Devices for Detecting Renal Disorders

Description

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application Ser. No. 61/327,389, filed Apr. 23, 2010, and U.S. provisional application Ser. No. 61/232,091, filed Aug. 7, 2009, each of which is hereby incorporated by reference in its entirety, and is related to U.S. patent application Ser. Nos. [Not Yet Assigned], entitled Methods and Devices for Detecting Obstructive Uropathy and Associated Disorders, Computer Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Damage, Methods and Devices for Detecting Kidney Transplant Rejection, Methods and Devices for Detecting Diabetic Nephropathy and Associated Disorders, and Methods and Devices for Detecting Glomerulonephritis and Associated Disorders, Attorney Docket Nos. 060075-, filed on the same date as this application, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention encompasses devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining renal disorders in a mammal using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.

BACKGROUND OF THE INVENTION

The urinary system, in particular the kidneys, perform several critical functions such as maintaining electrolyte balance and eliminating toxins from the bloodstream. In the human body, the pair of kidneys together process roughly 20% of the total cardiac output, amounting to about 1 L/min in a 70-kg adult male. Because compounds in circulation are concentrated in the kidney up to 1000-fold relative to the plasma concentration, the kidney is especially vulnerable to injury due to exposure to toxic compounds.

Renal disorders and disease are serious conditions that generally affect the function of the kidney. The disorders discussed herein may arise from a variety of causes, including intrinsic disease processes, such as inflammation and necrosis of the kidney. In addition, renal disorders may also arise from secondary sources including drugs that are toxic to the kidneys and alternative disease states that cause secondary adverse effects on the kidney, such as diabetes and hypertension. Prevention of renal disorders is largely dependent on early diagnosis of the condition. Existing diagnostic tests such as BUN and serum creatine tests typically detect only advanced stages of kidney damage. Other diagnostic tests such as kidney tissue biopsies or CAT scans have the advantage of enhanced sensitivity to earlier stages of kidney damage, but these tests are also generally costly, slow, and/or invasive.

A need exists in the art for a fast, simple, reliable, and sensitive method of detecting obstructive uropathy or an associated disorder. In a clinical setting, the early detection of kidney damage would help medical practitioners to diagnose and treat kidney damage more quickly and effectively.

SUMMARY OF THE INVENTION

The present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder in a mammal. In particular, the present invention provides methods and devices for diagnosing, monitoring, or determining a renal disorder using measured concentrations of a combination of three or more analytes in a test sample taken from the mammal.

In one aspect, the present invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising six antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, cystatin C, KIM-1, THP, and TIMP-1.

In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. It is also recognized that the assay device may include combinations of 6, 10, or 16 antibodies with antigenic determinants corresponding to the analytes disclosed herein.

In another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising: (a) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol; (b) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (c) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (d) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies.

In a further aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having a panel of biomarkers for diagnosing, monitoring, or determining a renal disorder comprising three or more antibodies immobilized on the contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; and (b) a collection apparatus suitable for collecting a sample of bodily fluid from the mammal.

In yet another aspect, the invention encompasses a kit for diagnosing, monitoring, or determining a renal disorder in a mammal, where the kit includes: (a) an assay device having (i) three or more capture antibodies, wherein the antigenic determinants of the capture antibodies are analytes associated with a renal disorder, wherein the analytes are selected from the group consisting of alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF; (ii) three or more capture agents comprising an antigenic moiety, wherein one of the capture agents is attached to each of the capture antibodies; (iii) three or more detection antibodies, wherein the antigenic determinant of the detection antibodies is the antigenic moiety; and (iv) three or more indicators, wherein each of the indicators is attached to one of the detection antibodies; and (b) a collection apparatus suitable for collecting a sample bodily fluid from the mammal.

In still another aspect, the invention encompasses an assay device for diagnosing, monitoring, or determining a renal disorder in a mammal, the device comprising a panel of biomarkers having sixteen antibodies immobilized on a contact surface, wherein the antigenic determinants of the antibodies are analytes associated with renal disorder, wherein the analytes are selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.

In a further aspect, the invention encompasses a platform for diagnosing, monitoring, or determining a renal disorder in a mammal, the platform comprising at least 6 antibodies selected from the group consisting of alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF.

Other aspects and iterations of the invention are described in more detail below.

DESCRIPTION OF FIGURES

FIG. 1 depicts four graphs comparing (A) the concentrations of alpha-1 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (B) the concentrations of beta-2 microglobulin in the urine of normal controls, kidney cancer patients, and patients with other cancer types; (C) the concentrations of NGAL in the urine of normal controls, kidney cancer patients, and patients with other cancer types; and (D) the concentrations of THP in the urine of normal controls, kidney cancer patients, and patients with other cancer types.

FIG. 2 shows the four different disease groups from which samples were analyzed, and a plot of two different estimations on eGFR outlining the distribution within each group.

FIG. 3 is a number of scatter plots of results on selected proteins in urine and plasma. The various groups are indicated as follows—control: blue, AA: red, DN: green, GN: yellow, OU: orange. (A) A1M in plasma, (B) cystatin C in plasma, (C) B2M in urine, (D) cystatin C in urine.

FIG. 4 depicts the multivariate analysis of the disease groups and their respective matched controls using plasma results. Relative importance shown using the random forest model.

FIG. 5 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish disease samples vs. normal samples. Disease encompasses analgesic abuse (AA), glomerulonephritis (GN), obstructive uropathy (OU), and diabetic nephropathy (DN). Normal=NL.

FIG. 6 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish disease (AA+GN+ON+DN) samples vs. normal samples from plasma (A) and urine (B and C).

FIG. 7 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. normal samples. Abbreviations as in FIG. 4.

FIG. 8 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. normal samples from plasma (A) and urine (B and C).

FIG. 9 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish analgesic abuse samples vs. diabetic nephropathy samples. Abbreviations as in FIG. 4.

FIG. 10 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish analgesic abuse samples vs. diabetic nephropathy samples from plasma (A) and urine (B and C).

FIG. 11 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish glomerulonephritis samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.

FIG. 12 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish glomerulonephritis samples vs. analgesic abuse samples from plasma (A) and urine (B and C).

FIG. 13 depicts three graphs showing the mean AUROC and its standard deviation (A) for plasma samples, and mean error rates (B) and mean AUROC (C) from urine samples for each classification method used to distinguish obstructive uropathy samples vs. analgesic abuse samples. Abbreviations as in FIG. 4.

FIG. 14 depicts three graphs showing the average importance of analytes and clinical variables from 100 bootstrap runs measured by random forest (A and B) or boosting (C) to distinguish obstructive uropathy samples vs. analgesic abuse samples from plasma (A) and urine (B and C).

DETAILED DESCRIPTION OF THE INVENTION

It has been discovered that a multiplexed panel of three or more, six or more, and preferably sixteen, biomarkers may be used to detect renal disorders. As used herein, the term “renal disorder” includes, but is not limited to glomerulonephritis, interstitial nephritis, tubular damage, vasculitis, glomerulosclerosis, diabetic nephropathy, analgesic nephropathy, and acute tubular necrosis. As used herein, the term “glomerulonephritis” refers to a disorder characterized by inflammation of the glomeruli. The term may encompass chronic glomerulonephritis, acute glomerulonephritis, primary glomerulonephritis, or secondary glomerulonephritis. As used herein, the term “diabetic nephropathy” refers to a disorder characterized by angiopathy of capillaries in the kidney glomeruli. The term encompasses Kimmelstiel-Wilson syndrome, or nodular diabetic glomerulosclerosis and intercapillary glomerulonephritis. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with diabetic nephropathy. As used herein, the phrase “a disorder associated with diabetic nephropathy” refers to a disorder that stems from angiopathy of capillaries in the kidney glomeruli. For instance, non-limiting examples of associated disorders may include nephritic syndrome, chronic kidney failure, and end-stage kidney disease. The devices of the present invention may also be used to detect secondary kidney damage or toxicity caused by exposure to a toxic compound including but not limited to therapeutic drugs, recreational drugs, medical imaging contrast agents, and toxins. Non-limiting examples of therapeutic drugs may include an analgesic (e.g. aspirin, acetaminophen, ibuprofen, naproxen sodium), an antibiotic (e.g. an aminoglycoside, a beta lactam (cephalosporins, penicillins, penems), rifampin, vancomycin, a sulfonamide, a fluoroquinolone, and a tetracycline), or a chemotherapy agent (e.g. Cisplatin (Platinol®), Carboplatin (Paraplatin®), Cytarabine (Cytosar-U®), Gemtuzumab ozogamicin (Mylotarg®), Gemcitabine (Gemzar®), Melphalan (Alkeran®), Ifosfamide (Ifex®), Methotrexate (Rheumatrex®), Interleukin-2 (Proleukin®), Oxaliplatin (Eloxatin®), Streptozocin (Zanosar®), Pemetrexed (Alimta®), Plicamycin (Mithracin®), and Trimetrexate (Neutrexin®). Further, the term renal disorder may include kidney damage due to kidney stones, ischemia, liver transplantation, heart transplantation, lung transplantation, or hypovolemia. Moreover, the devices of the current invention may be used to detect renal disorders including kidney damage cause by other disease states including but not limited to diabetes, hypertension, autoimmune diseases including lupus, Wegener's granulomatosis, Goodpasture syndrome, primary hyperoxaluria, kidney transplant rejection, sepsis, nephritis secondary to any infection of the kidney, rhabdomyolysis, multiple myeloma, and prostate disease.

In addition, the devices and systems of the current invention may be used to detect renal disorders including acute kidney transplant rejection or chronic allograft nephropathy. Importantly, the devices of the invention may be used to distinguish between an acute rejection reaction and a chronic allograft nephropathy. Alternatively, the devices of the present invention may be used to distinguish between a successful transplant and rejection. As used herein, the term “rejection” refers to a recipient response to a foreign antigen derived from the transplanted kidney. The phrase “acute rejection” refers to an immune related response to the foreign kidney. The response is primarily T-cell driven and originates from an HLC mismatch between the donor and recipient. The phrase “chronic allograft nephropathy” refers to a chronic inflammatory and immune response mediated reaction to a foreign kidney. Chronic allograft nephropathy may result in damage to the kidney manifested by diffuse interstitial fibrosis glomerular changes, typically membranous and sclerotic in nature, as well as intimal fibrosis of the blood vessels with tubular atrophy and loss of tubular structures.

Additionally, the present invention encompasses devices comprising biomarkers that may be used to detect a renal disorder associated with kidney transplant rejection. As used herein, the phrase “a disorder associated with kidney transplant rejection” refers to a disorder that stems from a host response to a foreign antigen derived from the transplated kidney. For instance, non-limiting examples of associated disorders may include chronic kidney failure and end-stage kidney disease.

The devices of the present invention may also be utilized to detect a renal disorder including obstructive uropathy or an associated disorder in a mammal that includes determining the presence or concentration of a combination of three or more sample analytes in a test sample containing the bodily fluid of the mammal. As used herein, the term “obstructive uropathy” refers to a structural or functional hindrance of normal urine flow. The term may encompass chronic unilateral obstructive uropathy, chronic bilateral obstructive uropathy, acute unilateral obstructive uropathy, or acute bilateral obstructive uropathy. Additionally, the present invention encompasses biomarkers that may be used to detect a disorder associated with obstructive uropathy. As used herein, the phrase “a disorder associated with obstructive uropathy” refers to a disorder that stems from a structural or functional hindrance of normal urine flow. For instance, non-limiting examples of associated disorders may include hydronephrosis and obstructive nephropathy. The measured concentrations of the combination of sample analytes is compared to the entries of a dataset in which each entry contains the minimum diagnostic concentrations of a combination of three of more analytes reflective of obstructive uropathy or an associated disorder. Other embodiments provide computer-readable media encoded with applications containing executable modules, systems that include databases and processing devices containing executable modules configured to diagnose, monitor, or determine a renal disorder in a mammal. Still other embodiments provide antibody-based devices for diagnosing, monitoring, or determining obstructive uropathy or an associated disorder in a mammal.

The biomarkers included in a multiplexed panel of the invention are analytes known in the art that may be detected in the urine, serum, plasma and other bodily fluids of mammals. As such, the analytes of the multiplexed panel may be readily extracted from the mammal in a test sample of bodily fluid. The concentrations of the analytes within the test sample may be measured using known analytical techniques such as a multiplexed antibody-based immunological assay. The combination of concentrations of the analytes in the test sample may be compared to empirically determined combinations of minimum diagnostic concentrations and combinations of diagnostic concentration ranges associated with healthy kidney function to determine whether a renal disorder is indicated in the mammal.

