising blood, blood serum, blood plasma, milk, mucous, saliva, sputum, semen, sweat and urine.
  • the bodily fluid is milk from a lactating bovine cow.
  • the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coii, Streptococcus dysgaiactiae, Streptococcus agalactiae, and Mycoplasma bovis.
  • the detection agent is selected from the group comprising metallic nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, and fluorescent molecules.
  • the detection agent comprises nanoparticles or nanoshells of metallic gold.
  • the pathogen or antigen of the pathogen is native. In other embodiments the pathogen or antigen of the pathogen is recombinant.
  • the visible colour change is the appearance of a black, red, green, blue or yellow colour.
  • the antibodies of the conjugate are chicken, goat, sheep or rabbit polyclonal antibodies or monoclonal antibodies. In some embodiments, the antibodies are anti-bovine Ig, IgG, IgM or IgA antibodies.
  • the device may be used for the detection of antibodies to two or more pathogens in a bodily fluid of an animal.
  • the device may comprise two or more detection bands, each detection band comprising a pathogen, or an antigen of a pathogen immobilised within the strip.
  • the antibodies of a pathogen in one detection band exhibit no cross-reactivity with antigens of a pathogen in another detection band.
  • a device particularly suited to the detection of early stage mastitis wherein the device is configured to enable detection of IgG antibodies.
  • the conjugate comprises anti-IgG antibodies, such as anti-bovine IgG antibodies.
  • the device comprises one or more immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
  • an LF device comprising anti- IgG antibodies is provided.
  • an LF device comprising an immunoglobulin-binding protein capable of binding IgG antibodies is provided. It is contemplated such a device is particularly suitable for the detection of early stage, and in some embodiments, preclinical or subclinical, mastitis.
  • a device particularly suited to the detection of late stage mastitis, persistent mastitis, or treatment-resistant mastitis wherein the device is configured to enable detection of bovine IgA antibodies.
  • the conjugate comprises anti-IgA antibodies, such as anti-bovine IgA antibodies.
  • the device comprises one or more immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
  • an LF device comprising anti- bovine IgA antibodies is provided.
  • an LF device comprising an immunoglobulin-binding protein capable of binding IgA antibodies is provided. It is contemplated such a device is particularly suitable for the detection of late stage mastitis, persistent mastitis, or treatment-resistant mastitis.
  • the invention provides a method for the detection of antibodies to a pathogen in a bodily fluid of an animal using a device of the invention.
  • the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coii, Streptococcus dysgaiactiae, Streptococcus agalactiae, and Mycoplasma bovis.
  • the detection of antibodies to Staphylococcus aureus and/or Streptococcus uberis in a sample of milk form a lactating bovine cow indicates the presence of mastitis in the cow.
  • the invention relates to a method of detecting mastitis in a subject.
  • the method comprises obtaining a biological sample from the subject, optionally processing the sample to provide a fluid sample capable of application to a device as described herein, and applying the sample to the device.
  • the method comprises contacting a fluid sample obtained from a subject, for example milk from a lactating mammal, to a device as described herein.
  • the method is a method for detecting mastitis in a lactating bovine cow using a device as described herein.
  • the method comprises obtaining milk from a lactating bovine, optionally defatting the milk, and applying the milk to the device, wherein the detection of antibodies to a pathogen, for example as indicated by a visible colour change or detection of a fluorescent signal, is indicative of mastitis in the bovine.
  • the method is a method of detecting early stage mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgG antibodies.
  • said conjugate comprises anti-IgG antibodies.
  • said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgG antibodies, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
  • the conjugate comprises Protein G or a Protein A/G fusion protein.
  • the method is a method of detecting preclinical or subclinical mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgG antibodies.
  • said conjugate comprises anti-IgG antibodies.
  • said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgG antibodies, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
  • the conjugate comprises Protein G or a Protein A/G fusion protein.
  • the method is a method of detecting late stage mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgA antibodies.
  • the conjugate comprises anti-IgA antibodies.
  • said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgA antibodies.
  • Another aspect of the present invention relates to a diagnostic kit comprising an LF device as described herein.
  • the diagnostic kit comprises an LF device, wherein the pathogen or an antigen of the pathogen at the detection band is or is from S. aureus or S. uberis.
  • the kit comprises a composition comprising one or more antibodies specific to the same pathogen as is present at the detection band or from which the antigen present at the detection band is derived.
  • Such an antibody composition is capable of use as a positive control.
  • a kit for the detection of S. aureus or S. uberis is provided, said kit comprising an LF device as described herein wherein the pathogen is S. aureus or S. uberis, and the kit comprises one or more antibodies reactive to S. aureus or S. uberis as a positive control.
  • a negative control may also be included, comprising an antibody that is not reactive to the pathogen at the detection band of from which the antigen present at the detection band is derived.
  • Figure 1 shows Mean ⁇ SEM of S. aureus (S.a.) and S. uberis (S.ub) and their negative controls (neg). Letters in common show highly significant differences (P ⁇ 0.001).
  • Figure 2 is a schematic representation of a LF device of the invention.
  • Figure 3 is a schematic representation of a test sample that is positive for S. uberis and S. aureus IgA.
  • Figure 4 is a schematic representation of a test sample that is positive for S. uberis IgA.
  • Figure 5 is a schematic representation of a test sample that is positive for S. aureus IgA.
  • Figure 6 is a schematic representation of a test sample that is negative for S. uberis and S. aureus IgA.
  • immunoglobulin or "Ig” means an antibody which is a large Y-shaped protein produced mainly by plasma cells used by the immune system to neutralise pathogens such as pathogenic bacteria and viruses.
  • Immunoglobulin A or "IgA” means a subclass of Ig that has an important role in the immune function of mucosal surfaces including the udder of bovine cows.
  • Immunoglobulin G or "IgG” means a subclass of Ig that is mostly found in circulating bodily fluids such as blood and lymph.
  • Immunoglobulin M or "IgM” means a subclass of Ig that is produced mainly in the spleen and is the first antibody to appear in response to initial exposure to an antigen.
  • antigen means a molecule having distinct surface features or epitopes capable of stimulating a specific immune response.
  • Antibodies immunoglobulins
  • Antigens maybe proteins, carbohydrates or lipids, although only protein antigens are classified as immunogens because carbohydrates and lipids cannot elicit an immune response on their own.
  • nanoparticles means uniform particles having a size of 1-200 nm.
  • nanoshells means nanoparticles that consist of a core and a metallic shell (usually gold).
  • the invention is based on the applicant's development of a rapid and effective LF device for the testing of bovine milk samples in order to determine whether the cow that produced the sample has a mastitis infection.
  • the test is based on the use of antibodies for the detection and identification of bacterial pathogens known to cause mastitis. It will be appreciated that the principle of using antibodies to detect the presence of a pathogen in a sample may also be employed for the detection of other animal and human diseases.
