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Title:  Methods of detecting early renal disease in animals
United States Patent: 
7,172,873
Issued: 
February 6, 2007

Inventors: 
McDonald; Thomas (Omaha, NE), Jensen; Wayne Arthur (Wellington, CO), Weber; Annika (Omaha, NE), Andrews; Janet S. (Fort Collins, CO)
Assignee: 
Heska Corporation (Loveland, CO)
Appl. No.: 
10/112,648
Filed: 
March 28, 2002


 

Pharm/Biotech Jobs


Abstract

The present invention provides a method for the detection of early renal disease in animals. The method includes the steps of (a) obtaining a sample from an animal to be tested and (b) determining the amount of albumin in the sample. An amount of albumin in the range of from 10 .mu.g/ml to about 300 .mu.g/ml indicates the presence of early renal disease. The present invention also provides antibodies to canine, feline and equine albumin which can be used to detect the presence of early renal disease.

SUMMARY OF THE INVENTION

The present invention relates to a method and kit for the detection of early renal disease in animals. Preferred animals to test for early renal disease are companion animals with dogs, cats and horses being the most preferred. Method and kit embodiments disclosed herein are based on the discovery that the presence of albumin in a sample from an animal, in the range of 10 .mu.g/ml to 300 .mu.g/ml can be used as an indicator of early renal disease. The most preferred sample to test is urine although any sample that is useful for measuring leakage of albumin from the glomerulus can be used. Any assay capable of detecting albumin may be used in the instant method or kit although preferred methods and kits employ immunologically-based assays, preferably single-step assays. The most preferred assay is an immunologically-based assay utilizing an anti-albumin antibody.

The present invention also provides isolated antibodies which can be used in detecting albumin levels in animal samples. Any antibody which binds albumin from the test animal can be used; preferred antibodies bind canine albumin and/or feline albumin and/or equine albumin. Preferred antibodies are TNB1, TNB3, TNB4, TNB5, TNB6, H352, H386, H387, H388, H389, H390, H391, H393, H394, H395, H396, H397, H398, H399, H400, H401, and H402. Also included are cultured cells which produce antibodies suitable for practicing the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to a novel method of detecting early renal disease in animals and to novel antibodies that selectively bind to albumin from one or more specie of animal. More particularly, the present invention relates to the discovery that the presence of microalbuminuria can be used to predict early renal disease in animals, particularly immune-mediated renal diseases. Therefore, the methods can also be useful for prescribing a treatment for an animal. Suitable treatment can be designed to delay or prevent the onset of late-stage renal disease. Examples of such treatment include, for example, pharmacological or dietary modification. The present invention is also useful in monitoring the effectiveness of a prescribed treatment.

A method of the present invention can be generally accomplished by:

(a) obtaining a sample from an animal; and

(b) determining the amount of albumin in the sample.

An amount of albumin in a range of from about 10 .mu.g/ml to about 300 .mu.g/ml in the sample is indicative of early renal disease.

It is to be noted that the term "a" entity or "an" entity refers to one or more of that entity. For example, a protein refers to one or more proteins or at least one protein. As such, the terms "a" "an" "one or more" and "at least one" can be used interchangeably herein. The terms "comprising," "including," and "having" can also be used interchangeably. In addition, the terms "amount" and "level" are also interchangeable and may be used to describe a concentration or a specific quantity. Furthermore, the term "selected from the group consisting of" refers to one or more members of the group in the list that follows, including mixtures (i.e. combinations) of two or more members.

As used herein, the term "renal disease" is defined as a dysfunction of the glomerular filtration process. Glomerular dysfunction may be transient or it may be chronic, depending on the underlying cause of the disease. One consequence of glomerular dysfunction is that proteins which are normally retained in the blood, leak through the glomerulus, into the glomerular filtrate and eventually into the urine. One example of a protein which may be present in urine due to glomerular dysfunction is albumin and its presence in urine at low levels has been termed microalbuminuria. The term "microalbuminuria," as used herein, refers to an amount of albumin that is present in a sample in a range from about 10 .mu.g/ml to about 300 .mu.g/ml when the sample is normalized to a specific gravity of 1.010. This is greater than the amount found in healthy animals which is typically low, i.e., less than 10 .mu.g/ml. Microalbuminuria may arise as a consequence of damage to the kidney resulting from, for example, immune-complex-mediated glomerulernephritis. As used herein, the term "late-stage renal disease" is used to define a state in which an animal has lost 70% or more of its renal function, with corresponding, elevated levels in the animal's serum metabolites, in particular blood-urea nitrogen (BUN) and serum creatinine levels. As used herein, the term "early renal disease" is defined as the presence of microalbuminuria in an animal in the absence of detectable changes in renal function (i.e. increased BUN, serum creatinine or decreased ability to concentrate urine). As such, an albumin level in a sample ranging from about 10 .mu.g/ml to about 300 .mu.g/ml when the sample is normalized to a specific gravity of 1.010 is indicative of early renal disease.