The analytes used as biomarkers in the multiplexed assay, methods of diagnosing, monitoring, or determining a renal disorder using measurements of the analytes, systems and applications used to analyze the multiplexed assay measurements, and antibody-based devices used to measure the analytes are described in detail below.

I. Analytes in Multiplexed Assay

One embodiment of the invention measures the concentrations of three or more, six or more, ten or more, and preferably sixteen, biomarker analytes within a test sample taken from a mammal and compares the measured analyte concentrations to minimum diagnostic concentrations to diagnose, monitor, or determine obstructive uropathy or an associated renal disorder in a mammal. In this aspect, the biomarker analytes are known in the art to occur in the urine, plasma, serum and other bodily fluids of mammals. The biomarker analytes are proteins that have known and documented associations with early renal damage in humans. As defined herein, the biomarker analytes include but are not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. A description of each biomarker analyte is given below. In one embodiment, the biomarker analytes include alpha-1-microglobulin, beta-2-microglobulin, cystatin-C, KIM-1, THP, and TIMP-1.

(a) Alpha-1 Microglobulin (A1M)

Alpha-1 microglobulin (A1M, Swiss-Prot Accession Number P02760) is a 26 kDa glycoprotein synthesized by the liver and reabsorbed in the proximal tubules. Elevated levels of A1M in human urine are indicative of glomerulotubular dysfunction. A1M is a member of the lipocalin super family and is found in all tissues. Alpha-1-microglobulin exists in blood in both a free form and complexed with immunoglobulin A (IgA) and heme. Half of plasma A1M exists in a free form, and the remainder exists in complexes with other molecules including prothrombin, albumin, immunoglobulin A and heme. Nearly all of the free A1M in human urine is reabsorbed by the megalin receptor in proximal tubular cells, where it is then catabolized. Small amounts of A1M are excreted in the urine of healthy humans. Increased A1M concentrations in human urine may be an early indicator of renal damage, primarily in the proximal tubule.

(b) Beta-2 Microglobulin (B2M)

Beta-2 microglobulin (B2M, Swiss-Prot Accession Number P61769) is a protein found on the surfaces of all nucleated cells and is shed into the blood, particularly by tumor cells and lymphocytes. Due to its small size, B2M passes through the glomerular membrane, but normally less than 1% is excreted due to reabsorption of B2M in the proximal tubules of the kidney. Therefore, high plasma levels of B2M occur as a result of renal failure, inflammation, and neoplasms, especially those associated with B-lymphocytes.

(c) Calbindin

Calbindin (Calbindin D-28K, Swiss-Prot Accession Number P05937) is a Ca-binding protein belonging to the troponin C superfamily. It is expressed in the kidney, pancreatic islets, and brain. Calbindin is found predominantly in subpopulations of central and peripheral nervous system neurons, in certain epithelial cells involved in Ca2+ transport such as distal tubular cells and cortical collecting tubules of the kidney, and in enteric neuroendocrine cells.

(d) Clusterin

Clusterin (Swiss-Prot Accession Number P10909) is a highly conserved protein that has been identified independently by many different laboratories and named SGP2, S35-S45, apolipoprotein J, SP-40, 40, ADHC-9, gp80, GPIII, and testosterone-repressed prostate message (TRPM-2). An increase in clusterin levels has been consistently detected in apoptotic heart, brain, lung, liver, kidney, pancreas, and retinal tissue both in vivo and in vitro, establishing clusterin as a ubiquitous marker of apoptotic cell loss. However, clusterin protein has also been implicated in physiological processes that do not involve apoptosis, including the control of complement-mediated cell lysis, transport of beta-amyloid precursor protein, shuttling of aberrant beta-amyloid across the blood-brain barrier, lipid scavenging, membrane remodeling, cell aggregation, and protection from immune detection and tumor necrosis factor induced cell death.

(e) Connective Tissue Growth Factor (CTGF)

Connective tissue growth factor (CTGF, Swiss-Prot Accession Number P29279) is a 349-amino acid cysteine-rich polypeptide belonging to the CCN family. In vitro studies have shown that CTGF is mainly involved in extracellular matrix synthesis and fibrosis. Up-regulation of CTGF mRNA and increased CTGF levels have been observed in various diseases, including diabetic nephropathy and cardiomyopathy, fibrotic skin disorders, systemic sclerosis, biliary atresia, liver fibrosis and idiopathic pulmonary fibrosis, and nondiabetic acute and progressive glomerular and tubulointerstitial lesions of the kidney. A recent cross-sectional study found that urinary CTGF may act as a progression promoter in diabetic nephropathy.

(f) Creatinine

Creatinine is a metabolite of creatine phosphate in muscle tissue, and is typically produced at a relatively constant rate by the body. Creatinine is chiefly filtered out of the blood by the kidneys, though a small amount is actively secreted by the kidneys into the urine. Creatinine levels in blood and urine may be used to estimate the creatinine clearance, which is representative of the overall glomerular filtration rate (GFR), a standard measure of renal function. Variations in creatinine concentrations in the blood and urine, as well as variations in the ratio of urea to creatinine concentration in the blood, are common diagnostic measurements used to assess renal function.

(g) Cystatin C (Cyst C)

Cystatin C (Cyst C, Swiss-Prot Accession Number P01034) is a 13 kDa protein that is a potent inhibitor of the C1 family of cysteine proteases. It is the most abundant extracellular inhibitor of cysteine proteases in testis, epididymis, prostate, seminal vesicles and many other tissues. Cystatin C, which is normally expressed in vascular wall smooth muscle cells, is severely reduced in both atherosclerotic and aneurismal aortic lesions.

(h) Epidermal Growth Factor (EGF)

Epidermal growth factor (EGF, Swiss-Prot Accession Number P07522) is a small protein that functions as a potent mitogen. EGF promotes cell growth and differentiation, is essential in embryogenesis, and is important in wound healing. It is produced by many normal cell types and is made in large amounts by certain types of tumors.

(i) Glutathione S-Transferase alpha (GST-alpha)

Glutathione S-transferase alpha (GST-alpha, Swiss-Prot Accession Number P08263) belongs to a family of enzymes that utilize glutathione in reactions contributing to the transformation of a wide range of compounds, including carcinogens, therapeutic drugs, and products of oxidative stress. These enzymes play a key role in the detoxification of such substances.

(j) Glutathione S-Transferase mu (GST-mu)

Glutathione S-transferase mu (GST-mu, Swiss-Prot Accession Number PO4905) functions in the detoxification of electrophilic compounds, including carcinogens, therapeutic drugs, environmental toxins and products of oxidative stress, by conjugation with glutathione. The genes encoding the mu class of enzymes are organized in a gene cluster on chromosome 1 p13.3 and are known to be highly polymorphic. Genetic variations in GST-mu can change a mammal's susceptibility to carcinogens and toxins as well as affect the toxicity and efficacy of certain drugs. Null mutations of this class mu gene have been linked with an increase in a number of cancers.

(k) Kidney Injury Molecule-1 (KIM-1)

Kidney injury molecule-1 (KIM-1, Swiss-Prot Accession Number Q96D42) is an immunoglobulin superfamily cell-surface protein highly upregulated on the surface of injured kidney epithelial cells. It is also known as TIM-1 (T-cell immunoglobulin mucin domain-1), as it is expressed at low levels by subpopulations of activated T-cells and hepatitis A virus cellular receptor-1 (HAVCR-1). KIM-1 is increased in expression more than any other protein in the injured kidney and is localized predominantly to the apical membrane of the surviving proximal epithelial cells.

(l) Microalbumin

Albumin is the most abundant plasma protein in humans and other mammals. Albumin is essential for maintaining the osmotic pressure needed for proper distribution of body fluids between intravascular compartments and body tissues. Healthy, normal kidneys typically filter out albumin from the urine. The presence of albumin in the urine may indicate damage to the kidneys. Albumin in the urine may also occur in patients with long-standing diabetes, especially type 1 diabetes. The amount of albumin eliminated in the urine has been used to differentially diagnose various renal disorders. For example, nephrotic syndrome usually results in the excretion of about 3.0 to 3.5 grams of albumin in human urine every 24 hours. Microalbuminuria, in which less than 300 mg of albumin is eliminated in the urine every 24 hours, may indicate the early stages of diabetic nephropathy.

(m) Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Neutrophil gelatinase-associated lipocalin (NGAL, Swiss-Prot Accession Number P80188) forms a disulfide bond-linked heterodimer with MMP-9. It mediates an innate immune response to bacterial infection by sequestrating iron. Lipocalins interact with many different molecules such as cell surface receptors and proteases, and play a role in a variety of processes such as the progression of cancer and allergic reactions.

(n) Osteopontin (OPN)

Osteopontin (OPN, Swiss-Prot Accession Number P10451) is a cytokine involved in enhancing production of interferon-gamma and IL-12, and inhibiting the production of IL-10. OPN is essential in the pathway that leads to type I immunity. OPN appears to form an integral part of the mineralized matrix. OPN is synthesized within the kidney and has been detected in human urine at levels that may effectively inhibit calcium oxalate crystallization. Decreased concentrations of OPN have been documented in urine from patients with renal stone disease compared with normal individuals.

(o) Tamm-Horsfall Protein (THP)

Tamm-Horsfall protein (THP, Swiss-Prot Accession Number P07911), also known as uromodulin, is the most abundant protein present in the urine of healthy subjects and has been shown to decrease in individuals with kidney stones. THP is secreted by the thick ascending limb of the loop of Henley. THP is a monomeric glycoprotein of ˜85 kDa with ˜30% carbohydrate moiety that is heavily glycosylated. THP may act as a constitutive inhibitor of calcium crystallization in renal fluids.

(p) Tissue Inhibitor of Metalloproteinase-1 (TIMP-1)

Tissue inhibitor of metalloproteinase-1 (TIMP-1, Swiss-Prot Accession Number P01033) is a major regulator of extracellular matrix synthesis and degradation. A certain balance of MMPs and TIMPs is essential for tumor growth and health. Fibrosis results from an imbalance of fibrogenesis and fibrolysis, highlighting the importance of the role of the inhibition of matrix degradation role in renal disease.

(q) Trefoil Factor 3 (TFF3)

Trefoil factor 3 (TFF3, Swiss-Prot Accession Number Q07654), also known as intestinal trefoil factor, belongs to a small family of mucin-associated peptides that include TFF1, TFF2, and TFF3. TFF3 exists in a 60-amino acid monomeric form and a 118-amino acid dimeric form. Under normal conditions TFF3 is expressed by goblet cells of the intestine and the colon. TFF3 expression has also been observed in the human respiratory tract, in human goblet cells and in the human salivary gland. In addition, TFF3 has been detected in the human hypothalamus.

(r) Vascular Endothelial Growth Factor (VEGF)

Vascular endothelial growth factor (VEGF, Swiss-Prot Accession Number P15692) is an important factor in the pathophysiology of neuronal and other tumors, most likely functioning as a potent promoter of angiogenesis. VEGF may also be involved in regulating blood-brain-barrier functions under normal and pathological conditions. VEGF secreted from the stromal cells may be responsible for the endothelial cell proliferation observed in capillary hemangioblastomas, which are typically composed of abundant microvasculature and primitive angiogenic elements represented by stromal cells.

(s) Vascular Endothelial Growth Factor A (VEGF A)

Vascular endothelial growth factor A (VEGF A, Swiss-Prot Accession Number Q00731) is a growth factor active in angiogenesis, vasculogenesis and endothelial cell growth. It induces endothelial cell proliferation, promotes cell migration, inhibits apoptosis, and induces permeabilization of blood vessles. It is important in the pathophysiology of neuronal and other tumors, likely functioning as a potent promoter of angiogenesis. Due to its influences on vascular permeability, VEGF A may be involved in altering blood-brain-barrier functions under normal and pathological conditions. The production and secretion of VEGF by mammalian retinal pigment epithelial cells may be important in the pathogenesis of ocular neovascularization.

(t) B-lymphocyte Chemoattractant (BLC)

B-lymphocyte chemoattractant (BLC, Swiss-Prot Accession Number 043927) is also referred to as C-X-C motif chemokine 13, Small-inducible cytokine B13, B lymphocyte chemoattractant, CXC chemokine BLC, and B cell-attracting chemokine 1. BLC functions as a potent chemoattractant for B lymphocytes, but not T lymphocytes, monocytes, or neutrophils. Its specific receptor BLR1 is a G protein-coupled receptor originally isolated from Burkitt's lymphoma cells. Among cells of the hematopoietic lineages, the expression of BRL1, now designated CXCR5, is restricted to B lymphocytes and a subpopulation of T helper memory cells.