  • Antibodies can be detected using several immunological assays such as ELISA, Western Blots and Lateral Flow tests.
  • ELISA tests have the advantage of being fast and the tests are amenable to high throughput and automation, while LF tests are especially suitable as point-of-care tests.
  • Immunoassays are highly suited to the diagnosis of chronic and subclinical diseases where antibody maturation has already occurred and antibody subclasses IgG and/or IgA are present. In contrast, IgM is produced shortly after infection. Therefore, the selection of antibody subclass for a specific test is critical and dependent on many factors such as pathogen, type and severity of infection.
  • the device of the invention detects antibodies to a bacterial pathogen in a bodily fluid of an animal.
  • the device comprises: a) a strip formed of a material enabling capillary flow of fluid along a portion of the strip;
  • sample pad located proximal to one end of the strip for receiving a sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
  • conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti- IgG, anti-M or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins, or a combination thereof, which antibodies or immunoglobulin-binding proteins are bound to a detection agent;
  • a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
  • the device comprises:
  • a strip formed of a material enabling capillary flow of fluid along a portion of the strip
  • sample pad located proximal to one end of the strip for receiving a sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
  • conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti- IgG, anti-IgM or anti-IgA antibodies specific to the animal, which antibodies are bound to a detection agent;
  • a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
  • the sample pad not only receives sample fluid for testing, but removes components from the fluid that might otherwise impeded capillary flow of the fluid through the strip or adversely affect detection of the antibodies at the detection band.
  • the components that may be removed by the sample pad include cellular material, fats, and particulate matter.
  • the sample pad provides an advantage because the milk would otherwise need to be treated or processed in some way before being subjected to immunoassay, such as an ELISA test, for the determination of whether the cow that poduced the milk had a specific mastitis infection.
  • immunoassay such as an ELISA test
  • the sample pad avoids any requirement for pre-treatment of the sample.
  • One specifically contemplated option for the sample pad as a CytoSepTM sample pad, but any other suitable sample pad may be used.
  • Nitrocellulose was found to be a suitable material from which the strip is made. Other materials may also be suitable provided they allow the desired capillary flow rate and enable suitable detection sensitivity.
  • Specifically contemplated strips (also referred to as membranes) were identified as FF80 HP and HF75 membranes.
  • the device of the invention may be used for the detection of a wide range of pathogen infections in humans and other animals.
  • the device is used for determining whether a bovine cow has a mastitis infection by detecting the presence of antibodies to a bacterial pathogen, such as S. aureus or S. uberis in a milk sample obtained from the cow.
  • the device is used for determining whether a bovine animal is infected with M. bovis.
  • the animal may alternatively be an ovine, caprine, equine, canine, feline, porcine, cervine, camelid or galline animal.
  • any bodily fluid that contains antibodies to a bacterial pathogen may be used as the fluid sample for testing using the device of the invention.
  • fluids include blood, blood serum, blood plasma, milk, mucous, saliva, sputum, semen, sweat and urine.
  • milk from a lactating bovine is the preferred choice of fluid for testing for bacterial pathogens associated with mastitis.
  • the device of the invention is capable of being used for detecting any bacterial, viral, fungal or parasitic pathogen provided the fluid sample tested contains antibodies to the pathogen.
  • pathogens include, but are not limited to, S. aureus, S. uberis, E. coii, S. dysgalactiae, S. agalactiae, and M. bovis.
  • the detection agent is any agent that provides a detectable change when contacted by antibodies present in the fluid sample.
  • the detection agent must incorporate or be bound to a moiety capable of binding an antibody from the animal from which the sample is obtained, such as anti-Ig, anti-IgG, ant-IgM or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins capable of binding antibodies from the animal.
  • the detectable change may be a visible colour change or observable fluorescence, or any other suitable change.
  • the detection agent is nanoparticles or nanoshells conjugated to anti-Ig, anti-IgG, anti-IgM or anti-IgA antibodies specific to the animal.
  • the detection agent is nanoparticles or nanoshells conjugated to immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof.
  • the nanoparticles or nanoshells may be metallic or non- metallic, but metallic gold nanoparticles or nanoshells are specifically contemplated.
  • the detection agent may be gold nanoparticles bound to polyclonal rabbit anti- bovine IgA antibody or polyclonal chicken anti-bovine IgG antibody.
  • the detection agent may be gold nanoparticles bound to Protein G, or to Protein A, or to a Protein A/G fusion protein.
  • the detection agent may be one or more enzymes or fluorescent molecules.
  • the detection band of the strip comprises a pathogen or an antigen of a pathogen immobilised within the strip.
  • Some devices of the invention may comprise two or more detection bands.
  • each band comprises cells of the pathogen, e.g. cells of S. aureus or S. uberis.
  • the cells would usually be UV-attenuated or sonicated and lysed, and may have been pre-treated with blocking serum to prevent non-specific binding of antibodies in the sample or on the detection reagent.
  • FIG. 2 shows a LF device (1) of the invention.
  • the device (1) has a specific sample pad (2) that makes milk (and other types of fluids) amenable to capillary flow, a conjugate pad (3) to which a detection agent is immobilised, a membrane (4), and a wicking pad (5) for receiving and holding fluid that has travelled by capillary flow from the sample pad (2) and through the conjugate pad (3) and the membrane (4).
  • a detection band (6) is shown which comprises immobilised S. uberis antigens.
  • Detection band (7) comprises immobilised S. aureus antigens.
  • Band (8) is a positive control band. Shown (schematically) on the conjugate pad (3) is a detection agent (9) representing a gold nanoparticle to which a Y- shaped antibody is bound.
  • the fluid then proceeds to detection band (7).
  • the detection agent binds to the S. aureus antigens in the detection band (7) and, again, the accumulation of gold nanoparticles gives a colour change.
  • the device (1) can be used for the detection of two pathogens that may be present in the fluid sample, in this case both S. uberis and S. aureus.
  • the antibodies of the pathogen in detection band (6) and the antibodies of the pathogen in detection band (7) can be selected so that there is no cross-reactivity between the two.
  • Figure 3 shows a LF device (1) of the invention where a test sample has been found to be positive for both S. uberis and S. aureus IgA. This is indicated by the dark colour of both detection band (6) and detection band (7), and the positive control band (8).
  • Figure 4 shows a LF device (1) of the invention where a test sample has been found to be positive for S. uberis IgA only. This is indicated by the dark colour of detection band (6), no colour change at detection band (7), and the dark colour of the positive control band (8).
  • Figure 5 shows a LF device (1) of the invention where a test sample has been found to be positive for S. aureus IgA only. This is indicated by no colour change at detection band (6), the dark colour of detection band (7), and the dark colour of the positive control band (8).