As used herein, the term "animal" is meant to encompass any non-human organism capable of developing early renal disease. Suitable animals to test for microalbuminuria include, but are not limited to companion animals (i.e. pets), food animals, work animals, or zoo animals. Preferred animals include, but are not limited to, cats, dogs, horses, ferrets and other Mustelids, cattle, sheep, swine, and rodents. More preferred animals include cats, dogs, horses and other companion animals, with cats, dogs and horses being even more preferred. As used herein, the term "companion animal" refers to any animal which a human regards as a pet. As used herein, a cat refers to any member of the cat family (i.e., Felidae), including domestic cats, wild cats and zoo cats. Examples of cats include, but are not limited to, domestic cats, lions, tigers, leopards, panthers, cougars, bobcats, lynx, jaguars, cheetahs, and servals. A preferred cat is a domestic cat. As used herein, a dog refers to any member of the family Canidae, including, but not limited to, domestic dogs, wild dogs, foxes, wolves, jackals, and coyotes and other members of the family Canidae. A preferred dog is a domestic dog. As used herein, a horse refers to any member of the family Equidae. An equid is a hoofed mammal and includes, but is not limited to, domestic horses and wild horses, such as, horses, asses, donkeys, and zebras. Preferred horses include domestic horses, including race horses.

In one embodiment of the present invention, a sample is obtained, or collected, from an animal to be tested for microalbuminuria. The animal may or may not be suspected of having early stage renal disease. A sample is any specimen obtained from the animal that can be used to measure albumin leakage from the glomerulus. A preferred sample is a bodily fluid that can be used to measure albumin leakage from the glomerulus. Those skilled in the art can readily identify appropriate samples.

Urine is particularly suitable as the sample. Urine samples can be collected from animals by methods known in the art, including, for example, collecting while the animal is voiding, or collecting by catheterization, or by cystocentesis. Urine may be refrigerated or frozen before assay, but is preferably assayed soon after collection.

Although not necessary for the present invention, the sample may be pre-treated as desired. For example, the sample can be normalized to a desired specific gravity. Normalizing the sample by appropriate dilution methods known in the art permits quantification of microalbuminuria independent of the concentration (e.g. specific gravity) of the sample. Although any desired specific gravity can be readily selected by those skilled in the art, a particularly suitable specific gravity is 1.010. If another specific gravity value is desired for normalizing a sample, those skilled in the art can readily determine the appropriate albumin amounts that would fall within the definition of microalbuminuria for the desired specific gravity.

After obtaining the sample, the level of albumin in that sample is determined. As used herein, the terms "determine," "determine the level of albumin," "determine the amount of albumin," "determine the albumin level," and the like are meant to encompass any technique which can be used to detect or measure the presence of albumin in a sample. Albumin is an example of an analyte. The term "analyte, as used herein, is used to describe any molecule or compound present in a sample. Such techniques may give qualitative or quantitative results. Albumin levels can be determined by detecting the entire albumin protein or by detecting fragments, degradation products or reaction products of albumin. In a preferred method, the level of albumin is determined using a suitable albumin-binding compound.