(u) Cluster of Differentiation Surface Receptors 40 (CD40)

Cluster of Differentiation Surface Receptors 40 (CD40, Swiss Prot Accession Number P25942) is also referred to TNFRSF5 (Tumor necrosis factor receptor superfamily member 5. CD40 is a member of the tumor necrosis factor-receptor superfamily of proteins. CD40 has been found to be essential in mediating a broad variety of immune and inflammatory responses including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation.

(v) Insulin-Like Growth Factor Binding Protein 2 (IGF BP2)

Insulin-like Growth Factor Binding Protein 2 (IGF BP2, Swiss Prot Accession Number P18065) functions to prolong the half-life of the insulin growth factors and have been shown to either inhibit or stimulate the growth promoting effects of the insulin growth factors on cell culture. Specifically, during development, insulin-like growth factor binding protein-2 is expressed in a number of tissues with the highest expression level found in the central nervous system. IGFBP-2 exhibits a 2-10 fold higher affinity for IGF II than for IGF I.

(w) Matrix Metalloproteinase-3 (MMP3)

Matrix Metalloproteinase-3 (MMP3, Swiss Prot Accession Number P08254) is also known as stromelysin-1 and Transin-1. MMP3 is involved in the breakdown of extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis. Most MMP's are secreted as inactive proproteins which are activated when cleaved by extracellular proteinases. MMP3 encodes an enzyme which degrades fibronectin, laminin, collagens III, IV, IX, and X, and cartilage proteoglycans. The enzyme is thought to be involved in wound repair, progression of atherosclerosis, and tumor initiation. MMP3 is part of a cluster of MMP genes which localize to chromosome 11q22.3.

(x) Peptide YY (PYY)

Peptide YY (PYY, Swiss-Prot Accession Number P10082) is also known as peptide tyrosine tyrosine and pancreatic peptide YY_3-36. Peptide YY exerts its action through neuropeptide Y receptors, inhibits gastric motility and increases water and electrolyte absorption in the colon. PYY may also suppress pancreatic secretion. It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal, and has been shown to reduce appetite. PYY works by slowing the gastric emptying; hence, it increases efficiency of digestion and nutrient absorption after meal. Research has also indicated that PYY may be useful in removing aluminum accumulated in the brain.

(y) Stem Cell Factor (SCF)

Stem Cell Factor (SCF, UniProtKB/TrEMBL Q13528) is also known as kit-ligand, KL, and steel factor. SCF functions SCF plays an important role in the hematopoiesis during embryonic development. Sites where hematopoiesis takes place, such as the fetal liver and bone marrow, all express SCF. SCF may serve as guidance cues that direct hematopoietic stem cells (HSCs) to their stem cell niche (the microenvironment in which a stem cell resides), and it plays an important role in HSC maintenance. Non-lethal point mutants on the c-Kit receptor can cause anemia, decreased fertility, and decreased pigmentation. During development, the presence of the SCF also plays an important role in the localization of melanocytes, cells that produce melanin and control pigmentation. In melanogenisis, melanoblasts migrate from the neural crest to their appropriate locations in the epidermis. Melanoblasts express the Kit receptor, and it is believed that SCF guides these cells to their terminal locations. SCF also regulates survival and proliferation of fully differentiated melanocytes in adults. In spermatogenesis, c-Kit is expressed in primordial germ cells, spermatogonia, and in primordial oocytes. It is also expressed in the primordial germ cells of females. SCF is expressed along the pathways that the germ cells use to reach their terminal destination in the body. It is also expressed in the final destinations for these cells. Like for melanoblasts, this helps guide the cells to their appropriate locations in the body

(z) Tumor Necrosis Factor Receptor Type II (TNF RII)

Tumor Necrosis Factor Receptor Type II (TNF RII, Swiss-Prot Accession Number P20333) is also known as p75, p80 TNF alpha receptor, and TNFRSF1B. TNF RII is a protein that in humans is encoded by the TNFRSF1B gene. The protein encoded by this gene is a member of the Tumor necrosis factor receptor superfamily, which also contains TNFRSF1A. The protein encoded by this gene is a member of the TNF-receptor superfamily. This protein and TNF-receptor 1 form a heterocomplex that mediates the recruitment of two anti-apoptotic proteins, c-IAP1 and c-IAP2, which possess E3 ubiquitin ligase activity. The function of IAPs in TNF-receptor signaling is unknown; however, c-IAP1 is thought to potentiate TNF-induced apoptosis by the ubiquitination and degradation of TNF-receptor-associated factor 2, which mediates anti-apoptotic signals. Knockout studies in mice also suggest a role of this protein in protecting neurons from apoptosis by stimulating antioxidative pathways.

(aa) AXL Oncogene

AXL (Swiss-Prot Accession Number P30530) is also known as UFO, ARK, and tyrosine-protein kinase receptor UFO. The protein encoded by AXL is a member of the receptor tyrosine kinase subfamily. Although it is similar to other receptor tyrosine kinases, the AXL protein represents a unique structure of the extracellular region that juxtaposes IgL and FNIII repeats. AXL transduces signals from the extracellular matrix into the cytoplasm by binding growth factors like vitamin K-dependent protein growth-arrest-specific gene 6. It is involved in the stimulation of cell proliferation. This receptor can also mediate cell aggregation by homophilic binding. AXL is a chronic myelogenous leukemia-associated oncogene and also associated with colon cancer and melanoma.

(bb) Eotaxin 3

Eotaxin 3 (Swiss-Prot Accession Number P51671) is also known as C-C motif chemokine 11 (CCL11), small inducible cytokine A11, and eosinophil chemotactic protein. Eotaxin 3 is a small cytokine belonging to the CC chemokine family that is also called Eotaxin-3, Macrophage inflammatory protein 4-alpha (MIP-4-alpha), Thymic stroma chemokine-1 (TSC-1), and IMAC. It is expressed by several tissues including heart, lung and ovary, and in endothelial cells that have been stimulated with the cytokine interleukin 4.[1][2] CCL26 is chemotactic for eosinophils and basophils and elicits its effects by binding to the cell surface chemokine receptor CCR3.

(cc) Fatty Acid Binding Protein (FABP)

Fatty Acid Binding Protein (FABP, Swiss-Prot Accession Number Q01469) is also known as epidermal-type fatty acid binding protein, and fatty-acid binding protein 5. This gene encodes the fatty acid binding protein found in epidermal cells, and was first identified as being upregulated in psoriasis tissue. Fatty acid binding proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. It is thought that FABPs roles include fatty acid uptake, transport, and metabolism.

(dd) Basic Fibroblast Growth Factor (FGF basic)

Basic Fibroblast Growth Factor (FGF basic, Swiss-Prot Accession NumberP09038) is also known as heparin-binding growth factor. In normal tissue, basic fibroblast growth factor is present in basement membranes and in the subendothelial extracellular matrix of blood vessels. It stays membrane-bound as long as there is no signal peptide. It has been hypothesized that, during both wound healing of normal tissues and tumor development, the action of heparan sulfate-degrading enzymes activates FGF basic, thus mediating the formation of new blood vessels. Additionally, FGF basic is a critical component of human embryonic stem cell culture medium; the growth factor is necessary for the cells to remain in an undifferentiated state, although the mechanisms by which it does this are poorly defined. It has been demonstrated to induce gremlin expression which in turn is known to inhibit the induction of differentiation by bone morphogenetic proteins. It is necessary in mouse-feeder cell dependent culture systems, as well as in feeder and serum-free culture systems.

(ee) Myoglobin

Myoglobin (Swiss-Prot Accession Number P02144) is released from damaged muscle tissue (rhabdomyolysis), which has very high concentrations of myoglobin. The released myoglobin is filtered by the kidneys but is toxic to the renal tubular epithelium and so may cause acute renal failure. Myoglobin is a sensitive marker for muscle injury, making it a potential marker for heart attack in patients with chest pain.

(ff) Resistin (RETN)

Resistin (RETN, UniProtKB/TrEMBL Q76B53) is theorized to participate in the inflammatory response. Resistin has also been shown to increase transcriptional events leading to an increased expression of several pro-inflammatory cytokines including (but not limited to) interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), and tumor necrosis factor-α (TNF-α) in an NF-KB-mediated fashion. It has also been demonstrated that resistin upregulates intracellular adhesion molecule-1 (ICAM1) vascular cell-adhesion molecule-1 (VCAM1) and CCL2, all of which are occupied in chemotactic pathways involved in leukocyte recruitment to sites of infection. Resistin itself can be upregulated by interleukins and also by microbial antigens such as lipopolysaccharide, which are recognized by leukocytes. Taken together, because resistin is reputed to contribute to insulin resistance, results such as those mentioned suggest that resistin may be a link in the well-known association between inflammation and insulin resistance. In fact, recent data have shown positive correlations between obesity, insulin resistance, and chronic inflammation which is believed to be directed in part by resistin signaling.

(gg) Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor 3 (TRAIL R3)

TRAIL R3 (Swiss-Prot Accession Number P83626 (mouse)) is also known as tumor necrosis factor-related apoptosis-inducing ligand receptor 3, and tumor necrosis factor receptor mouse homolog. TRAIL R3 is a decoy receptor for TRAIL, a member of the tumor necrosis factor family. In several cell types decoy receptors inhibit TRAIL-induced apoptosis by binding TRAIL and thus preventing its binding to proapoptotic TRAIL receptors.

(hh) Endothelin 1 (ET1)

Endothelin 1 (ET1, UniProtKB/TrEMBL Q6FH53) is also known as EDN1 and EDN1 protein. Endothelin 1 is a protein that constricts blood vessels and raises blood pressure. It is normally kept in balance by other mechanisms, but when over-expressed, it contributes to high blood pressure (hypertension) and heart disease. Endothelin 1 peptides and receptors are implicated in the pathogenesis of a number of disease states, including cancer and heart disease.

(ii) Neuronal Cell Adhesion Molecule (NrCAM)

Neuronal Cell Adhesion Molecule (NrCAM, UniProtKB/TrEMBL Q14CA1) encodes a neuronal cell adhesion molecule with multiple immunoglobulin-like C2-type domains and fibronectin type-III domains. This ankyrin-binding protein is involved in neuron-neuron adhesion and promotes directional signaling during axonal cone growth. This gene is also expressed in non-neural tissues and may play a general role in cell-cell communication via signaling from its intracellular domain to the actin cytoskeleton during directional cell migration. Allelic variants of this gene have been associated with autism and addiction vulnerability.

(jj) Tenascin C (TN-C)

Tenascin C (TN-C, UniProt/TrEMBL Q99857) has anti-adhesive properties, causing cells in tissue culture to become rounded after it is added to the medium. One mechanism to explain this may come from its ability to bind to the extracellular matrix glycoprotein fibronectin and block fibronectin's interactions with specific syndecans. The expression of tenascin-C in the stroma of certain tumors is associated with a poor prognosis.

(kk) Vascular Cell Adhesion Molecule 1 (VCAM1)

Vascular Cell Adhesion Molecule 1 (VCAM1, Swiss-Prot Accession Number P19320) is also known as vascular cell adhesion protein 1. VCAM1 mediates the adhesion of lymphocytes, monocytes, eosinophils, and basophils to vascular endothelium. It also functions in leukocyte-endothelial cell signal transduction, and it may play a role in the development of atherosclerosis and rheumatoid arthritis. Upregulation of VCAM-1 in endothelial cells by cytokines occurs as a result of increased gene transcription (e.g., in response to Tumor necrosis factor-alpha (TNF-α) and Interleukin-1 (IL-1)) and through stabilization of Messenger RNA (mRNA) (e.g., Interleukin-4 (IL-4)). The promoter region of the VCAM-1 gene contains functional tandem NF-κB (nuclear factor-kappa B) sites. The sustained expression of VCAM-1 lasts over 24 hours. Primarily, the VCAM-1 protein is an endothelial ligand for VLA-4 (Very Late Antigen-4 or α4β1) of the β1 subfamily of integrins, and for integrin α4β7. VCAM-1 expression has also been observed in other cell types (e.g., smooth muscle cells). It has also been shown to interact with EZR and Moesin. Certain melanoma cells can use VCAM-1 to adhere to the endothelium, and VCAM-1 may participate in monocyte recruitment to atherosclerotic sites.