  • Figure 6 shows a LF device (1) of the invention where a test sample has been found to be negative for both S. uberis and S. aureus IgA. This is indicated by no colour change at both detection band (6) and detection band (7), and the dark colour of positive control band (8).
  • Example 1 shows that infected animals have significantly elevated IgA antibody titres when compared to culture-negative samples. This provides strong evidence that IgA is indicative of the pathogen that causes mastitis, and that IgA is a suitable antibody subclass for detection in a LF test.
  • Example 2 shows that the identification of the most suitable membrane is critical for the performance of the LF test.
  • Slow flowing membranes such as FF170 HP, that have a very small pore size are not suitable for milk samples, as capillary flow is blocked by components usually found in milk.
  • fast flowing membranes such as Prima 40
  • the most suitable membranes for milk samples were identified as FF80 HP and HF75 which proved to be sensitive and provided the desired capillary flow properties for milk samples.
  • Example 3 shows that better sensitivity can be achieved by using 150 nm nanoshells in comparison with 40 nm nanoparticles.
  • Example 4 shows that an anti-rabbit antibody serves as an ideal positive control by binding directly to the host antibody that is bound to the detection particle.
  • the detection particle consisted of a rabbit anti-bovine IgA antibody conjugated to 150 nm nanoshells. Bovine colostrum was found to have insufficient IgA levels to consistently give strong positive signals.
  • Example 5 shows that the selection of the most suitable conjugate pad is important for the overal performace of the device. The strongest signals were obtained for conjugate pads 8951, T5NM and 6613H for milk samples while milk failed to elute the conjugate from pads 8649 and 6613.
  • Example 6 shows that a amount of lysed bacterial antigen immobilised on the membrane should be a minimum of 1 pi OD600 0.55 to produce strong signals. Because S. aureus is known for its ability to non-specifically bind to antibodies, a specific S. aureus strain was found to have a reduced ability for non-specific binding. This strain was therefore used for the test development.
  • Milk samples from cows with elevated cell counts were collected from four farms in New Zealand. Milk samples from the first three farms were used for the development of antibody subclass specific ELISA assays for IgG, IgA and Ig.
  • Antigen-specific IgA reactivity was tested with S. aureus or S. uberis antigens in 64 samples with known elevated somatic cell counts (SCC) and the test evaluated against bacterial culture results. The culture results showed that 17 samples were positve for S. aureus, 16 samples were positive for S. uberis, 9 samples had mixed cultures of S. aureus and S. uberis, and 40 samples were culture-negative.
  • Figure 1 demonstrates that Mean ⁇ SEM of ODs for S. aureus culture positive samples was significantly higher than for S. aureus culture negative samples (P ⁇ 0.001). Similarly, Mean ⁇ SEM of ODs for S. uberis culture positive samples was significantly higher than for S. uberis culture negative samples (P ⁇ 0.001). The statistical difference was calculated using the non-parametric Mann-Whitney test. From these studies, antigen-specific IgA was identified as a reliable marker for S. aureus and S. uberis mastitis.
  • FF80 HP and HF75 membranes were then assessed using milk samples with high and low levels of antibodies for S. aureus and S. uberis, that were also culture-positive for both pathogens, a sample that was antibody and culture-positive for S. aureus, and a negative sample.
  • Rabbit anti-bovine IgA antibodies conjugated to functionalised 150 nm nanoshells were used for detection. The results are shown in Table 2. FF80 and HF75 were found to be equally sensitive for S. aureus, but FF80 was found to be more sensitive for S. uberis.
  • Colostrum is known to contain IgG, but also IgA antibodies.
  • the amount of IgA in 0.5 pi of colostrum was found to be at the limit of detection and gave inconsistent results.
  • An anti-rabbit antibody that would directly bind to the antibody 150 nm nanoshell conjugate was tested and gave consistently strong positive signals (Table 3), then 1 pi and 0.5 mI of an anti- rabbit antibody and 1 mI of colostrum were immobilised on HT75 membranes and tested for binding to rabbit anti-S. aureus 150 nm nanoshell conjugates.
  • conjugate pads were compared for their ability to hold and release dried conjugate (Rb anti-bovine IgA 150 nm nanoshells).
  • Conjugate was diluted 1 : 10 in 2 mM borate buffer substituted with 10% sucrose.
  • Conjugate pads were saturated with the reagent, dried for 2 h at 37°C and then fitted on to FF80 membranes. CytosepTM 1663 and a wicking pad were fitted at the proximal and distal ends of the strips respectively. Comparisions were made with a milk sample known to be antibody-positive for S. aureus which was deposited on the sample pad. The following conjugate pads were tested : 8649, 8951, 6615, 6613, T5NM, 6613H.
  • S. aureus and S. uberis were isolated from milk samples and propagated on sheep blood agar plates. Overnight cultures were taken off plates, resuspended in PBS supplemented with 0.1% sodium azide and sonicated for 2 min to solubilise bacteria. The S. aureus milk strain was found to non-specifically bind to antibodies in milk in ELISA assays. A S. aureus strain (strain A) exhibited a lower ability for non-specific binding and consequently this strain was used in LF assays. S. aureus lysates with an OD600 nm of 0.55 were found to be suitable for LF. 0.5 pi of the lysate was used for capture (Table 5). The test was performed with a milk sample that was ELISA positive for IgA and visualisation with a rabbit- anti S. aureus 150 nm nanoshell conjugate. Table 5: Identification of optimal antigen concentrations for immobilisation on strips
  • Example 7 Assembling and testing of prototype LF test for S. aureus and S. uberis IgA
  • a prototype test strip was assembled using the following components: FF80 nitrocellulose membrane fitted with a conjugate pad 8951 (loaded with 150 nm nanoshells conjugaed to a rabbit anti-bovine IgA antibody) and CytosepTM as a sample membrane at one end.
  • the sample pad overlapped the conjugate by 2 mm and the conjugate pad the nitrocellulose membrane by 2 mm in order to facilitate capillary flow.
  • a wicking pad was fitted at the other end to promote capillary flow and to receive and hold fluid.

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Abstract

A lateral flow device for the detection of antibodies to a bacterial pathogen in a bodily fluid of an animal. The device can be used for the rapid determination of infection, for example the presence of mastitis in a bovine cow.

Description

DEVICE FOR DETECTION OF ANTIBODIES TO A PATHOGEN
TECHNICAL FIELD
The invention relates to a device for the detection of antibodies to a pathogen in a bodily fluid of an animal. In particular, the invention relates to a lateral flow device that can be used for the rapid determination of infection in an animal, for example mastitis in a bovine cow.
BACKGROUND OF THE INVENTION
The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field or in any particular jurisdiction.