As used herein, the terms "albumin-binding molecule", "albumin-binding compound", "anti-albumin compound", and the like are used interchangeably and refer to any molecule which binds to albumin and forms a stable complex. A preferred albumin-binding compound is one which selectively binds albumin from an animal. The term "selectively binds albumin" means to preferentially bind to albumin as opposed to binding other proteins unrelated to albumin. A particularly useful albumin-binding compound is a anti-albumin antibody. As used herein, the terms "anti-albumin antibody," "antibody to albumin," "antibody to animal albumin," "antibody having specificity for albumin from animals," "animal albumin antibody," and the like refer to an antibody that preferentially binds albumin from one or more animals. A particularly suitable anti-albumin antibody preferentially binds to canine, feline and/or equine albumin as opposed to binding to different, unrelated canine, feline or equine proteins. Another particularly suitable anti-albumin antibody preferentially binds to canine albumin as opposed to binding to a different, unrelated canine protein. Another particularly suitable antibody to companion animal albumin preferentially binds to feline albumin as opposed to binding to a different, unrelated feline protein. Another particularly suitable antibody to companion animal albumin preferentially binds to equine albumin as opposed to binding to a different, unrelated equine protein.

The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to albumin of one ore more animal species. Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes on albumin. A suitable method to produce antibodies effective for use in the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof to produce the antibodies and (b) recovering the antibodies. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay. Methods to produce such antibodies are known in the art and are described in detail in Harlow et al., Antibodies, a Laboratory Manual (Cold Spring Harbor Labs Press, 1988), incorporated by reference herein in its entirety, and include immunizing animals to produce preparations of polyclonal antibodies that are recovered from, for example, ascites fluid and purified by methods known in the art to yield preparations that are reactive to animal albumin. Many species have proteins sharing closely related sequences and therefore it may be difficult using standard immunization protocols to produce antibodies which recognize a protein from only one specie. Therefore, modification of standard methods used to produce antibodies, such as, for example, subtractive hybridization techniques, are also contemplated. Such modifications can be those known to those skilled in the art or additionally modified techniques as disclosed within this application. In another method, antibodies for use in the present invention are produced recombinantly using techniques disclosed in Sambrook et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Labs Press, 1989), incorporated by reference herein in its entirety.

As noted previously, other suitable methods include producing monoclonal antibodies. Briefly, monoclonal antibodies are produced from the fusion of spleen cells from an immunized animal and myeloma cells to produce a hybridoma. Hybridomas can be screened for production of the proper antibody, then cultured and the antibodies harvested. As used herein, the term "cultured cell" refers to hybridomas or any cell which produces an antibody. Methods to produce and screen such hybridomas are described in Harlow, et al., supra. Methods to prepare an antigen so that antibodies produced will be reactive with animal albumin are known in the art and are described, for example, in Harlow, et al., supra. Preparation of the antigen material for injection into the animal includes any technique known in the art, and include, for example, using the full-length protein, using peptides selected from immunogenic regions of the protein, modifying the antigen by methods such as, for example, dinitrophenol coupling, arsynyl coupling, denaturation of the antigen, coupling antigen to protein carriers such as, for example, keyhole limpet hemacyanin, peptides containing Class II- T-cell receptor binding sites, to beads, and any other method known in the art. See Harlow, et al., supra.

The anti-albumin antibodies of the present invention can include multifunctional antibodies, for example a bifunctional antibody having at least one functional portion that specifically binds to animal albumin. Such multifunctional antibodies can include, for example, a chimeric molecule comprising a portion of the molecule that binds to animal albumin and a second portion that enables the chimeric molecule to be bound to a substrate or to be detected in such a manner that the binding to the albumin is unimpaired. Examples of suitable second portions include but are not limited to a fragment of an immunoglobulin molecule, a fluorescent protein or an enzyme.

In addition to anti-albumin antibodies, albumin-binding molecules can also include proteins and peptides that bind to albumin. Such proteins and peptides may be from natural, recombinant or synthetic sources and may or may not be purified. Examples of non-antibody, albumin-binding, proteins include, but are not limited to, the 42-kilodalton (kDa) Protein A from Staphlococcus aureus, Protein G from S. aureus and Eschericia coli, the rat 60-kDa albumin binding protein (gp60) and the human renal tubule cubilin protein. The use of functional homologues of such proteins, from these or other species, for the detection of albumin is also contemplated. Hybrids or fusions of albumin-binding proteins which retain their albumin-binding ability may also be used. In such hybrids, the albumin-binding portion of the protein would be joined to a second portion which allows the hybrid to be bound to a substrate or to be detected. Examples of suitable second portions include, but are not limited to, a fragment of an immunoglobulin molecule, an epitope tag, a fluorescent protein or an enzyme.