(ll) Cortisol

Cortisol (Swiss-Prot Accession Number P08185) is also known as corticosteroid-binding globulin, transcortin, and Serpin A6. Cortisol is a steroid hormone or glucocorticoid produced by the adrenal gland. It is released in response to stress, and to a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis, suppress the immune system, and aid in fat, protein and carbohydrate metabolism. It also decreases bone formation. In addition, cortisol can weaken the activity of the immune system. Cortisol prevents proliferation of T-cells by rendering the interleukin-2 producer T-cells unresponsive to interleukin-1 (IL-1), and unable to produce the T-cell growth factor. Cortisol also has a negative feedback effect on interleukin-1. IL-1 must be especially useful in combating some diseases; however, endotoxin bacteria have gained an advantage by forcing the hypothalamus to increase cortisol levels via forcing secretion of CRH hormone, thus antagonizing IL-1 in this case. The suppressor cells are not affected by GRMF, so that the effective set point for the immune cells may be even higher than the set point for physiological processes. It reflects leukocyte redistribution to lymph nodes, bone marrow, and skin.

II. Combinations of Analytes Measured by Multiplexed Assay

The device for diagnosing, monitoring, or determining a renal disorder involves determining the presence or concentrations of a combination of sample analytes in a test sample. The combinations of sample analytes, as defined herein, are any group of three or more analytes selected from the biomarker analytes, including but not limited to alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, VEGF, BLC, CD40, IGF BP2, MMP3, peptide YY, stem cell factor, TNF RII, AXL, Eotaxin 3, FABP, FGF basic, myoglobin, resistin, TRAIL R3, endothelin 1, NrCAM, Tenascin C, VCAM1, and cortisol. In one embodiment, the combination of analytes may be selected to provide a group of analytes associated with renal disorder in a mammal.

In one embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, or all sixteen of the sixteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table A.