Lateral flow (LF) tests are membrane-based immunoassay tests. LF devices have a test strip consisting of a membrane, such as porous paper or a sintered polymer, which enables capillary flow of a sample and detection reagents. The membrane also holds substances that form test and control lines. One end of the membrane is fitted with a sample pad that contacts another pad used to store the detection conjugate. The other end of the membrane has a wicking pad that holds fluids and reagents that have migrated via capillary action through the membrane. LF devices exist for a wide range of targets including the detection of infectious agents, metabolic molecules, antibodies, toxins and drugs for use in human and veterinary applications. Products for detecting targets that are present in the environment or in agriculture are also in use.
LF tests are typically modelled on existing immunoassay formats and can be sandwich assays or competitive assays. Many assay variations are possible, but a positive signal is usually achieved by the specific accumulation of a detection complex at the test line.
LF devices usually need to be specifically developed in an iterative process for the desired application. Each component of the test strip of a LF device requires specific evaluation followed by testing to determine the optimal combination of components. The identification of the most suitable membrane is an important step in the test strip development as this determines the capillary flow properties that need to be compatible with the type of sample to be tested. Samples therefore may need to be pre-treated in order to achieve the required capillary flow properties. For instance, viscous or highly concentrated samples, such as serum, may need to be diluted in a suitable buffer, corpuscular bodies such as cells and aggregates may need to be removed by centrifugation or fat globules may need to be removed by flotation for the sample to be amenable to capillary flow. Additional treatments such as the adjustment of pH or salt concentrations that enhance antibody/ligand binding may also be required for optimal performance of the test. Sample pre-treatment can be a time-consuming process and often requires processing in a laboratory. This substantially extends the time from sampling to obtaining results which delays important treatment decisions. The delay in commencing treatment may be detrimental to the outcome of a disease. It would therefore be an advantage if some sample pre-treatment steps could be directly integrated in the assay.
Mastitis is a world-wide problem in every dairy industry. Approximately 15% of cows in New Zealand develop clinical mastitis. Current diagnostic procedures typically consist of aseptic sampling of milk in a sterile vessel, transportation to a specialised laboratory, followed by bacteriological examination. The entire process takes a minimum of 48 hours, but more often 72 hours or more are required before culture results are available for determining the most suitable treatment option. This delay in identifying the causal agent can mean the course of treatment is inappropriate or ineffective, the infection is prolonged, and farmers suffer higher economic losses than they would have otherwise. A rapid point-of-care test would be advantageous because informed treatment and management decisions could be made immediately after detection of the causal agent.
The use of milk samples in LF assays is inherently difficult because milk contains cells and fat that may reduce capillary flow and therefore the effectiveness of the assay. Furthermore, mastitic milk has highly elevated somatic cell counts (SCC), and may contain debris and bacterial conglomerates that prevent effective capillary flow.
The applicant has now developed a rapid and effective LF device for the testing of bovine milk samples in order to determine whether the cow that produced the sample has a specific mastitis infection. The test is based on the use of Immunoglobulin A (IgA) antibodies for the detection of bacterial pathogens known to cause mastitis. It will be appreciated that the principle of using IgA antibodies to detect the presence of a pathogen in a sample may also be employed for the detection of other animal and human diseases.
It is therefore an object of the invention to provide a device for the detection of antibodies to a pathogen in a bodily fluid of an animal, or at least to provide a useful alternative to existing devices.
SUMMARY OF THE INVENTION
In one aspect, the invention provides a device for the detection of antibodies to a bacterial pathogen in a bodily fluid of an animal, the device comprising :
a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti-IgG, anti-M or anti-IgA antibodies specific to the animal, which antibodies are bound to a detection agent;
d) a detection band comprising the pathogen, or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and
e) a wicking pad for receiving and retaining sample after passing through the detection band.
In some embodiments of the invention, the antibodies are Immunoglobulin A (IgA), Immunoglobulin G (IgG), or Immunoglobulin M (IgM) antibodies. In certain embodiments, the antibodies are IgA antibodies. In other specifically contemplated embodiments, the antibodies are IgG antibodies.
In one embodiment, the anti-Ig, anti-IgG, anti-M or anti-IgA antibodies comprising the conjugate are partially or fully substituted for one or more immunoglobulin binding proteins, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion. For example, the conjugate comprises Protein G or a Protein A/G fusion protein.
Accordingly, in one embodiment, the device comprises
a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti-IgG, anti-M or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins, or a combination thereof, which antibodies or immunoglobulin-binding proteins are bound to a detection agent; d) a detection band comprising the pathogen, or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
In some embodiments of the invention, the one or more components removed from the sample by filter membrane of the sample pad are cellular material, fats, or particulate matter. For example, the sample pad is a CytoSep™ sample pad.
In specifically contemplated embodiments of the invention, the sample pad enables the sample to be applied to the strip without prior processing of the sample.
The strip may be formed from any suitable material that allows capillary transport of a fluid. For example, the strip is formed from a nitrocellulose material.
In some embodiments of the invention, the animal is a human or is a bovine, ovine, caprine, equine, canine, feline, porcine, cervine, camelid or galline animal. In certain specifically contemplated embodiments, the animal is a human. In other specifically contemplated embodiments, the animal is a bovine animal. In some embodiments, the bovine animal is a lactating bovine cow.
In various embodiment, the bodily fluid is selected from the group comprising blood, blood serum, blood plasma, milk, mucous, saliva, sputum, semen, sweat and urine. In some embodiments, the bodily fluid is milk from a lactating bovine cow.
In some embodiments, the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coii, Streptococcus dysgaiactiae, Streptococcus agalactiae, and Mycoplasma bovis.
In some embodiments, the detection agent is selected from the group comprising metallic nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, and fluorescent molecules. For example, the detection agent comprises nanoparticles or nanoshells of metallic gold.
In some embodiments, the pathogen or antigen of the pathogen is native. In other embodiments the pathogen or antigen of the pathogen is recombinant.
In some embodiments, the visible colour change is the appearance of a black, red, green, blue or yellow colour.
In some embodiments, the antibodies of the conjugate are chicken, goat, sheep or rabbit polyclonal antibodies or monoclonal antibodies. In some embodiments, the antibodies are anti-bovine Ig, IgG, IgM or IgA antibodies. In some embodiments, the device may be used for the detection of antibodies to two or more pathogens in a bodily fluid of an animal. For example, the device may comprise two or more detection bands, each detection band comprising a pathogen, or an antigen of a pathogen immobilised within the strip. In certain specifically contemplated embodiments, the antibodies of a pathogen in one detection band exhibit no cross-reactivity with antigens of a pathogen in another detection band.
In one example, a device particularly suited to the detection of early stage mastitis is provided, wherein the device is configured to enable detection of IgG antibodies. For example, the conjugate comprises anti-IgG antibodies, such as anti-bovine IgG antibodies. In another example, the device comprises one or more immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
Accordingly, in a specifically contemplated example, an LF device comprising anti- IgG antibodies is provided. In another specifically contemplated example, an LF device comprising an immunoglobulin-binding protein capable of binding IgG antibodies is provided. It is contemplated such a device is particularly suitable for the detection of early stage, and in some embodiments, preclinical or subclinical, mastitis.