An albumin-binding molecule used in the present invention can be contained in a formulation. For example, an antibody can be combined with a buffer in which the antibody is solubilized, and/or with a carrier. Suitable buffers and carriers are known to those skilled in the art. Examples of suitable buffers include any buffer in which an albumin-binding molecule can function to selectively bind to albumin, such as, but not limited to, phosphate buffered saline, water, saline, phosphate buffer, HEPES buffer (N-2-hydroxyethylpiperazine-N'-2-ethansulfonic acid buffered saline) TES buffer (Tris-EDTA buffered saline), Tris buffer and TAE buffer (Tris-acetate-EDTA). Examples of carriers include, but are not limited to, polymeric matrices, toxoids, and serum albumins, such as bovine serum albumin. Carriers can be combined with an albumin-binding molecule or conjugated (i.e. attached) to an albumin-binding molecule in such a manner as to not substantially interfere with the ability of the albumin-binding molecule to selectively bind to albumin. In addition, suitable formulations of the present invention can include not only the albumin-binding molecule to specie-specific albumin, but also one or more additional antigens or antibodies useful for detecting albumin.

As used herein, the term "contacting" refers to the introduction of a sample putatively containing albumin to an albumin-binding compound, for example, by combining or mixing the sample with the albumin-binding compound. When albumin is present in the sample, an albumin-compound complex is then formed; such complex formation refers to the ability of an anti-albumin compound to selectively bind to the albumin in order to form a stable complex that can be detected. Detection can be qualitative, quantitative, or semi-quantitative. Binding albumin in the sample to the albumin-binding compound is accomplished under conditions suitable to form a complex. Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art. Binding can be measured using a variety of methods standard in the art including, but not limited to, enzyme immunoassays (e.g., ELISA), immunoprecipitations, immunoblot assays and other immunoassays as described, for example, in Sambrook et al., supra, and Harlow, et al., supra. These references also provide examples of complex formation conditions.

In one embodiment, an albumin/albumin-binding compound complex, also referred to herein as an albumin-compound complex, can be formed in solution. In another embodiment, an albumin/albumin-binding compound complex can be formed in which the albumin or the albumin-binding compound is immobilized on (e.g., coated onto) a substrate. Immobilization techniques are known to those skilled in the art. Suitable substrate materials include, but are not limited to, plastic, glass, gel, celluloid, fabric, paper, and particulate materials. Examples of substrate materials include, but are not limited to, latex, polystyrene, nylon, nitrocellulose, agarose, cotton, PVDF (polyvinylidene-fluoride), and magnetic resin. Suitable shapes for substrate material include, but are not limited to, a well (e.g., microtiter dish well), a microtiter plate, a dipstick, a strip, a bead, a lateral flow apparatus, a membrane, a filter, a tube, a dish, a celluloid-type matrix, a magnetic particle, and other particulates. Particularly preferred substrates include, for example, an ELISA plate, a dipstick, an immunodot strip, a radioimmunoassay plate, an agarose bead, a plastic bead, a latex bead, a sponge, a cotton thread, a plastic chip, an immunoblot membrane, an immunoblot paper and a flow-through membrane. In one embodiment, a substrate, such as a particulate, can include a detectable marker. For descriptions of examples of substrate materials, see, for example, Kemeny, D. M. (1991) A Practical Guide to ELISA, Pergamon Press, Elmsford, N.Y. pp 33 44, and Price, C. and Newman, D. eds. Principles and Practice of Immunoassay, 2.sup.nd edition (1997) Stockton Press, NY, N.Y., both of which are incorporated herein by reference in their entirety.

In a preferred embodiment, an anti-albumin compound is immobilized on a substrate, such as a microtiter dish well, a dipstick, an immunodot strip, or a lateral flow apparatus. A sample collected from an animal is applied to the substrate and incubated under conditions suitable (i.e., sufficient) to allow for anti-albumin compound-albumin complex formation bound to the substrate (i.e., albumin in the sample binds to the anti-albumin compound immobilized on the substrate).