	TABLE A
	alpha-1 microglobulin	beta-2 microglobulin	calbindin
	alpha-1 microglobulin	beta-2 microglobulin	clusterin
	alpha-1 microglobulin	beta-2 microglobulin	CTGF
	alpha-1 microglobulin	beta-2 microglobulin	creatinine
	alpha-1 microglobulin	beta-2 microglobulin	cystatin C
	alpha-1 microglobulin	beta-2 microglobulin	GST-alpha
	alpha-1 microglobulin	beta-2 microglobulin	KIM-1
	alpha-1 microglobulin	beta-2 microglobulin	microalbumin
	alpha-1 microglobulin	beta-2 microglobulin	NGAL
	alpha-1 microglobulin	beta-2 microglobulin	osteopontin
	alpha-1 microglobulin	beta-2 microglobulin	THP
	alpha-1 microglobulin	beta-2 microglobulin	TIMP-1
	alpha-1 microglobulin	beta-2 microglobulin	TFF-3
	alpha-1 microglobulin	beta-2 microglobulin	VEGF
	alpha-1 microglobulin	calbindin	clusterin
	alpha-1 microglobulin	calbindin	CTGF
	alpha-1 microglobulin	calbindin	creatinine
	alpha-1 microglobulin	calbindin	cystatin C
	alpha-1 microglobulin	calbindin	GST-alpha
	alpha-1 microglobulin	calbindin	KIM-1
	alpha-1 microglobulin	calbindin	microalbumin
	alpha-1 microglobulin	calbindin	NGAL
	alpha-1 microglobulin	calbindin	osteopontin
	alpha-1 microglobulin	calbindin	THP
	alpha-1 microglobulin	calbindin	TIMP-1
	alpha-1 microglobulin	calbindin	TFF-3
	alpha-1 microglobulin	calbindin	VEGF
	alpha-1 microglobulin	clusterin	CTGF
	alpha-1 microglobulin	clusterin	creatinine
	alpha-1 microglobulin	clusterin	cystatin C
	alpha-1 microglobulin	clusterin	GST-alpha
	alpha-1 microglobulin	clusterin	KIM-1
	alpha-1 microglobulin	clusterin	microalbumin
	alpha-1 microglobulin	clusterin	NGAL
	alpha-1 microglobulin	clusterin	osteopontin
	alpha-1 microglobulin	clusterin	THP
	alpha-1 microglobulin	clusterin	TIMP-1
	alpha-1 microglobulin	clusterin	TFF-3
	alpha-1 microglobulin	clusterin	VEGF
	alpha-1 microglobulin	CTGF	creatinine
	alpha-1 microglobulin	CTGF	cystatin C
	alpha-1 microglobulin	CTGF	GST-alpha
	alpha-1 microglobulin	CTGF	KIM-1
	alpha-1 microglobulin	CTGF	microalbumin
	alpha-1 microglobulin	CTGF	NGAL
	alpha-1 microglobulin	CTGF	osteopontin
	alpha-1 microglobulin	CTGF	THP
	alpha-1 microglobulin	CTGF	TIMP-1
	alpha-1 microglobulin	CTGF	TFF-3
	alpha-1 microglobulin	CTGF	VEGF
	alpha-1 microglobulin	creatinine	cystatin C
	alpha-1 microglobulin	creatinine	GST-alpha
	alpha-1 microglobulin	creatinine	KIM-1
	alpha-1 microglobulin	creatinine	microalbumin
	alpha-1 microglobulin	creatinine	NGAL
	alpha-1 microglobulin	creatinine	osteopontin
	alpha-1 microglobulin	creatinine	THP
	alpha-1 microglobulin	creatinine	TIMP-1
	alpha-1 microglobulin	creatinine	TFF-3
	alpha-1 microglobulin	creatinine	VEGF
	alpha-1 microglobulin	cystatin C	GST-alpha
	alpha-1 microglobulin	cystatin C	KIM-1
	alpha-1 microglobulin	cystatin C	microalbumin
	alpha-1 microglobulin	cystatin C	NGAL
	alpha-1 microglobulin	cystatin C	osteopontin
	alpha-1 microglobulin	cystatin C	THP
	alpha-1 microglobulin	cystatin C	TIMP-1
	alpha-1 microglobulin	cystatin C	TFF-3
	alpha-1 microglobulin	cystatin C	VEGF
	alpha-1 microglobulin	GST-alpha	KIM-1
	alpha-1 microglobulin	GST-alpha	microalbumin
	alpha-1 microglobulin	GST-alpha	NGAL
	alpha-1 microglobulin	GST-alpha	osteopontin
	alpha-1 microglobulin	GST-alpha	THP
	alpha-1 microglobulin	GST-alpha	TIMP-1
	alpha-1 microglobulin	GST-alpha	TFF-3
	alpha-1 microglobulin	GST-alpha	VEGF
	alpha-1 microglobulin	KIM-1	microalbumin
	alpha-1 microglobulin	KIM-1	NGAL
	alpha-1 microglobulin	KIM-1	osteopontin
	alpha-1 microglobulin	KIM-1	THP
	alpha-1 microglobulin	KIM-1	TIMP-1
	alpha-1 microglobulin	KIM-1	TFF-3
	alpha-1 microglobulin	KIM-1	VEGF
	alpha-1 microglobulin	microalbumin	NGAL
	alpha-1 microglobulin	microalbumin	osteopontin
	alpha-1 microglobulin	microalbumin	THP
	alpha-1 microglobulin	microalbumin	TIMP-1
	alpha-1 microglobulin	microalbumin	TFF-3
	alpha-1 microglobulin	microalbumin	VEGF
	alpha-1 microglobulin	NGAL	osteopontin
	alpha-1 microglobulin	NGAL	THP
	alpha-1 microglobulin	NGAL	TIMP-1
	alpha-1 microglobulin	NGAL	TFF-3
	alpha-1 microglobulin	NGAL	VEGF
	alpha-1 microglobulin	osteopontin	THP
	alpha-1 microglobulin	osteopontin	TIMP-1
	alpha-1 microglobulin	osteopontin	TFF-3
	alpha-1 microglobulin	osteopontin	VEGF
	alpha-1 microglobulin	THP	TIMP-1
	alpha-1 microglobulin	THP	TFF-3
	alpha-1 microglobulin	THP	VEGF
	alpha-1 microglobulin	TIMP-1	TFF-3
	alpha-1 microglobulin	TIMP-1	VEGF
	alpha-1 microglobulin	TFF-3	VEGF
	beta-2 microglobulin	calbindin	clusterin
	beta-2 microglobulin	calbindin	CTGF
	beta-2 microglobulin	calbindin	creatinine
	beta-2 microglobulin	calbindin	cystatin C
	beta-2 microglobulin	calbindin	GST-alpha
	beta-2 microglobulin	calbindin	KIM-1
	beta-2 microglobulin	calbindin	microalbumin
	beta-2 microglobulin	calbindin	NGAL
	beta-2 microglobulin	calbindin	osteopontin
	beta-2 microglobulin	calbindin	THP
	beta-2 microglobulin	calbindin	TIMP-1
	beta-2 microglobulin	calbindin	TFF-3
	beta-2 microglobulin	calbindin	VEGF
	beta-2 microglobulin	clusterin	CTGF
	beta-2 microglobulin	clusterin	creatinine
	beta-2 microglobulin	clusterin	cystatin C
	beta-2 microglobulin	clusterin	GST-alpha
	beta-2 microglobulin	clusterin	KIM-1
	beta-2 microglobulin	clusterin	microalbumin
	beta-2 microglobulin	clusterin	NGAL
	beta-2 microglobulin	clusterin	osteopontin
	beta-2 microglobulin	clusterin	THP
	beta-2 microglobulin	clusterin	TIMP-1
	beta-2 microglobulin	clusterin	TFF-3
	beta-2 microglobulin	clusterin	VEGF
	beta-2 microglobulin	CTGF	creatinine
	beta-2 microglobulin	CTGF	cystatin C
	beta-2 microglobulin	CTGF	GST-alpha
	beta-2 microglobulin	CTGF	KIM-1
	beta-2 microglobulin	CTGF	microalbumin
	beta-2 microglobulin	CTGF	NGAL
	beta-2 microglobulin	CTGF	osteopontin
	beta-2 microglobulin	CTGF	THP
	beta-2 microglobulin	CTGF	TIMP-1
	beta-2 microglobulin	CTGF	TFF-3
	beta-2 microglobulin	CTGF	VEGF
	beta-2 microglobulin	creatinine	cystatin C
	beta-2 microglobulin	creatinine	GST-alpha
	beta-2 microglobulin	creatinine	KIM-1
	beta-2 microglobulin	creatinine	microalbumin
	beta-2 microglobulin	creatinine	NGAL
	beta-2 microglobulin	creatinine	osteopontin
	beta-2 microglobulin	creatinine	THP
	beta-2 microglobulin	creatinine	TIMP-1
	beta-2 microglobulin	creatinine	TFF-3
	beta-2 microglobulin	creatinine	VEGF
	beta-2 microglobulin	cystatin C	GST-alpha
	beta-2 microglobulin	cystatin C	KIM-1
	beta-2 microglobulin	cystatin C	microalbumin
	beta-2 microglobulin	cystatin C	NGAL
	beta-2 microglobulin	cystatin C	osteopontin
	beta-2 microglobulin	cystatin C	THP
	beta-2 microglobulin	cystatin C	TIMP-1
	beta-2 microglobulin	cystatin C	TFF-3
	beta-2 microglobulin	cystatin C	VEGF
	beta-2 microglobulin	GST-alpha	KIM-1
	beta-2 microglobulin	GST-alpha	microalbumin
	beta-2 microglobulin	GST-alpha	NGAL
	beta-2 microglobulin	GST-alpha	osteopontin
	beta-2 microglobulin	GST-alpha	THP
	beta-2 microglobulin	GST-alpha	TIMP-1
	beta-2 microglobulin	GST-alpha	TFF-3
	beta-2 microglobulin	GST-alpha	VEGF
	beta-2 microglobulin	KIM-1	microalbumin
	beta-2 microglobulin	KIM-1	NGAL
	beta-2 microglobulin	KIM-1	osteopontin
	beta-2 microglobulin	KIM-1	THP
	beta-2 microglobulin	KIM-1	TIMP-1
	beta-2 microglobulin	KIM-1	TFF-3
	beta-2 microglobulin	KIM-1	VEGF
	beta-2 microglobulin	microalbumin	NGAL
	beta-2 microglobulin	microalbumin	osteopontin
	beta-2 microglobulin	microalbumin	THP
	beta-2 microglobulin	microalbumin	TIMP-1
	beta-2 microglobulin	microalbumin	TFF-3
	beta-2 microglobulin	microalbumin	VEGF
	beta-2 microglobulin	NGAL	osteopontin
	beta-2 microglobulin	NGAL	THP
	beta-2 microglobulin	NGAL	TIMP-1
	beta-2 microglobulin	NGAL	TFF-3
	beta-2 microglobulin	NGAL	VEGF
	beta-2 microglobulin	osteopontin	THP
	beta-2 microglobulin	osteopontin	TIMP-1
	beta-2 microglobulin	osteopontin	TFF-3
	beta-2 microglobulin	osteopontin	VEGF
	beta-2 microglobulin	THP	TIMP-1
	beta-2 microglobulin	THP	TFF-3
	beta-2 microglobulin	THP	VEGF
	beta-2 microglobulin	TIMP-1	TFF-3
	beta-2 microglobulin	TIMP-2	VEGF
	beta-2 microglobulin	TFF-3	VEGF
	calbindin	clusterin	CTGF
	calbindin	clusterin	creatinine
	calbindin	clusterin	cystatin C
	calbindin	clusterin	GST-alpha
	calbindin	clusterin	KIM-1
	calbindin	clusterin	microalbumin
	calbindin	clusterin	NGAL
	calbindin	clusterin	osteopontin
	calbindin	clusterin	THP
	calbindin	clusterin	TIMP-1
	calbindin	clusterin	TFF-3
	calbindin	clusterin	VEGF
	calbindin	CTGF	creatinine
	calbindin	CTGF	cystatin C
	calbindin	CTGF	GST-alpha
	calbindin	CTGF	KIM-1
	calbindin	CTGF	microalbumin
	calbindin	CTGF	NGAL
	calbindin	CTGF	osteopontin
	calbindin	CTGF	THP
	calbindin	CTGF	TIMP-1
	calbindin	CTGF	TFF-3
	calbindin	CTGF	VEGF
	calbindin	creatinine	cystatin C
	calbindin	creatinine	GST-alpha
	calbindin	creatinine	KIM-1
	calbindin	creatinine	microalbumin
	calbindin	creatinine	NGAL
	calbindin	creatinine	osteopontin
	calbindin	creatinine	THP
	calbindin	creatinine	TIMP-1
	calbindin	creatinine	TFF-3
	calbindin	creatinine	VEGF
	calbindin	cystatin C	GST-alpha
	calbindin	cystatin C	KIM-1
	calbindin	cystatin C	microalbumin
	calbindin	cystatin C	NGAL
	calbindin	cystatin C	osteopontin
	calbindin	cystatin C	THP
	calbindin	cystatin C	TIMP-1
	calbindin	cystatin C	TFF-3
	calbindin	cystatin C	VEGF
	calbindin	GST-alpha	KIM-1
	calbindin	GST-alpha	microalbumin
	calbindin	GST-alpha	NGAL
	calbindin	GST-alpha	osteopontin
	calbindin	GST-alpha	THP
	calbindin	GST-alpha	TIMP-1
	calbindin	GST-alpha	TFF-3
	calbindin	GST-alpha	VEGF
	calbindin	KIM-1	microalbumin
	calbindin	KIM-1	NGAL
	calbindin	KIM-1	osteopontin
	calbindin	KIM-1	THP
	calbindin	KIM-1	TIMP-1
	calbindin	KIM-1	TFF-3
	calbindin	KIM-1	VEGF
	calbindin	microalbumin	NGAL
	calbindin	microalbumin	osteopontin
	calbindin	microalbumin	THP
	calbindin	microalbumin	TIMP-1
	calbindin	microalbumin	TFF-3
	calbindin	microalbumin	VEGF
	calbindin	NGAL	osteopontin
	calbindin	NGAL	THP
	calbindin	NGAL	TIMP-1
	calbindin	NGAL	TFF-3
	calbindin	NGAL	VEGF
	calbindin	osteopontin	THP
	calbindin	osteopontin	TIMP-1
	calbindin	osteopontin	TFF-3
	calbindin	osteopontin	VEGF
	calbindin	THP	TIMP-1
	calbindin	THP	TFF-3
	calbindin	THP	VEGF
	calbindin	TIMP-1	TFF-3
	calbindin	TIMP-1	VEGF
	calbindin	TFF-3	VEGF
	clusterin	CTGF	creatinine
	clusterin	CTGF	cystatin C
	clusterin	CTGF	GST-alpha
	clusterin	CTGF	KIM-1
	clusterin	CTGF	microalbumin
	clusterin	CTGF	NGAL
	clusterin	CTGF	osteopontin
	clusterin	CTGF	THP
	clusterin	CTGF	TIMP-1
	clusterin	CTGF	TFF-3
	clusterin	CTGF	VEGF
	clusterin	creatinine	cystatin C
	clusterin	creatinine	GST-alpha
	clusterin	creatinine	KIM-1
	clusterin	creatinine	microalbumin
	clusterin	creatinine	NGAL
	clusterin	creatinine	osteopontin
	clusterin	creatinine	THP
	clusterin	creatinine	TIMP-1
	clusterin	creatinine	TFF-3
	clusterin	creatinine	VEGF
	clusterin	cystatin C	GST-alpha
	clusterin	cystatin C	KIM-1
	clusterin	cystatin C	microalbumin
	clusterin	cystatin C	NGAL
	clusterin	cystatin C	osteopontin
	clusterin	cystatin C	THP
	clusterin	cystatin C	TIMP-1
	clusterin	cystatin C	TFF-3
	clusterin	cystatin C	VEGF
	clusterin	GST-alpha	KIM-1
	clusterin	GST-alpha	microalbumin
	clusterin	GST-alpha	NGAL
	clusterin	GST-alpha	osteopontin
	clusterin	GST-alpha	THP
	clusterin	GST-alpha	TIMP-1
	clusterin	GST-alpha	TFF-3
	clusterin	GST-alpha	VEGF
	clusterin	KIM-1	microalbumin
	clusterin	KIM-1	NGAL
	clusterin	KIM-1	osteopontin
	clusterin	KIM-1	THP
	clusterin	KIM-1	TIMP-1
	clusterin	KIM-1	TFF-3
	clusterin	KIM-1	VEGF
	clusterin	microalbumin	NGAL
	clusterin	microalbumin	osteopontin
	clusterin	microalbumin	THP
	clusterin	microalbumin	TIMP-1
	clusterin	microalbumin	TFF-3
	clusterin	microalbumin	VEGF
	clusterin	NGAL	osteopontin
	clusterin	NGAL	THP
	clusterin	NGAL	TIMP-1
	clusterin	NGAL	TFF-3
	clusterin	NGAL	VEGF
	clusterin	osteopontin	THP
	clusterin	osteopontin	TIMP-1
	clusterin	osteopontin	TFF-3
	clusterin	osteopontin	VEGF
	clusterin	THP	TIMP-1
	clusterin	THP	TFF-3
	clusterin	THP	VEGF
	clusterin	TIMP-1	TFF-3
	clusterin	TIMP-1	VEGF
	clusterin	TFF-3	VEGF
	CTGF	creatinine	cystatin C
	CTGF	creatinine	GST-alpha
	CTGF	creatinine	KIM-1
	CTGF	creatinine	microalbumin
	CTGF	creatinine	NGAL
	CTGF	creatinine	osteopontin
	CTGF	creatinine	THP
	CTGF	creatinine	TIMP-1
	CTGF	creatinine	TFF-3
	CTGF	creatinine	VEGF
	CTGF	cystatin C	GST-alpha
	CTGF	cystatin C	KIM-1
	CTGF	cystatin C	microalbumin
	CTGF	cystatin C	NGAL
	CTGF	cystatin C	osteopontin
	CTGF	cystatin C	THP
	CTGF	cystatin C	TIMP-1
	CTGF	cystatin C	TFF-3
	CTGF	cystatin C	VEGF
	CTGF	GST-alpha	KIM-1
	CTGF	GST-alpha	microalbumin
	CTGF	GST-alpha	NGAL
	CTGF	GST-alpha	osteopontin
	CTGF	GST-alpha	THP
	CTGF	GST-alpha	TIMP-1
	CTGF	GST-alpha	TFF-3
	CTGF	GST-alpha	VEGF
	CTGF	KIM-1	microalbumin
	CTGF	KIM-1	NGAL
	CTGF	KIM-1	osteopontin
	CTGF	KIM-1	THP
	CTGF	KIM-1	TIMP-1
	CTGF	KIM-1	TFF-3
	CTGF	KIM-1	VEGF
	CTGF	microalbumin	NGAL
	CTGF	microalbumin	osteopontin
	CTGF	microalbumin	THP
	CTGF	microalbumin	TIMP-1
	CTGF	microalbumin	TFF-3
	CTGF	microalbumin	VEGF
	CTGF	NGAL	osteopontin
	CTGF	NGAL	THP
	CTGF	NGAL	TIMP-1
	CTGF	NGAL	TFF-3
	CTGF	NGAL	VEGF
	CTGF	osteopontin	THP
	CTGF	osteopontin	TIMP-1
	CTGF	osteopontin	TFF-3
	CTGF	osteopontin	VEGF
	CTGF	THP	TIMP-1
	CTGF	THP	TFF-3
	CTGF	THP	VEGF
	CTGF	TIMP-1	TFF-3
	CTGF	TIMP-1	VEGF
	CTGF	TFF-3	VEGF
	creatinine	cystatin C	GST-alpha
	creatinine	cystatin C	KIM-1
	creatinine	cystatin C	microalbumin
	creatinine	cystatin C	NGAL
	creatinine	cystatin C	osteopontin
	creatinine	cystatin C	THP
	creatinine	cystatin C	TIMP-1
	creatinine	cystatin C	TFF-3
	creatinine	cystatin C	VEGF
	creatinine	GST-alpha	KIM-1
	creatinine	GST-alpha	microalbumin
	creatinine	GST-alpha	NGAL
	creatinine	GST-alpha	osteopontin
	creatinine	GST-alpha	THP
	creatinine	GST-alpha	TIMP-1
	creatinine	GST-alpha	TFF-3
	creatinine	GST-alpha	VEGF
	creatinine	KIM-1	microalbumin
	creatinine	KIM-1	NGAL
	creatinine	KIM-1	osteopontin
	creatinine	KIM-1	THP
	creatinine	KIM-1	TIMP-1
	creatinine	KIM-1	TFF-3
	creatinine	KIM-1	VEGF
	creatinine	microalbumin	NGAL
	creatinine	microalbumin	osteopontin
	creatinine	microalbumin	THP
	creatinine	microalbumin	TIMP-1
	creatinine	microalbumin	TFF-3
	creatinine	microalbumin	VEGF
	creatinine	NGAL	osteopontin
	creatinine	NGAL	THP
	creatinine	NGAL	TIMP-1
	creatinine	NGAL	TFF-3
	creatinine	NGAL	VEGF
	creatinine	osteopontin	THP
	creatinine	osteopontin	TIMP-1
	creatinine	osteopontin	TFF-3
	creatinine	osteopontin	VEGF
	creatinine	THP	TIMP-1
	creatinine	THP	TFF-3
	creatinine	THP	VEGF
	creatinine	TIMP-1	TFF-3
	creatinine	TIMP-1	VEGF
	creatinine	TFF-3	VEGF
	cystatin C	GST-alpha	KIM-1
	cystatin C	GST-alpha	microalbumin
	cystatin C	GST-alpha	NGAL
	cystatin C	GST-alpha	osteopontin
	cystatin C	GST-alpha	THP
	cystatin C	GST-alpha	TIMP-1
	cystatin C	GST-alpha	TFF-3
	cystatin C	GST-alpha	VEGF
	cystatin C	KIM-1	microalbumin
	cystatin C	KIM-1	NGAL
	cystatin C	KIM-1	osteopontin
	cystatin C	KIM-1	THP
	cystatin C	KIM-1	TIMP-1
	cystatin C	KIM-1	TFF-3
	cystatin C	KIM-1	VEGF
	cystatin C	microalbumin	NGAL
	cystatin C	microalbumin	osteopontin
	cystatin C	microalbumin	THP
	cystatin C	microalbumin	TIMP-1
	cystatin C	microalbumin	TFF-3
	cystatin C	microalbumin	VEGF
	cystatin C	NGAL	osteopontin
	cystatin C	NGAL	THP
	cystatin C	NGAL	TIMP-1
	cystatin C	NGAL	TFF-3
	cystatin C	NGAL	VEGF
	cystatin C	osteopontin	THP
	cystatin C	osteopontin	TIMP-1
	cystatin C	osteopontin	TFF-3
	cystatin C	osteopontin	VEGF
	cystatin C	THP	TIMP-1
	cystatin C	THP	TFF-3
	cystatin C	THP	VEGF
	cystatin C	TIMP-1	TFF-3
	cystatin C	TIMP-1	VEGF
	cystatin C	TFF-3	VEGF
	GST-alpha	KIM-1	microalbumin
	GST-alpha	KIM-1	NGAL
	GST-alpha	KIM-1	osteopontin
	GST-alpha	KIM-1	THP
	GST-alpha	KIM-1	TIMP-1
	GST-alpha	KIM-1	TFF-3
	GST-alpha	KIM-1	VEGF
	GST-alpha	microalbumin	NGAL
	GST-alpha	microalbumin	osteopontin
	GST-alpha	microalbumin	THP
	GST-alpha	microalbumin	TIMP-1
	GST-alpha	microalbumin	TFF-3
	GST-alpha	microalbumin	VEGF
	GST-alpha	NGAL	osteopontin
	GST-alpha	NGAL	THP
	GST-alpha	NGAL	TIMP-1
	GST-alpha	NGAL	TFF-3
	GST-alpha	NGAL	VEGF
	GST-alpha	osteopontin	THP
	GST-alpha	osteopontin	TIMP-1
	GST-alpha	osteopontin	TFF-3
	GST-alpha	osteopontin	VEGF
	GST-alpha	THP	TIMP-1
	GST-alpha	THP	TFF-3
	GST-alpha	THP	VEGF
	GST-alpha	TIMP-1	TFF-3
	GST-alpha	TIMP-1	VEGF
	GST-alpha	TFF-3	VEGF
	KIM-1	microalbumin	NGAL
	KIM-1	microalbumin	osteopontin
	KIM-1	microalbumin	THP
	KIM-1	microalbumin	TIMP-1
	KIM-1	microalbumin	TFF-3
	KIM-1	microalbumin	VEGF
	KIM-1	NGAL	osteopontin
	KIM-1	NGAL	THP
	KIM-1	NGAL	TIMP-1
	KIM-1	NGAL	TFF-3
	KIM-1	NGAL	VEGF
	KIM-1	osteopontin	THP
	KIM-1	osteopontin	TIMP-1
	KIM-1	osteopontin	TFF-3
	KIM-1	osteopontin	VEGF
	KIM-1	THP	TIMP-1
	KIM-1	THP	TFF-3
	KIM-1	THP	VEGF
	KIM-1	TIMP-1	TFF-3
	KIM-1	TIMP-1	VEGF
	KIM-1	TFF-3	VEGF
	microalbumin	NGAL	osteopontin
	microalbumin	NGAL	THP
	microalbumin	NGAL	TIMP-1
	microalbumin	NGAL	TFF-3
	microalbumin	NGAL	VEGF
	microalbumin	osteopontin	THP
	microalbumin	osteopontin	TIMP-1
	microalbumin	osteopontin	TFF-3
	microalbumin	osteopontin	VEGF
	microalbumin	THP	TIMP-1
	microalbumin	THP	TFF-3
	microalbumin	THP	VEGF
	microalbumin	TIMP-1	TFF-3
	microalbumin	TIMP-1	VEGF
	microalbumin	TFF-3	VEGF
	NGAL	osteopontin	THP
	NGAL	osteopontin	TIMP-1
	NGAL	osteopontin	TFF-3
	NGAL	osteopontin	VEGF
	NGAL	THP	TIMP-1
	NGAL	THP	TFF-3
	NGAL	THP	VEGF
	NGAL	TIMP-1	TFF-3
	NGAL	TIMP-1	VEGF
	NGAL	TFF-3	VEGF
	osteopontin	THP	TIMP-1
	osteopontin	THP	TFF-3
	osteopontin	THP	VEGF
	osteopontin	TIMP-1	TFF-3
	osteopontin	TIMP-1	VEGF
	osteopontin	TFF-3	VEGF
	THP	TIMP-1	TFF-3
	THP	TIMP-1	VEGF
	THP	TFF-3	VEGF
	TIMP-1	TFF-3	VEGF