In another example, a device particularly suited to the detection of late stage mastitis, persistent mastitis, or treatment-resistant mastitis is provided, wherein the device is configured to enable detection of bovine IgA antibodies. For example, the conjugate comprises anti-IgA antibodies, such as anti-bovine IgA antibodies. In another example, the device comprises one or more immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion.
Accordingly, in a specifically contemplated example, an LF device comprising anti- bovine IgA antibodies is provided. In another specifically contemplated example, an LF device comprising an immunoglobulin-binding protein capable of binding IgA antibodies is provided. It is contemplated such a device is particularly suitable for the detection of late stage mastitis, persistent mastitis, or treatment-resistant mastitis.
In a second aspect, the invention provides a method for the detection of antibodies to a pathogen in a bodily fluid of an animal using a device of the invention.
In some embodiments, the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coii, Streptococcus dysgaiactiae, Streptococcus agalactiae, and Mycoplasma bovis.
In some embodiments, the detection of antibodies to Staphylococcus aureus and/or Streptococcus uberis in a sample of milk form a lactating bovine cow indicates the presence of mastitis in the cow. Accordingly, in another aspect, the invention relates to a method of detecting mastitis in a subject. In one embodiment, the method comprises obtaining a biological sample from the subject, optionally processing the sample to provide a fluid sample capable of application to a device as described herein, and applying the sample to the device. In one embodiment, the method comprises contacting a fluid sample obtained from a subject, for example milk from a lactating mammal, to a device as described herein.
In one embodiment, the method is a method for detecting mastitis in a lactating bovine cow using a device as described herein. For example, the method comprises obtaining milk from a lactating bovine, optionally defatting the milk, and applying the milk to the device, wherein the detection of antibodies to a pathogen, for example as indicated by a visible colour change or detection of a fluorescent signal, is indicative of mastitis in the bovine.
In one embodiment, the method is a method of detecting early stage mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgG antibodies. In one example, said conjugate comprises anti-IgG antibodies. In another example, said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgG antibodies, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion. For example, the conjugate comprises Protein G or a Protein A/G fusion protein.
In one embodiment, the method is a method of detecting preclinical or subclinical mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgG antibodies. In one example, said conjugate comprises anti-IgG antibodies. In another example, said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgG antibodies, such as Protein A, Protein G, or a combination or derivative thereof, such as both Protein A and Protein G, or a Protein A/G fusion. For example, the conjugate comprises Protein G or a Protein A/G fusion protein.
In another embodiment, the method is a method of detecting late stage mastitis, the method comprising obtaining a fluid sample from a subject, and applying the sample to a device as described herein, wherein the device comprises a conjugate capable of binding IgA antibodies. In one example, said conjugate comprises anti-IgA antibodies. In another example, said conjugate comprises one or more immunoglobulin-binding proteins capable of binding IgA antibodies.
Another aspect of the present invention relates to a diagnostic kit comprising an LF device as described herein.
In one embodiment, the diagnostic kit comprises an LF device, wherein the pathogen or an antigen of the pathogen at the detection band is or is from S. aureus or S. uberis. In one embodiment, the kit comprises a composition comprising one or more antibodies specific to the same pathogen as is present at the detection band or from which the antigen present at the detection band is derived. Such an antibody composition is capable of use as a positive control. For example, in one embodiment, a kit for the detection of S. aureus or S. uberis is provided, said kit comprising an LF device as described herein wherein the pathogen is S. aureus or S. uberis, and the kit comprises one or more antibodies reactive to S. aureus or S. uberis as a positive control.
Optionally, a negative control may also be included, comprising an antibody that is not reactive to the pathogen at the detection band of from which the antigen present at the detection band is derived.
It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7).
Other objects, aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows Mean ± SEM of S. aureus (S.a.) and S. uberis (S.ub) and their negative controls (neg). Letters in common show highly significant differences (P<0.001). Figure 2 is a schematic representation of a LF device of the invention.
Figure 3 is a schematic representation of a test sample that is positive for S. uberis and S. aureus IgA.
Figure 4 is a schematic representation of a test sample that is positive for S. uberis IgA. Figure 5 is a schematic representation of a test sample that is positive for S. aureus IgA. Figure 6 is a schematic representation of a test sample that is negative for S. uberis and S. aureus IgA.
DETAILED DESCRIPTION
Definitions
As used in this specification, the words "comprises", "comprising", and similar words, are not to be interpreted in an exclusive or exhaustive sense. In other words, they are intended to mean "including, but not limited to". The term "Immunoglobulin" or "Ig" means an antibody which is a large Y-shaped protein produced mainly by plasma cells used by the immune system to neutralise pathogens such as pathogenic bacteria and viruses.
The term "Immunoglobulin A" or "IgA" means a subclass of Ig that has an important role in the immune function of mucosal surfaces including the udder of bovine cows.
The term "Immunoglobulin G" or "IgG" means a subclass of Ig that is mostly found in circulating bodily fluids such as blood and lymph.
The term "Immunoglobulin M" or "IgM" means a subclass of Ig that is produced mainly in the spleen and is the first antibody to appear in response to initial exposure to an antigen.
The term "antigen" means a molecule having distinct surface features or epitopes capable of stimulating a specific immune response. Antibodies (immunoglobulins) are produced by the immune system in response to exposure to antigens. Antigens maybe proteins, carbohydrates or lipids, although only protein antigens are classified as immunogens because carbohydrates and lipids cannot elicit an immune response on their own.
The term "nanoparticles" means uniform particles having a size of 1-200 nm.
The term "nanoshells" means nanoparticles that consist of a core and a metallic shell (usually gold).
The invention is based on the applicant's development of a rapid and effective LF device for the testing of bovine milk samples in order to determine whether the cow that produced the sample has a mastitis infection. The test is based on the use of antibodies for the detection and identification of bacterial pathogens known to cause mastitis. It will be appreciated that the principle of using antibodies to detect the presence of a pathogen in a sample may also be employed for the detection of other animal and human diseases.
The detection of antigen specific antibodies in samples has long been recognised as being reflective of infections. This approach has been used for many diagnostic tests including for testing milk samples. Antibodies can be detected using several immunological assays such as ELISA, Western Blots and Lateral Flow tests. ELISA tests have the advantage of being fast and the tests are amenable to high throughput and automation, while LF tests are especially suitable as point-of-care tests.
Immunoassays are highly suited to the diagnosis of chronic and subclinical diseases where antibody maturation has already occurred and antibody subclasses IgG and/or IgA are present. In contrast, IgM is produced shortly after infection. Therefore, the selection of antibody subclass for a specific test is critical and dependent on many factors such as pathogen, type and severity of infection.