In accordance with the present invention, once formed, an albumin-binding molecule/albumin complex is detected. As used herein, the term "detecting complex formation" refers to identifying the presence of albumin-binding compound complexed to albumin. If complexes are formed, the amount of complexes formed can, but need not be, quantified. Complex formation, or selective binding, between a putative albumin-composition with an albumin-binding compound can be measured (i.e., detected, determined) using a variety of methods standard in the art (see, for example, Sambrook et al. supra.), examples of which are disclosed herein. A complex can be detected in a variety of ways including, but not limited to use of one or more of the following assays: an enzyme-linked immunoassay, a competitive enzyme-linked immunoassay, a radioimmunoassay, a fluorescence immunoassay, a chemiluminescent assay, a lateral flow assay, a flow-through assay, an agglutination assay, a particulate-based assay (e.g., using particulates such as, but not limited to, magnetic particles or plastic polymers, such as latex or polystyrene beads), an immunoprecipitation assay, a BioCore.TM. assay (e.g., using colloidal gold), an immunodot assay (e.g., CMG's Immunodot System, Fribourg, Switzerland), and an immunoblot assay (e.g., a western blot), an phosphorescence assay, a flow-through assay, a chromatography assay, a PAGe-based assay, a surface plasmon resonance assay, a spectrophotometric assay, a particulate-based assay, and an electronic sensory assay. Such assays are well known to those skilled in the art.

Assays can be used to give qualitative or quantitative results depending on how they are used. The assay results can be based on detecting the entire albumin molecule or fragments, degradation products or reaction products of albumin. Some assays, such as agglutination, particulate separation, and immunoprecipitation, can be observed visually (e.g., either by eye or by a machines, such as a densitometer or spectrophotometer) without the need for a detectable marker.

In other assays, conjugation (i.e., attachment) of a detectable marker to the anti-albumin compound or to a reagent that selectively binds to the anti-albumin compound aids in detecting complex formation. A detectable marker can be conjugated to the anti-albumin compound or reagent at a site that does not interfere with ability of the anti-albumin compound to bind albumin. Methods of conjugation are known to those of skill in the art. Examples of detectable markers include, but are not limited to, a radioactive label, a fluorescent label, a chemiluminescent label, a chromophoric label, an enzyme label, a phosphorescent label, an electronic label; a metal sol label, a colored bead, a physical label, or a ligand. A ligand refers to a molecule that binds selectively to another molecule. Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or ImmunoPure.RTM. NeutrAvidin).

In one embodiment, an animal albumin-compound complex can be detected by contacting a sample with a specific compound-antibody conjugated to a detectable marker. A detectable marker can be conjugated to an anti-albumin antibody or other compound which binds the albumin-binding-compound in such a manner as not to block the ability of the anti-compound antibody or other compound to bind to the canine albumin-binding compound being detected. Preferred detectable markers include, but are not limited to, fluorescein, a radioisotope, a phosphatase (e.g., alkaline phosphatase), biotin, avidin, a peroxidase (e.g., horseradish peroxidase), beta-galactosidase, and biotin-related compounds or avidin-related compounds (e.g., streptavidin or ImmunoPure.RTM. NeutrAvidin).

In another embodiment, a complex is detected by contacting the complex with an indicator molecule. Suitable indicator molecules include molecules that can bind to the albumin/ albumin-binding molecule complex or to the albumin. As such, an indicator molecule can comprise, for example, an albumin-binding reagent, such as an antibody. Preferred indicator molecules that are antibodies include, for example, antibodies reactive with the antibodies from species of animal in which the anti-albumin antibodies are produced. An indicator molecule itself can be attached to a detectable marker of the present invention. For example, an antibody can be conjugated to biotin, horseradish peroxidase, alkaline phosphatase or fluorescein.

The present invention can further comprise one or more layers and/or types of secondary molecules or other binding molecules capable of detecting the presence of an indicator molecule. For example, an untagged (i.e., not conjugated to a detectable marker) secondary antibody that selectively binds to an indicator molecule can be bound to a tagged (i.e., conjugated to a detectable marker) tertiary antibody that selectively binds to the secondary antibody. Suitable secondary antibodies, tertiary antibodies and other secondary or tertiary molecules can be readily selected by those skilled in the art. Preferred tertiary molecules can also be selected by those skilled in the art based upon the characteristics of the secondary molecule. The same strategy can be applied for subsequent layers.

Preferably, the indicator molecule is conjugated to a detectable marker. A developing agent is added, if required, and the substrate is submitted to a detection device for analysis. In some protocols, washing steps are added after one or both complex formation steps in order to remove excess reagents. If such steps are used, they involve conditions known to those skilled in the art such that excess reagents are removed but the complex is retained.