In one exemplary embodiment, the combination of sample analytes may include creatinine, KIM-1, and THP. In another exemplary embodiment, the combination of sample analytes may include microalbumin, creatinine, and KIM-1. In yet another exemplary embodiment, the combination of sample analytes may include creatinine, THP, and A1M. In still another exemplary embodiment, the combination of sample analytes may include microalbumin, TIMP-1, and osteopontin.

In still another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of obstructive uropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy include three or more biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of obstructive uropathy includes six biomarker analytes, including creatinine, THP, A1M, clusterin, NGAL, and osteopontin.

In yet another embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of glomerulonephritis. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In an additional embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephritis include three or more biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of glomerulonephropathy includes six biomarker analytes, including creatinine, KIM-1, TIMP-1, alpha-1 microglobulin, THP, and osteopontin.

In an additional embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of kidney damage or toxicity. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In anotherembodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include three or more biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of kidney damage or toxicity include six biomarker analytes, including creatinine, KIM-1, THP, osteopontin, NGAL, and TIMP-1.

In a further embodiment, the devices and systems of the current invention may be used to diagnose, monitor or determine the presence of diabetic nephropathy. The combination of sample analytes may include any three of the biomarker analytes previously discussed. In another embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include three or more biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin. In a further embodiment, the devices and systems to diagnose, monitor or determine the presence of diabetic nephropathy include six biomarker analytes, including microalbumin, alpha-1 microglobulin, NGAL, KIM-1, THP, and clusterin.

In another embodiment, the devices and systems of the current invention detect the combination of sample analytes, and may include any three of the biomarker analytes discussed previously to diagnose kidney transplant rejection or other associated disease as discussed previously. In other embodiments, the combination of sample analytes may be any four, any five, any six, any seven, any eight, any nine, any ten, any eleven, any twelve, any thirteen, any fourteen, any fifteen, any sixteen, any seventeen, any eighteen, or any nineteen biomarker analytes. In another embodiment, the combination of sample analytes may comprise a combination listed in Table B.