The device of the invention detects antibodies to a bacterial pathogen in a bodily fluid of an animal.
In one aspect, the device comprises: a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti- IgG, anti-M or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins, or a combination thereof, which antibodies or immunoglobulin-binding proteins are bound to a detection agent;
d) a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
In a particular embodiment, the device comprises:
a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti- IgG, anti-IgM or anti-IgA antibodies specific to the animal, which antibodies are bound to a detection agent;
d) a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band. The sample pad not only receives sample fluid for testing, but removes components from the fluid that might otherwise impeded capillary flow of the fluid through the strip or adversely affect detection of the antibodies at the detection band. The components that may be removed by the sample pad include cellular material, fats, and particulate matter. For the purpose of detecting antibodies to a pathogen, such as S. aureus, that are present in a milk sample, the sample pad provides an advantage because the milk would otherwise need to be treated or processed in some way before being subjected to immunoassay, such as an ELISA test, for the determination of whether the cow that poduced the milk had a specific mastitis infection. The sample pad avoids any requirement for pre-treatment of the sample. One specifically contemplated option for the sample pad as a CytoSep™ sample pad, but any other suitable sample pad may be used.
Nitrocellulose was found to be a suitable material from which the strip is made. Other materials may also be suitable provided they allow the desired capillary flow rate and enable suitable detection sensitivity. Specifically contemplated strips (also referred to as membranes) were identified as FF80 HP and HF75 membranes.
It will be appreciated that the device of the invention may be used for the detection of a wide range of pathogen infections in humans and other animals. For example, in a specifically contemplated embodiment of the invention, the device is used for determining whether a bovine cow has a mastitis infection by detecting the presence of antibodies to a bacterial pathogen, such as S. aureus or S. uberis in a milk sample obtained from the cow. In another specifically contemplated embodiment, the device is used for determining whether a bovine animal is infected with M. bovis. The animal may alternatively be an ovine, caprine, equine, canine, feline, porcine, cervine, camelid or galline animal.
As will be appreciated, any bodily fluid that contains antibodies to a bacterial pathogen may be used as the fluid sample for testing using the device of the invention. Such fluids include blood, blood serum, blood plasma, milk, mucous, saliva, sputum, semen, sweat and urine. In the context of this disclosure, milk from a lactating bovine is the preferred choice of fluid for testing for bacterial pathogens associated with mastitis.
The device of the invention is capable of being used for detecting any bacterial, viral, fungal or parasitic pathogen provided the fluid sample tested contains antibodies to the pathogen. Such pathogens include, but are not limited to, S. aureus, S. uberis, E. coii, S. dysgalactiae, S. agalactiae, and M. bovis.
The detection agent is any agent that provides a detectable change when contacted by antibodies present in the fluid sample. Thus, in practice, the detection agent must incorporate or be bound to a moiety capable of binding an antibody from the animal from which the sample is obtained, such as anti-Ig, anti-IgG, ant-IgM or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins capable of binding antibodies from the animal. The detectable change may be a visible colour change or observable fluorescence, or any other suitable change. In various specifically contemplated embodiments of the invention, the detection agent is nanoparticles or nanoshells conjugated to anti-Ig, anti-IgG, anti-IgM or anti-IgA antibodies specific to the animal. In other specifically contemplated embodiments of the invention, the detection agent is nanoparticles or nanoshells conjugated to immunoglobulin-binding protein, such as Protein A, Protein G, or a combination or derivative thereof. The nanoparticles or nanoshells may be metallic or non- metallic, but metallic gold nanoparticles or nanoshells are specifically contemplated. For example, the detection agent may be gold nanoparticles bound to polyclonal rabbit anti- bovine IgA antibody or polyclonal chicken anti-bovine IgG antibody. In another example, the detection agent may be gold nanoparticles bound to Protein G, or to Protein A, or to a Protein A/G fusion protein. In other embodiments, the detection agent may be one or more enzymes or fluorescent molecules.
The detection band of the strip comprises a pathogen or an antigen of a pathogen immobilised within the strip. Some devices of the invention may comprise two or more detection bands. In specifically contemplated embodiments, each band comprises cells of the pathogen, e.g. cells of S. aureus or S. uberis. The cells would usually be UV-attenuated or sonicated and lysed, and may have been pre-treated with blocking serum to prevent non- specific binding of antibodies in the sample or on the detection reagent.
Figure 2 shows a LF device (1) of the invention. The device (1) has a specific sample pad (2) that makes milk (and other types of fluids) amenable to capillary flow, a conjugate pad (3) to which a detection agent is immobilised, a membrane (4), and a wicking pad (5) for receiving and holding fluid that has travelled by capillary flow from the sample pad (2) and through the conjugate pad (3) and the membrane (4). A detection band (6) is shown which comprises immobilised S. uberis antigens. Detection band (7) comprises immobilised S. aureus antigens. Band (8) is a positive control band. Shown (schematically) on the conjugate pad (3) is a detection agent (9) representing a gold nanoparticle to which a Y- shaped antibody is bound.
When a fluid sample (not shown) is deposited on the sample pad (2), or the sample pad (2) is dipped into the fluid sample, fluid travels from the sample pad (2) by capillary flow to the conjugate pad (3) where it comes into contact with the detection agent. The fluid mobilises the detection agent and is transported to the detection band (6). The detection agent binds to the S. uberis antigens in the detection band (6) and the accumulation of gold nanoparticles gives a positive visual signal, i.e. a colour change (black in the case of 150 nm nanoshells). The colour change at detection band (6) would indicate the presence of S. uberis in the fluid sample.
Under the continued influence of capillary flow, the fluid then proceeds to detection band (7). The detection agent binds to the S. aureus antigens in the detection band (7) and, again, the accumulation of gold nanoparticles gives a colour change. Thus, the device (1) can be used for the detection of two pathogens that may be present in the fluid sample, in this case both S. uberis and S. aureus. The antibodies of the pathogen in detection band (6) and the antibodies of the pathogen in detection band (7) can be selected so that there is no cross-reactivity between the two.
Figure 3 shows a LF device (1) of the invention where a test sample has been found to be positive for both S. uberis and S. aureus IgA. This is indicated by the dark colour of both detection band (6) and detection band (7), and the positive control band (8).
Figure 4 shows a LF device (1) of the invention where a test sample has been found to be positive for S. uberis IgA only. This is indicated by the dark colour of detection band (6), no colour change at detection band (7), and the dark colour of the positive control band (8).
Figure 5 shows a LF device (1) of the invention where a test sample has been found to be positive for S. aureus IgA only. This is indicated by no colour change at detection band (6), the dark colour of detection band (7), and the dark colour of the positive control band (8).
Figure 6 shows a LF device (1) of the invention where a test sample has been found to be negative for both S. uberis and S. aureus IgA. This is indicated by no colour change at both detection band (6) and detection band (7), and the dark colour of positive control band (8).