One embodiment to detect microalbuminuria involves the use of a lateral flow assay, examples of which are described in U.S. Pat. No. 5,424,193, issued Jun. 13, 1995, by Pronovost et al.; U.S. Pat. No. 5,415,994, issued May 16, 1995, by Imrich et al; WO 94/29696, published Dec. 22, 1994, by Miller et al.; and WO 94/01775, published Jan. 20, 1994, by Pawlak et al.; all of which are incorporated by reference herein. A lateral flow assay is an example of a single-step assay. In a single-step assay, once the sample has been obtained and made ready for testing, only a single action is necessary on the part of the user to detect the present of an analyte. For example, the sample, in whole or part, can be applied to a device which then measures analyte in the sample. In one embodiment, a sample is placed in a lateral flow apparatus that includes the following components: (a) a support structure defining a flow path; (b) a labeling reagent comprising a bead conjugated to a specific antibody, the labeling reagent being impregnated within the support structure in a labeling zone; and (c) a capture reagent. Preferred antibodies include those disclosed herein. The capture reagent is located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The support structure comprises a material that does not impede the flow of the beads from the labeling zone to the capture zone. Suitable materials for use as a support structure include ionic (i.e., anionic or cationic) material. Examples of such a material include, but are not limited to, nitrocellulose, PVDF, or carboxymethylcellulose. The support structure defines a flow path that is lateral and is divided into zones, namely a labeling zone and a capture zone. The apparatus can further include a sample receiving zone located along the flow path, preferably upstream of the labeling reagent. The flow path in the support structure is created by contacting a portion of the support structure downstream of the capture zone, preferably at the end of the flow path, to an absorbent capable of absorbing excess liquid from the labeling and capture zones.

In another embodiment, a lateral flow apparatus used to detect albumin includes: (a) a support structure defining a flow path; (b) a labeling reagent comprising a anti-albumin antibody as described above, the labeling reagent impregnated within the support structure in a labeling zone; and (c) a capture reagent, the capture reagent being located downstream of the labeling reagent within a capture zone fluidly connected to the labeling zone in such a manner that the labeling reagent can flow from the labeling zone into the capture zone. The apparatus preferably also includes a sample receiving zone located along the flow path, preferably upstream of the labeling reagent. The apparatus preferably also includes an absorbent located at the end of the flow path. One preferred embodiment includes a capture reagent comprising anti-canine albumin antibody.

Once the albumin level has been measured, an assessment of whether early renal disease is present can then be made. Assessing the presence of early renal disease means comparing the level of albumin in the test sample to the level found in healthy animals. The presence of microalbuminuria in the sample, in the absence of changes in renal function, is indicative of early renal disease. As used herein, the term "indicative of early renal disease" is means sufficient glomerular dysfunction is present to allow albumin to pass into the urine in the range of from about 10 .mu.g/ml to about 300 .mu.g/ml. The amount of albumin present in the sample may vary depending on the amount of damage present but in early renal disease, the albumin level is higher than that found in healthy animals but lower than that detectable by current methods used to measure proteinuria. In the present invention, a determination of early renal disease is made when the level of albumin in the sample is determined to be in the range of from about 10 .mu.g/ml to about 300 .mu.g/ml. The upper range of albumin levels can also be about 25 .mu.g/ml, about 50 .mu.g/ml, about 75 .mu.g/ml, about 100 .mu.g/ml, about 125 .mu.g/ml, about 150 .mu.g/ml, about 175 .mu.g/ml, about 200 .mu.g/ml, about 225 .mu.g/ml, about 250 .mu.g/ml, about 275 .mu.g/ml, or about 300 .mu.g/ml. The level of albumin in the sample may vary depending on the severity of the damage to the kidney. Preferred embodiments of the present inventions can detect albumin when about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% of kidney function is lost. A more preferred embodiment can detect microalbuminuria in time for medical intervention which may then delay or prevent the onset of late-stage renal disease. Such intervention may, for example, include, but is not limited to the use of pharmacological compounds or dietary modifications to delay or prevent the progression of renal disease.