	TABLE B
	BLC	CD40	IGF BP2
	BLC	CD40	MMP3
	BLC	CD40	peptide YY
	BLC	CD40	stem cell factor
	BLC	CD40	TNF RII
	BLC	CD40	AXL
	BLC	CD40	Eotaxin 3
	BLC	CD40	FABP
	BLC	CD40	FGF basic
	BLC	CD40	myoglobin
	BLC	CD40	resistin
	BLC	CD40	TRAIL R3
	BLC	CD40	endothilin 1
	BLC	CD40	NrCAM
	BLC	CD40	Tenascin C
	BLC	CD40	VCAM1
	BLC	CD40	cortisol
	BLC	IGF BP2	MMP3
	BLC	IGF BP2	peptide YY
	BLC	IGF BP2	stem cell factor
	BLC	IGF BP2	TNF RII
	BLC	IGF BP2	AXL
	BLC	IGF BP2	Eotaxin 3
	BLC	IGF BP2	FABP
	BLC	IGF BP2	FGF basic
	BLC	IGF BP2	myoglobin
	BLC	IGF BP2	resistin
	BLC	IGF BP2	TRAIL R3
	BLC	IGF BP2	endothilin 1
	BLC	IGF BP2	NrCAM
	BLC	IGF BP2	Tenascin C
	BLC	IGF BP2	VCAM1
	BLC	IGF BP2	cortisol
	BLC	MMP3	peptide YY
	BLC	MMP3	stem cell factor
	BLC	MMP3	TNF RII
	BLC	MMP3	AXL
	BLC	MMP3	Eotaxin 3
	BLC	MMP3	FABP
	BLC	MMP3	FGF basic
	BLC	MMP3	myoglobin
	BLC	MMP3	resistin
	BLC	MMP3	TRAIL R3
	BLC	MMP3	endothilin 1
	BLC	MMP3	NrCAM
	BLC	MMP3	Tenascin C
	BLC	MMP3	VCAM1
	BLC	MMP3	cortisol
	BLC	peptide YY	stem cell factor
	BLC	peptide YY	TNF RII
	BLC	peptide YY	AXL
	BLC	peptide YY	Eotaxin 3
	BLC	peptide YY	FABP
	BLC	peptide YY	FGF basic
	BLC	peptide YY	myoglobin
	BLC	peptide YY	resistin
	BLC	peptide YY	TRAIL R3
	BLC	peptide YY	endothilin 1
	BLC	peptide YY	NrCAM
	BLC	peptide YY	Tenascin C
	BLC	peptide YY	VCAM1
	BLC	peptide YY	cortisol
	BLC	stem cell factor	TNF RII
	BLC	stem cell factor	AXL
	BLC	stem cell factor	Eotaxin 3
	BLC	stem cell factor	FABP
	BLC	stem cell factor	FGF basic
	BLC	stem cell factor	myoglobin
	BLC	stem cell factor	resistin
	BLC	stem cell factor	TRAIL R3
	BLC	stem cell factor	endothilin 1
	BLC	stem cell factor	NrCAM
	BLC	stem cell factor	Tenascin C
	BLC	stem cell factor	VCAM1
	BLC	stem cell factor	cortisol
	BLC	TNF RII	AXL
	BLC	TNF RII	Eotaxin 3
	BLC	TNF RII	FABP
	BLC	TNF RII	FGF basic
	BLC	TNF RII	myoglobin
	BLC	TNF RII	resistin
	BLC	TNF RII	TRAIL R3
	BLC	TNF RII	endothilin 1
	BLC	TNF RII	NrCAM
	BLC	TNF RII	Tenascin C
	BLC	TNF RII	VCAM1
	BLC	TNF RII	cortisol
	BLC	AXL	Eotaxin 3
	BLC	AXL	FABP
	BLC	AXL	FGF basic
	BLC	AXL	myoglobin
	BLC	AXL	resistin
	BLC	AXL	TRAIL R3
	BLC	AXL	endothilin 1
	BLC	AXL	NrCAM
	BLC	AXL	Tenascin C
	BLC	AXL	VCAM1
	BLC	AXL	cortisol
	BLC	Eotaxin 3	FABP
	BLC	Eotaxin 3	FGF basic
	BLC	Eotaxin 3	myoglobin
	BLC	Eotaxin 3	resistin
	BLC	Eotaxin 3	TRAIL R3
	BLC	Eotaxin 3	endothilin 1
	BLC	Eotaxin 3	NrCAM
	BLC	Eotaxin 3	Tenascin C
	BLC	Eotaxin 3	VCAM1
	BLC	Eotaxin 3	cortisol
	BLC	FABP	FGF basic
	BLC	FABP	myoglobin
	BLC	FABP	resistin
	BLC	FABP	TRAIL R3
	BLC	FABP	endothilin 1
	BLC	FABP	NrCAM
	BLC	FABP	Tenascin C
	BLC	FABP	VCAM1
	BLC	FABP	cortisol
	BLC	FGF basic	myoglobin
	BLC	FGF basic	resistin
	BLC	FGF basic	TRAIL R3
	BLC	FGF basic	endothilin 1
	BLC	FGF basic	NrCAM
	BLC	FGF basic	Tenascin C
	BLC	FGF basic	VCAM1
	BLC	FGF basic	cortisol
	BLC	myoglobin	resistin
	BLC	myoglobin	TRAIL R3
	BLC	myoglobin	endothilin 1
	BLC	myoglobin	NrCAM
	BLC	myoglobin	Tenascin C
	BLC	myoglobin	VCAM1
	BLC	myoglobin	cortisol
	BLC	resistin	TRAIL R3
	BLC	resistin	endothilin 1
	BLC	resistin	NrCAM
	BLC	resistin	Tenascin C
	BLC	resistin	VCAM1
	BLC	resistin	cortisol
	BLC	TRAIL R3	endothilin 1
	BLC	TRAIL R3	NrCAM
	BLC	TRAIL R3	Tenascin C
	BLC	TRAIL R3	VCAM1
	BLC	TRAIL R3	cortisol
	BLC	endothilin 1	NrCAM
	BLC	endothilin 1	Tenascin C
	BLC	endothilin 1	VCAM1
	BLC	endothilin 1	cortisol
	BLC	NrCAM	Tenascin C
	BLC	NrCAM	VCAM1
	BLC	NrCAM	cortisol
	BLC	Tenascin C	VCAM1
	BLC	Tenascin C	cortisol
	BLC	VCAM1	cortisol
	CD40	IGF BP2	MMP3
	CD40	IGF BP2	peptide YY
	CD40	IGF BP2	stem cell factor
	CD40	IGF BP2	TNF RII
	CD40	IGF BP2	AXL
	CD40	IGF BP2	Eotaxin 3
	CD40	IGF BP2	FABP
	CD40	IGF BP2	FGF basic
	CD40	IGF BP2	myoglobin
	CD40	IGF BP2	resistin
	CD40	IGF BP2	TRAIL R3
	CD40	IGF BP2	endothilin 1
	CD40	IGF BP2	NrCAM
	CD40	IGF BP2	Tenascin C
	CD40	IGF BP2	VCAM1
	CD40	IGF BP2	cortisol
	CD40	MMP3	peptide YY
	CD40	MMP3	stem cell factor
	CD40	MMP3	TNF RII
	CD40	MMP3	AXL
	CD40	MMP3	Eotaxin 3
	CD40	MMP3	FABP
	CD40	MMP3	FGF basic
	CD40	MMP3	myoglobin
	CD40	MMP3	resistin
	CD40	MMP3	TRAIL R3
	CD40	MMP3	endothilin 1
	CD40	MMP3	NrCAM
	CD40	MMP3	Tenascin C
	CD40	MMP3	VCAM1
	CD40	MMP3	cortisol
	CD40	peptide YY	stem cell factor
	CD40	peptide YY	TNF RII
	CD40	peptide YY	AXL
	CD40	peptide YY	Eotaxin 3
	CD40	peptide YY	FABP
	CD40	peptide YY	FGF basic
	CD40	peptide YY	myoglobin
	CD40	peptide YY	resistin
	CD40	peptide YY	TRAIL R3
	CD40	peptide YY	endothilin 1
	CD40	peptide YY	NrCAM
	CD40	peptide YY	Tenascin C
	CD40	peptide YY	VCAM1
	CD40	peptide YY	cortisol
	CD40	stem cell factor	TNF RII
	CD40	stem cell factor	AXL
	CD40	stem cell factor	Eotaxin 3
	CD40	stem cell factor	FABP
	CD40	stem cell factor	FGF basic
	CD40	stem cell factor	myoglobin
	CD40	stem cell factor	resistin
	CD40	stem cell factor	TRAIL R3
	CD40	stem cell factor	endothilin 1
	CD40	stem cell factor	NrCAM
	CD40	stem cell factor	Tenascin C
	CD40	stem cell factor	VCAM1
	CD40	stem cell factor	cortisol
	CD40	TNF RII	AXL
	CD40	TNF RII	Eotaxin 3
	CD40	TNF RII	FABP
	CD40	TNF RII	FGF basic
	CD40	TNF RII	myoglobin
	CD40	TNF RII	resistin
	CD40	TNF RII	TRAIL R3
	CD40	TNF RII	endothilin 1
	CD40	TNF RII	NrCAM
	CD40	TNF RII	Tenascin C
	CD40	TNF RII	VCAM1
	CD40	TNF RII	cortisol
	CD40	AXL	Eotaxin 3
	CD40	AXL	FABP
	CD40	AXL	FGF basic
	CD40	AXL	myoglobin
	CD40	AXL	resistin
	CD40	AXL	TRAIL R3
	CD40	AXL	endothilin 1
	CD40	AXL	NrCAM
	CD40	AXL	Tenascin C
	CD40	AXL	VCAM1
	CD40	AXL	cortisol
	CD40	Eotaxin 3	FABP
	CD40	Eotaxin 3	FGF basic
	CD40	Eotaxin 3	myoglobin
	CD40	Eotaxin 3	resistin
	CD40	Eotaxin 3	TRAIL R3
	CD40	Eotaxin 3	endothilin 1
	CD40	Eotaxin 3	NrCAM
	CD40	Eotaxin 3	Tenascin C
	CD40	Eotaxin 3	VCAM1
	CD40	Eotaxin 3	cortisol
	CD40	FABP	FGF basic
	CD40	FABP	myoglobin
	CD40	FABP	resistin
	CD40	FABP	TRAIL R3
	CD40	FABP	endothilin 1
	CD40	FABP	NrCAM
	CD40	FABP	Tenascin C
	CD40	FABP	VCAM1
	CD40	FABP	cortisol
	CD40	FGF basic	myoglobin
	CD40	FGF basic	resistin
	CD40	FGF basic	TRAIL R3
	CD40	FGF basic	endothilin 1
	CD40	FGF basic	NrCAM
	CD40	FGF basic	Tenascin C
	CD40	FGF basic	VCAM1
	CD40	FGF basic	cortisol
	CD40	myoglobin	resistin
	CD40	myoglobin	TRAIL R3
	CD40	myoglobin	endothilin 1
	CD40	myoglobin	NrCAM
	CD40	myoglobin	Tenascin C
	CD40	myoglobin	VCAM1
	CD40	myoglobin	cortisol
	CD40	resistin	TRAIL R3
	CD40	resistin	endothilin 1
	CD40	resistin	NrCAM
	CD40	resistin	Tenascin C
	CD40	resistin	VCAM1
	CD40	resistin	cortisol
	CD40	TRAIL R3	endothilin 1
	CD40	TRAIL R3	NrCAM
	CD40	TRAIL R3	Tenascin C
	CD40	TRAIL R3	VCAM1
	CD40	TRAIL R3	cortisol
	CD40	endothilin 1	NrCAM
	CD40	endothilin 1	Tenascin C
	CD40	endothilin 1	VCAM1
	CD40	endothilin 1	cortisol
	CD40	NrCAM	Tenascin C
	CD40	NrCAM	VCAM1
	CD40	NrCAM	cortisol
	CD40	Tenascin C	VCAM1
	CD40	Tenascin C	cortisol
	CD40	VCAM1	cortisol
	IGF BP2	MMP3	peptide YY
	IGF BP2	MMP3	stem cell factor
	IGF BP2	MMP3	TNF RII
	IGF BP2	MMP3	AXL
	IGF BP2	MMP3	Eotaxin 3
	IGF BP2	MMP3	FABP
	IGF BP2	MMP3	FGF basic
	IGF BP2	MMP3	myoglobin
	IGF BP2	MMP3	resistin
	IGF BP2	MMP3	TRAIL R3
	IGF BP2	MMP3	endothilin 1
	IGF BP2	MMP3	NrCAM
	IGF BP2	MMP3	Tenascin C
	IGF BP2	MMP3	VCAM1
	IGF BP2	MMP3	cortisol
	IGF BP2	peptide YY	stem cell factor
	IGF BP2	peptide YY	TNF RII
	IGF BP2	peptide YY	AXL
	IGF BP2	peptide YY	Eotaxin 3
	IGF BP2	peptide YY	FABP
	IGF BP2	peptide YY	FGF basic
	IGF BP2	peptide YY	myoglobin
	IGF BP2	peptide YY	resistin
	IGF BP2	peptide YY	TRAIL R3
	IGF BP2	peptide YY	endothilin 1
	IGF BP2	peptide YY	NrCAM
	IGF BP2	peptide YY	Tenascin C
	IGF BP2	peptide YY	VCAM1
	IGF BP2	peptide YY	cortisol
	IGF BP2	stem cell factor	TNF RII
	IGF BP2	stem cell factor	AXL
	IGF BP2	stem cell factor	Eotaxin 3
	IGF BP2	stem cell factor	FABP
	IGF BP2	stem cell factor	FGF basic
	IGF BP2	stem cell factor	myoglobin
	IGF BP2	stem cell factor	resistin
	IGF BP2	stem cell factor	TRAIL R3
	IGF BP2	stem cell factor	endothilin 1
	IGF BP2	stem cell factor	NrCAM
	IGF BP2	stem cell factor	Tenascin C
	IGF BP2	stem cell factor	VCAM1
	IGF BP2	stem cell factor	cortisol
	IGF BP2	TNF RII	AXL
	IGF BP2	TNF RII	Eotaxin 3
	IGF BP2	TNF RII	FABP
	IGF BP2	TNF RII	FGF basic
	IGF BP2	TNF RII	myoglobin
	IGF BP2	TNF RII	resistin
	IGF BP2	TNF RII	TRAIL R3
	IGF BP2	TNF RII	endothilin 1
	IGF BP2	TNF RII	NrCAM
	IGF BP2	TNF RII	Tenascin C
	IGF BP2	TNF RII	VCAM1
	IGF BP2	TNF RII	cortisol
	IGF BP2	AXL	Eotaxin 3
	IGF BP2	AXL	FABP
	IGF BP2	AXL	FGF basic
	IGF BP2	AXL	myoglobin
	IGF BP2	AXL	resistin
	IGF BP2	AXL	TRAIL R3
	IGF BP2	AXL	endothilin 1
	IGF BP2	AXL	NrCAM
	IGF BP2	AXL	Tenascin C
	IGF BP2	AXL	VCAM1
	IGF BP2	AXL	cortisol
	IGF BP2	Eotaxin 3	FABP
	IGF BP2	Eotaxin 3	FGF basic
	IGF BP2	Eotaxin 3	myoglobin
	IGF BP2	Eotaxin 3	resistin
	IGF BP2	Eotaxin 3	TRAIL R3
	IGF BP2	Eotaxin 3	endothilin 1
	IGF BP2	Eotaxin 3	NrCAM
	IGF BP2	Eotaxin 3	Tenascin C
	IGF BP2	Eotaxin 3	VCAM1
	IGF BP2	Eotaxin 3	cortisol
	IGF BP2	FABP	FGF basic
	IGF BP2	FABP	myoglobin
	IGF BP2	FABP	resistin
	IGF BP2	FABP	TRAIL R3
	IGF BP2	FABP	endothilin 1
	IGF BP2	FABP	NrCAM
	IGF BP2	FABP	Tenascin C
	IGF BP2	FABP	VCAM1
	IGF BP2	FABP	cortisol
	IGF BP2	FGF basic	myoglobin
	IGF BP2	FGF basic	resistin
	IGF BP2	FGF basic	TRAIL R3
	IGF BP2	FGF basic	endothilin 1
	IGF BP2	FGF basic	NrCAM
	IGF BP2	FGF basic	Tenascin C
	IGF BP2	FGF basic	VCAM1
	IGF BP2	FGF basic	cortisol
	IGF BP2	myoglobin	resistin