Example 1 shows that infected animals have significantly elevated IgA antibody titres when compared to culture-negative samples. This provides strong evidence that IgA is indicative of the pathogen that causes mastitis, and that IgA is a suitable antibody subclass for detection in a LF test.
Example 2 shows that the identification of the most suitable membrane is critical for the performance of the LF test. Slow flowing membranes, such as FF170 HP, that have a very small pore size are not suitable for milk samples, as capillary flow is blocked by components usually found in milk. In contrast, fast flowing membranes, such as Prima 40, may not have the desired sensitivity. The most suitable membranes for milk samples were identified as FF80 HP and HF75 which proved to be sensitive and provided the desired capillary flow properties for milk samples.
Example 3 shows that better sensitivity can be achieved by using 150 nm nanoshells in comparison with 40 nm nanoparticles.
Example 4 shows that an anti-rabbit antibody serves as an ideal positive control by binding directly to the host antibody that is bound to the detection particle. In this case the detection particle consisted of a rabbit anti-bovine IgA antibody conjugated to 150 nm nanoshells. Bovine colostrum was found to have insufficient IgA levels to consistently give strong positive signals. Example 5 shows that the selection of the most suitable conjugate pad is important for the overal performace of the device. The strongest signals were obtained for conjugate pads 8951, T5NM and 6613H for milk samples while milk failed to elute the conjugate from pads 8649 and 6613.
Example 6 shows that a amount of lysed bacterial antigen immobilised on the membrane should be a minimum of 1 pi OD600 0.55 to produce strong signals. Because S. aureus is known for its ability to non-specifically bind to antibodies, a specific S. aureus strain was found to have a reduced ability for non-specific binding. This strain was therefore used for the test development.
The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.
EXAMPLES
Example 1 - Identification of antibody subclass for mastitis
Milk samples from cows with elevated cell counts were collected from four farms in New Zealand. Milk samples from the first three farms were used for the development of antibody subclass specific ELISA assays for IgG, IgA and Ig.
Antigen-specific IgA reactivity was tested with S. aureus or S. uberis antigens in 64 samples with known elevated somatic cell counts (SCC) and the test evaluated against bacterial culture results. The culture results showed that 17 samples were positve for S. aureus, 16 samples were positive for S. uberis, 9 samples had mixed cultures of S. aureus and S. uberis, and 40 samples were culture-negative. Figure 1 demonstrates that Mean ± SEM of ODs for S. aureus culture positive samples was significantly higher than for S. aureus culture negative samples (P<0.001). Similarly, Mean ± SEM of ODs for S. uberis culture positive samples was significantly higher than for S. uberis culture negative samples (P<0.001). The statistical difference was calculated using the non-parametric Mann-Whitney test. From these studies, antigen-specific IgA was identified as a reliable marker for S. aureus and S. uberis mastitis.
Example 2: Identification of membrane for use with milk samples
A selection of commercially available membranes was tested for performance with milk samples (Table 1). S. aureus and S. uberis lysate antigens were immobilised onto the membranes by drying at 37°C. Milk samples that were found to have antibodies to S. aureus and S. uberis by ELISA were used as test samples. Phosphate buffered saline (PBS) was used as a negative control. Detection used rabbit anti-bovine IgA antibody conjugated to 150 nm nanoshells (nanoComposix). The results were ranked as -, (+), +, ++, or + + + (negative, questionable, weak, medium or highly positive). Table 1: Membrane ID and flow rates
Figure imgf000015_0001
This experiment showed that slow flowing membrans (FF12 HP and FF170 HP) are not suitable for testing milk samples. Fast flowing membranes, such as Prima 40, FF80 HP and HF75, were found to perform well with the sensitivity of HF75 slighlty outperforming FF80 HP
Prima 40 membranes but also showing a low level of non-specific binding.
The performance of FF80 HP and HF75 membranes was then assessed using milk samples with high and low levels of antibodies for S. aureus and S. uberis, that were also culture-positive for both pathogens, a sample that was antibody and culture-positive for S. aureus, and a negative sample. Rabbit anti-bovine IgA antibodies conjugated to functionalised 150 nm nanoshells were used for detection. The results are shown in Table 2. FF80 and HF75 were found to be equally sensitive for S. aureus, but FF80 was found to be more sensitive for S. uberis.
Table 2: Comparison of FF80 and HF75 membranes
Figure imgf000015_0002
Figure imgf000016_0001
Example 3: Identification of detection reagent
In intial experiments, 40 nm gold nanoparticles to which anti-bovine antibodies were passively absorbed were used for detection. These conjugates were found to not have the required high level of sensitivity and stability. Functionalised 150 nm nanoshells to which antibodies were covalently attached (as per protocol by nanoComposix) were then tested and found to have improved sensitivity by at least 10-fold. Consequently, 150 nm nanoshells were deployed for test development. These results were obtained using a LF detection method for specific numbers of S. aureus. This test method utilised an anti-S. aureus monoclonal antibody immobilised at the test line and another anti-S. aureus antibody conjugated to the detection reagent. The detection limit for specific numbers of S. aureus suspended in a buffer was determined and found to be 5xl05 CFU for 150 nm nanoshells and 5xl06 CFU for 40 nm gold nanoparticles. Example 4: Identification of a positive control
Colostrum is known to contain IgG, but also IgA antibodies. The amount of IgA in 0.5 pi of colostrum was found to be at the limit of detection and gave inconsistent results. An anti-rabbit antibody that would directly bind to the antibody 150 nm nanoshell conjugate was tested and gave consistently strong positive signals (Table 3), then 1 pi and 0.5 mI of an anti- rabbit antibody and 1 mI of colostrum were immobilised on HT75 membranes and tested for binding to rabbit anti-S. aureus 150 nm nanoshell conjugates.
Table 3: Identification of a positive control
Figure imgf000016_0002
Example 5: Identification of conjugate pad
Several conjugate pads were compared for their ability to hold and release dried conjugate (Rb anti-bovine IgA 150 nm nanoshells). Conjugate was diluted 1 : 10 in 2 mM borate buffer substituted with 10% sucrose. Conjugate pads were saturated with the reagent, dried for 2 h at 37°C and then fitted on to FF80 membranes. Cytosep™ 1663 and a wicking pad were fitted at the proximal and distal ends of the strips respectively. Comparisions were made with a milk sample known to be antibody-positive for S. aureus which was deposited on the sample pad. The following conjugate pads were tested : 8649, 8951, 6615, 6613, T5NM, 6613H. The strongest signals were obtained for conjugate pads 8951, T5NM and 6613H where an anti-rabbit antibody served as a positive control and an ELISA-positive milk sample as a positive sample control (Table 4). The results were ranked as -, (+), +, ++, or + + + (negative, questionable, weak, medium or highly positive).