One embodiment of the present invention is a "dipstick" device which can detect microalbuminuria in animals. Dipsticks may be constructed in a variety of ways that partly depend on the way in which they will be used. They may be held directly in a sample (e.g., a urine stream), dipped directly in sample contained in a collection vessel, or have sample applied to a strip contained in a plastic cassette or platform. Another example of a dipstick is a "flow-through" device, an example of which is a heterogenous immunometric assay system based on a capture antibody immobilized onto a membrane attached to an absorbent reservoir. A "bead" refers to a particulate substrate composed of a matrix such as latex or polystyrene, which can be covalently or non-covalently cross-linked to a detection molecule. A preferred embodiment of the "dipstick" assay is an immunometric system, described in U.S. Pat. No. 5,656,502, issued on Aug. 12, 1997, to MacKay and Fredrickson, and U.S. Pat. No. 6,001,658, issued Dec. 14, 1999 to Fredrickson, both incorporated herein by reference. Particularly preferred is an ImmunoDip.TM. device available from Diagnostic Chemicals Ltd., PEI, CA.

Non-immunological methods may also be used. In order to detect microalbuminuria, methods such as preconcentration of the urine in order to concentrate albumin may be used to increase sensitivity of the test to protein. Such non-immunological methods include, for example, urine electrophoresis, where detection of microalbuminuria can be determined by methods known in the art, and include, for example, protein staining. In another embodiment, a protein based albumin test may be used to determine microalbuminuria on a preconcentrated sample of urine from an animal.

The methods of the present invention can be used to detect nephropathy in a canid, felid, equid, or other animal, particularly when the nephropathy is glomerulonephropathy, and especially glomerulonephritis. More specifically, the microalbuminuria measurement is correlated to the presence of early renal disease in a target animal. As used herein, the term "nephropathy" and/or "renal disease" refers to any disease of the kidneys, and may include, for example, nephritis of the glomerular, tubular, or interstitial renal tissues.

Such early stage nephropathy can result from many different causes, including, for example, allergy, cancer, parasitic, viral, or bacterial infection of any tissue in the animal, exposure to renal toxins, immune-mediated diseases, such as systemic lupus erythematosus and vasculitis, malignancy, Vitamin D3 rodenticides, pyelonephritis, leptospirosis, urinary tract obstruction, chronic inflammatory disease, pyoderma, pancreatitis, prostatitis, immune-mediated diseases, dental disease, high blood pressure, or diabetes. As used herein, an "infectious agent" is one that infects animals and include, but are not limited to, viruses, bacteria, fungi, endoparasites and ectoparasites. Examples of viral infectious agents include, but are not limited to, adenoviruses, caliciviruses, coronaviruses, distemper viruses, hepatitis viruses, herpesviruses, immunodeficiency viruses, infectious peritonitis viruses, leukemia viruses, oncogenic viruses, papilloma viruses, parainfluenza viruses, parvoviruses, rabies viruses, and reoviruses, as well as other cancer-causing or cancer-related viruses. Examples of bacterial infectious agents include, but are not limited to, Actinomyces, Bacillus, Bacteroides, Bartonella, Bordetella, Borrelia, Brucella, Campylobacter, Capnocytophaga, Clostridium, Corynebacterium, Coxiella, Dermatophilus, Ehrlichia, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemobartonella, Helicobacter, Klebsiella, L-form bacteria, Leptospira, Listeria, Mycobacteria, Mycoplasma, Neorickettsia, Nocardia, Pasteurella, Peptococcus, Peptostreptococcus, Proteus, Pseudomonas, Rickettsia, Rochalimaea, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia. Examples of fungal infectious agents include, but are not limited to, Absidia, Acremonium, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastomyces, Candida, Chlamydia, Coccidioides, Conidiobolus, Cryptococcus, Curvalaria, Epidermophyton, Exophiala, Geotrichum, Histoplasma, Madurella, Malassezia, Microsporum, Moniliella, Mortierella, Mucor, Paecilomyces, Penicillium, Phialemoniuin, Phialophora; Prototheca, Pseudallescheria, Pseudomicrodochium, Pythium, Rhinosporidium, Rhizopus, Scolecobasidium, Sporothrix, Stemphylium, Trichophyton, Trichosporon, and Xylohypha. Examples of protozoan parasite infectious agents include, but are not limited to, Babesia, Balantidium, Besnoitia, Cryptosporidium, Eimeria, Encephalitozoon, Entamoeba, Giardia, Hammondia, Hepatozoon, Isospora, Leishmania, Microsporidia, Neospora, Nosema, Pentatrichomonas, Plasmodium, Pneumocystis, Sarcocystis, Schistosoma, Theileria, Toxoplasma, and Trypanosoma. Examples of helminth parasite infectious agents include, but are not limited to, Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus, Ascaris, Brugia, Bunostomum, Capillaria, Chabertia, Cooperia, Crenosoma, Dictyocaulus, Dioctophyme, Dipetalonema, Diphyllohothrium, Diplydium, Dirofilaria, Dracunculus, Enterobius, Filaroides, Haemonchus, Lagochilascaris, Loa, Mansonella, Muellerius, Nanophyetus, Necator, Nematodirus, Oesophagostomuim, Onchocerca, Opisthorchis, Ostertagia, Parafilaria, Paragonimus, Parascaris, Physaloptera, Protostrongylus, Setaria, Spirocerca, Spirometra, Stephanofilaria, Strongyloides, Strongylus, Thelazia, Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris, Unicinaria, and Wuchereria. Examples of ectoparasite infectious agents include, but are not limited to, fleas, ticks, including hard ticks and soft ticks, flies such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats, ants, spiders, lice, mites, and true bugs, such as bed bugs and kissing bugs.