Table 4: Identification of best-performing conjugate pads
Figure imgf000017_0001
Example 6: Preparation of antigens
Strains of S. aureus and S. uberis were isolated from milk samples and propagated on sheep blood agar plates. Overnight cultures were taken off plates, resuspended in PBS supplemented with 0.1% sodium azide and sonicated for 2 min to solubilise bacteria. The S. aureus milk strain was found to non-specifically bind to antibodies in milk in ELISA assays. A S. aureus strain (strain A) exhibited a lower ability for non-specific binding and consequently this strain was used in LF assays. S. aureus lysates with an OD600 nm of 0.55 were found to be suitable for LF. 0.5 pi of the lysate was used for capture (Table 5). The test was performed with a milk sample that was ELISA positive for IgA and visualisation with a rabbit- anti S. aureus 150 nm nanoshell conjugate. Table 5: Identification of optimal antigen concentrations for immobilisation on strips
Figure imgf000018_0001
Example 7: Assembling and testing of prototype LF test for S. aureus and S. uberis IgA
A prototype test strip was assembled using the following components: FF80 nitrocellulose membrane fitted with a conjugate pad 8951 (loaded with 150 nm nanoshells conjugaed to a rabbit anti-bovine IgA antibody) and Cytosep™ as a sample membrane at one end. The sample pad overlapped the conjugate by 2 mm and the conjugate pad the nitrocellulose membrane by 2 mm in order to facilitate capillary flow. A wicking pad was fitted at the other end to promote capillary flow and to receive and hold fluid.
Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

Claims
2. A device for the detection of antibodies to a bacterial pathogen in a bodily fluid of an animal, the device comprising :
a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action through the strip from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti- IgG, anti-M or anti-IgA antibodies specific to the animal, or one or more immunoglobulin-binding proteins, or a combination thereof, which antibodies or immunoglobulin-binding proteins are bound to a detection agent;
d) a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
3. The device of claim 1, wherein the device comprises:
a) a strip formed of a material enabling 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 sample of the fluid, wherein the sample pad comprises a filter membrane for removal of one or more components from the sample;
c) a conjugate pad located in the strip so that the sample flows under capillary action from the sample pad to the conjugate pad and mobilises a conjugate contained in the conjugate pad, the conjugate comprising anti-Ig, anti-IgG or anti-IgA antibodies specific to the animal, which antibodies are bound to a detection agent; d) a detection band comprising the pathogen or an antigen of the pathogen immobilised within the strip along a band located substantially perpendicular to the direction of flow of the sample along the strip so that when the mobilised conjugate in the sample contacts the pathogen or antigen of the pathogen in the detection band the presence of the antibodies to the pathogen in the sample is indicated by a visible colour change or detection of a fluorescence signal; and e) a wicking pad for receiving and retaining sample after passing through the detection band.
4. The device as claimed in claim 1 or claim 2 wherein the antibodies are Immunoglobulin A (IgA), Immunoglobulin G (IgG), or Immunoglobulin M (IgM) antibodies.
5. The device as claimed in any one of claims 1 to 3 wherein the antibodies are IgA antibodies.
6. The device as claimed in any one of claims 1 to 3 wherein the antibodies are IgG antibodies.
7. The device as claimed in any one of claims 1 to 5 wherein the immunoglobulin-binding protein is Protein G, or a Protein A/G fusion protein.
8. The device as claimed in any one of claims 1 to 6 wherein the one or more components removed from the sample by filter membrane of the sample pad are cellular material, fats, or particulate matter.
9. The device as claimed in any one of claims 1 to 7 wherein the sample pad is a CytoSepTM sample pad.
10. The device as claimed in any one of claims 1 to 8 wherein the sample pad enables the sample to be applied to the strip without prior processing of the sample.
11. The device as claimed in in any one of claims 1 to 9 wherein the strip is formed from a nitrocellulose material.
12. The device as claimed in any one of claims 1 to 10 wherein the animal is a human or is a bovine, ovine, caprine, equine, canine, feline, porcine, cervine, camelid or galline animal.
13. The device as claimed in any one of claims 1 to 11 wherein the animal is a bovine animal.
14. The device as claimed in claim 12 wherein the bovine animal is a lactating bovine cow.
15. The device as claimed in any one of claims 1 to 13 wherein the bodily fluid is selected from the group comprising blood, blood serum, blood plasma, milk, processed milk, mucous, saliva, sputum, semen, sweat and urine.
16. The device as claimed in 14 wherein the bodily fluid is milk from a lactating bovine cow.
17. The device as claimed in any one of claims 1 to 15 wherein the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coli, Streptococcus dysgalactiae, Streptococcus agalactiae, and Mycoplasma bovis.
18. The device as claimed in any one of claims 1 to 16 wherein the detection agent is selected from the group comprising metallic nanoparticles or nanoshells, non-metallic nanoparticles or nanoshells, enzymes, and fluorescent molecules.
19. The device as claimed in claim 17 wherein the detection agent comprises nanoparticles or nanoshells of metallic gold.
20. The device as claimed in any one of claims 1 to 18 wherein the pathogen or antigen of the pathogen is native or recombinant.
21. The device as claimed in any one of claims 1 to 19 wherein the visible colour change is the appearance of a black, red, green, blue or yellow colour.
22. The device as claimed in any one of claims 1 to 20 wherein the antibodies of the conjugate are chicken, goat, sheep or rabbit monoclonal antibodies or polyclonal antibodies.
23. The device as claimed in claim 21 wherein the antibodies are anti-bovine IgG or anti- bovine IgA antibodies.
24. The device as claimed in any one of claims 1 to 22 for the detection of antibodies to two or more pathogens in a bodily fluid of an animal.
25. The device as claimed in claim 23 comprising two or more detection bands, each detection band comprising a pathogen or an antigen of a pathogen immobilised within the strip.
26. The device as claimed in claim 24 wherein antibodies of a pathogen in one detection band exhibits no cross-reactivity with antigens of a pathogen in another detection band.
27. A method for the detection of antibodies to a pathogen in a bodily fluid of an animal using a device of any one of claims 1 to 25.
28. The method as claimed in claim 26 wherein the pathogen is selected from the group comprising Staphylococcus aureus, Streptococcus uberis, Escherichia coli, Streptococcus dysgalactiae, Streptococcus agalactiae, and Mycoplasma bovis.
29. The method as claimed in claim 27 wherein the detection of antibodies to Staphylococcus aureus and/or Streptococcus uberis in a sample of milk form a lactating bovine cow indicates the presence of mastitis in the cow.
30. A method for detecting mastitis in a lactating bovine cow using a device of any one of claims 1 to 25.
31. The method of claim 29, wherein the device comprises a conjugate capable of binding IgG antibodies.
32. The method of claim 30, wherein the device comprises a conjugate comprising an immunoglobulin-binding protein capable of binding bovine IgG antibodies.
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