The present invention may also be used to measure multiple analytes. Other analytes may be any analyte which can be detected in sample suitable for use in detecting early renal disease. Additional analytes can be used to detect, for example, infectious disease or inborn errors of metabolism.

The present invention also relates to antibodies that bind to albumin from an animal being tested. A preferred antibody is one which detects albumin levels when the amount in the sample is about 50 .mu.g/ml, more preferably 25 .mu.g/ml, more preferably 10 .mu.g/ml. Another preferred antibody is one which detects albumin levels when the amount in the sample is about 50 .mu.g/ml, more preferably about 25 .mu.g/ml, more preferably about 10 .mu.g/ml and the detection method is a dipstick device described in U.S. Pat. No. 6,001,658. A preferred antibody is one which competes with any of the monoclonal antibodies TNB1, TNB3, TNB4, TNB5, TNB6, H352, H386, H387, H388, H389, H390, H391, H393, H394, H395, H396, H397, H398, H399, H400, H401, or H402 for selective binding to animal albumin, preferably canine albumin. Another preferred embodiment is an antibody which binds to the same or related epitope, as defined by sequence homology, bound by the antibodies TNB3, TNB6 and H402. A preferred antibody is selected from the group consisting of TNB1, TNB3, TNB4, TNB5, TNB6, H352, H386, H387, H388, H389, H390, H391, H393, H394, H395, H396, H397, H398, H399, H400, H401, and H402More preferred is an antibody selected from the group consisting of TNB3, TNB6 and H402. As used herein, the terms "compete" and "inhibit selective binding" refer to the ability of an antibody to prevent another antibody from binding to the same protein as described in the included examples.

The present invention also includes kits suitable for detecting animal albumin using the methods disclosed herein. Suitable means of detection include the techniques disclosed herein, utilizing compounds that bind the desired animal albumin, such as, for example, an anti-albumin antibody. As such, a kit can also comprise a detectable marker, such as an antibody that selectively binds to the albumin binding compound or other indicator molecules. The kit can also contain associated components, such as, but not limited to, buffers, labels, containers, inserts, tubings, vials, syringes and the like.

The present invention is based on a surprising discovery that microalbuminuria in canids can be used as a marker to predict the development of renal disease in nondiabetic dogs as well as diabetic dogs because microalbuminuria does not clearly have predictive value in nondiabetic human patients. Similar uses are contemplated in other animals. However, despite this surprising discovery, until the present invention, effective methods to detect microalbuminuria in dogs did not exist.


Claim 1 of 41 Claims

1. A method to detect early renal disease in a canid, a feud or an equid comprising: (a) obtaining a urine sample from said canid, feud or equid; and (b) determining the amount of albumin in said sample, wherein an amount of albumin in a range from about 10 .mu.g/ml to about 300 .mu.g/ml in the sample, when the specific gravity of the sample is normalized to 1.010, is indicative of early renal disease.

 

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