	IGF BP2	myoglobin	TRAIL R3
	IGF BP2	myoglobin	endothilin 1
	IGF BP2	myoglobin	NrCAM
	IGF BP2	myoglobin	Tenascin C
	IGF BP2	myoglobin	VCAM1
	IGF BP2	myoglobin	cortisol
	IGF BP2	resistin	TRAIL R3
	IGF BP2	resistin	endothilin 1
	IGF BP2	resistin	NrCAM
	IGF BP2	resistin	Tenascin C
	IGF BP2	resistin	VCAM1
	IGF BP2	resistin	cortisol
	IGF BP2	TRAIL R3	endothilin 1
	IGF BP2	TRAIL R3	NrCAM
	IGF BP2	TRAIL R3	Tenascin C
	IGF BP2	TRAIL R3	VCAM1
	IGF BP2	TRAIL R3	cortisol
	IGF BP2	endothilin 1	NrCAM
	IGF BP2	endothilin 1	Tenascin C
	IGF BP2	endothilin 1	VCAM1
	IGF BP2	endothilin 1	cortisol
	IGF BP2	NrCAM	Tenascin C
	IGF BP2	NrCAM	VCAM1
	IGF BP2	NrCAM	cortisol
	IGF BP2	Tenascin C	VCAM1
	IGF BP2	Tenascin C	cortisol
	IGF BP2	VCAM1	cortisol
	MMP3	peptide YY	stem cell factor
	MMP3	peptide YY	TNF RII
	MMP3	peptide YY	AXL
	MMP3	peptide YY	Eotaxin 3
	MMP3	peptide YY	FABP
	MMP3	peptide YY	FGF basic
	MMP3	peptide YY	myoglobin
	MMP3	peptide YY	resistin
	MMP3	peptide YY	TRAIL R3
	MMP3	peptide YY	endothilin 1
	MMP3	peptide YY	NrCAM
	MMP3	peptide YY	Tenascin C
	MMP3	peptide YY	VCAM1
	MMP3	peptide YY	cortisol
	MMP3	stem cell factor	TNF RII
	MMP3	stem cell factor	AXL
	MMP3	stem cell factor	Eotaxin 3
	MMP3	stem cell factor	FABP
	MMP3	stem cell factor	FGF basic
	MMP3	stem cell factor	myoglobin
	MMP3	stem cell factor	resistin
	MMP3	stem cell factor	TRAIL R3
	MMP3	stem cell factor	endothilin 1
	MMP3	stem cell factor	NrCAM
	MMP3	stem cell factor	Tenascin C
	MMP3	stem cell factor	VCAM1
	MMP3	stem cell factor	cortisol
	MMP3	TNF RII	AXL
	MMP3	TNF RII	Eotaxin 3
	MMP3	TNF RII	FABP
	MMP3	TNF RII	FGF basic
	MMP3	TNF RII	myoglobin
	MMP3	TNF RII	resistin
	MMP3	TNF RII	TRAIL R3
	MMP3	TNF RII	endothilin 1
	MMP3	TNF RII	NrCAM
	MMP3	TNF RII	Tenascin C
	MMP3	TNF RII	VCAM1
	MMP3	TNF RII	cortisol
	MMP3	AXL	Eotaxin 3
	MMP3	AXL	FABP
	MMP3	AXL	FGF basic
	MMP3	AXL	myoglobin
	MMP3	AXL	resistin
	MMP3	AXL	TRAIL R3
	MMP3	AXL	endothilin 1
	MMP3	AXL	NrCAM
	MMP3	AXL	Tenascin C
	MMP3	AXL	VCAM1
	MMP3	AXL	cortisol
	MMP3	Eotaxin 3	FABP
	MMP3	Eotaxin 3	FGF basic
	MMP3	Eotaxin 3	myoglobin
	MMP3	Eotaxin 3	resistin
	MMP3	Eotaxin 3	TRAIL R3
	MMP3	Eotaxin 3	endothilin 1
	MMP3	Eotaxin 3	NrCAM
	MMP3	Eotaxin 3	Tenascin C
	MMP3	Eotaxin 3	VCAM1
	MMP3	Eotaxin 3	cortisol
	MMP3	FABP	FGF basic
	MMP3	FABP	myoglobin
	MMP3	FABP	resistin
	MMP3	FABP	TRAIL R3
	MMP3	FABP	endothilin 1
	MMP3	FABP	NrCAM
	MMP3	FABP	Tenascin C
	MMP3	FABP	VCAM1
	MMP3	FABP	cortisol
	MMP3	FGF basic	myoglobin
	MMP3	FGF basic	resistin
	MMP3	FGF basic	TRAIL R3
	MMP3	FGF basic	endothilin 1
	MMP3	FGF basic	NrCAM
	MMP3	FGF basic	Tenascin C
	MMP3	FGF basic	VCAM1
	MMP3	FGF basic	cortisol
	MMP3	myoglobin	resistin
	MMP3	myoglobin	TRAIL R3
	MMP3	myoglobin	endothilin 1
	MMP3	myoglobin	NrCAM
	MMP3	myoglobin	Tenascin C
	MMP3	myoglobin	VCAM1
	MMP3	myoglobin	cortisol
	MMP3	resistin	TRAIL R3
	MMP3	resistin	endothilin 1
	MMP3	resistin	NrCAM
	MMP3	resistin	Tenascin C
	MMP3	resistin	VCAM1
	MMP3	resistin	cortisol
	MMP3	TRAIL R3	endothilin 1
	MMP3	TRAIL R3	NrCAM
	MMP3	TRAIL R3	Tenascin C
	MMP3	TRAIL R3	VCAM1
	MMP3	TRAIL R3	cortisol
	MMP3	endothilin 1	NrCAM
	MMP3	endothilin 1	Tenascin C
	MMP3	endothilin 1	VCAM1
	MMP3	endothilin 1	cortisol
	MMP3	NrCAM	Tenascin C
	MMP3	NrCAM	VCAM1
	MMP3	NrCAM	cortisol
	MMP3	Tenascin C	VCAM1
	MMP3	Tenascin C	cortisol
	MMP3	VCAM1	cortisol
	peptide YY	stem cell factor	TNF RII
	peptide YY	stem cell factor	AXL
	peptide YY	stem cell factor	Eotaxin 3
	peptide YY	stem cell factor	FABP
	peptide YY	stem cell factor	FGF basic
	peptide YY	stem cell factor	myoglobin
	peptide YY	stem cell factor	resistin
	peptide YY	stem cell factor	TRAIL R3
	peptide YY	stem cell factor	endothilin 1
	peptide YY	stem cell factor	NrCAM
	peptide YY	stem cell factor	Tenascin C
	peptide YY	stem cell factor	VCAM1
	peptide YY	stem cell factor	cortisol
	peptide YY	TNF RII	AXL
	peptide YY	TNF RII	Eotaxin 3
	peptide YY	TNF RII	FABP
	peptide YY	TNF RII	FGF basic
	peptide YY	TNF RII	myoglobin
	peptide YY	TNF RII	resistin
	peptide YY	TNF RII	TRAIL R3
	peptide YY	TNF RII	endothilin 1
	peptide YY	TNF RII	NrCAM
	peptide YY	TNF RII	Tenascin C
	peptide YY	TNF RII	VCAM1
	peptide YY	TNF RII	cortisol
	peptide YY	AXL	Eotaxin 3
	peptide YY	AXL	FABP
	peptide YY	AXL	FGF basic
	peptide YY	AXL	myoglobin
	peptide YY	AXL	resistin
	peptide YY	AXL	TRAIL R3
	peptide YY	AXL	endothilin 1
	peptide YY	AXL	NrCAM
	peptide YY	AXL	Tenascin C
	peptide YY	AXL	VCAM1
	peptide YY	AXL	cortisol
	peptide YY	Eotaxin 3	FABP
	peptide YY	Eotaxin 3	FGF basic
	peptide YY	Eotaxin 3	myoglobin
	peptide YY	Eotaxin 3	resistin
	peptide YY	Eotaxin 3	TRAIL R3
	peptide YY	Eotaxin 3	endothilin 1
	peptide YY	Eotaxin 3	NrCAM
	peptide YY	Eotaxin 3	Tenascin C
	peptide YY	Eotaxin 3	VCAM1
	peptide YY	Eotaxin 3	cortisol
	peptide YY	FABP	FGF basic
	peptide YY	FABP	myoglobin
	peptide YY	FABP	resistin
	peptide YY	FABP	TRAIL R3
	peptide YY	FABP	endothilin 1
	peptide YY	FABP	NrCAM
	peptide YY	FABP	Tenascin C
	peptide YY	FABP	VCAM1
	peptide YY	FABP	cortisol
	peptide YY	FGF basic	myoglobin
	peptide YY	FGF basic	resistin
	peptide YY	FGF basic	TRAIL R3
	peptide YY	FGF basic	endothilin 1
	peptide YY	FGF basic	NrCAM
	peptide YY	FGF basic	Tenascin C
	peptide YY	FGF basic	VCAM1
	peptide YY	FGF basic	cortisol
	peptide YY	myoglobin	resistin
	peptide YY	myoglobin	TRAIL R3
	peptide YY	myoglobin	endothilin 1
	peptide YY	myoglobin	NrCAM
	peptide YY	myoglobin	Tenascin C
	peptide YY	myoglobin	VCAM1
	peptide YY	myoglobin	cortisol
	peptide YY	resistin	TRAIL R3
	peptide YY	resistin	endothilin 1
	peptide YY	resistin	NrCAM
	peptide YY	resistin	Tenascin C
	peptide YY	resistin	VCAM1
	peptide YY	resistin	cortisol
	peptide YY	TRAIL R3	endothilin 1
	peptide YY	TRAIL R3	NrCAM
	peptide YY	TRAIL R3	Tenascin C
	peptide YY	TRAIL R3	VCAM1
	peptide YY	TRAIL R3	cortisol
	peptide YY	endothilin 1	NrCAM
	peptide YY	endothilin 1	Tenascin C
	peptide YY	endothilin 1	VCAM1
	peptide YY	endothilin 1	cortisol
	peptide YY	NrCAM	Tenascin C
	peptide YY	NrCAM	VCAM1
	peptide YY	NrCAM	cortisol
	peptide YY	Tenascin C	VCAM1
	peptide YY	Tenascin C	cortisol
	peptide YY	VCAM1	cortisol
	stem cell factor	TNF RII	AXL
	stem cell factor	TNF RII	Eotaxin 3
	stem cell factor	TNF RII	FABP
	stem cell factor	TNF RII	FGF basic
	stem cell factor	TNF RII	myoglobin
	stem cell factor	TNF RII	resistin
	stem cell factor	TNF RII	TRAIL R3
	stem cell factor	TNF RII	endothilin 1
	stem cell factor	TNF RII	NrCAM
	stem cell factor	TNF RII	Tenascin C
	stem cell factor	TNF RII	VCAM1
	stem cell factor	TNF RII	cortisol
	stem cell factor	AXL	Eotaxin 3
	stem cell factor	AXL	FABP
	stem cell factor	AXL	FGF basic
	stem cell factor	AXL	myoglobin
	stem cell factor	AXL	resistin
	stem cell factor	AXL	TRAIL R3
	stem cell factor	AXL	endothilin 1
	stem cell factor	AXL	NrCAM
	stem cell factor	AXL	Tenascin C
	stem cell factor	AXL	VCAM1
	stem cell factor	AXL	cortisol
	stem cell factor	Eotaxin 3	FABP
	stem cell factor	Eotaxin 3	FGF basic
	stem cell factor	Eotaxin 3	myoglobin
	stem cell factor	Eotaxin 3	resistin
	stem cell factor	Eotaxin 3	TRAIL R3
	stem cell factor	Eotaxin 3	endothilin 1
	stem cell factor	Eotaxin 3	NrCAM
	stem cell factor	Eotaxin 3	Tenascin C
	stem cell factor	Eotaxin 3	VCAM1
	stem cell factor	Eotaxin 3	cortisol
	stem cell factor	FABP	FGF basic
	stem cell factor	FABP	myoglobin
	stem cell factor	FABP	resistin
	stem cell factor	FABP	TRAIL R3
	stem cell factor	FABP	endothilin 1
	stem cell factor	FABP	NrCAM
	stem cell factor	FABP	Tenascin C
	stem cell factor	FABP	VCAM1
	stem cell factor	FABP	cortisol
	stem cell factor	FGF basic	myoglobin
	stem cell factor	FGF basic	resistin
	stem cell factor	FGF basic	TRAIL R3
	stem cell factor	FGF basic	endothilin 1
	stem cell factor	FGF basic	NrCAM
	stem cell factor	FGF basic	Tenascin C
	stem cell factor	FGF basic	VCAM1
	stem cell factor	FGF basic	cortisol
	stem cell factor	myoglobin	resistin
	stem cell factor	myoglobin	TRAIL R3
	stem cell factor	myoglobin	endothilin 1
	stem cell factor	myoglobin	NrCAM
	stem cell factor	myoglobin	Tenascin C
	stem cell factor	myoglobin	VCAM1
	stem cell factor	myoglobin	cortisol
	stem cell factor	resistin	TRAIL R3
	stem cell factor	resistin	endothilin 1
	stem cell factor	resistin	NrCAM
	stem cell factor	resistin	Tenascin C
	stem cell factor	resistin	VCAM1
	stem cell factor	resistin	cortisol
	stem cell factor	TRAIL R3	endothilin 1
	stem cell factor	TRAIL R3	NrCAM
	stem cell factor	TRAIL R3	Tenascin C
	stem cell factor	TRAIL R3	VCAM1
	stem cell factor	TRAIL R3	cortisol
	stem cell factor	endothilin 1	NrCAM
	stem cell factor	endothilin 1	Tenascin C
	stem cell factor	endothilin 1	VCAM1
	stem cell factor	endothilin 1	cortisol
	stem cell factor	NrCAM	Tenascin C
	stem cell factor	NrCAM	VCAM1
	stem cell factor	NrCAM	cortisol
	stem cell factor	Tenascin C	VCAM1
	stem cell factor	Tenascin C	cortisol
	stem cell factor	VCAM1	cortisol
	TNF RII	AXL	Eotaxin 3
	TNF RII	AXL	FABP
	TNF RII	AXL	FGF basic
	TNF RII	AXL	myoglobin
	TNF RII	AXL	resistin
	TNF RII	AXL	TRAIL R3
	TNF RII	AXL	endothilin 1
	TNF RII	AXL	NrCAM
	TNF RII	AXL	Tenascin C
	TNF RII	AXL	VCAM1
	TNF RII	AXL	cortisol
	TNF RII	Eotaxin 3	FABP
	TNF RII	Eotaxin 3	FGF basic
	TNF RII	Eotaxin 3	myoglobin
	TNF RII	Eotaxin 3	resistin
	TNF RII	Eotaxin 3	TRAIL R3
	TNF RII	Eotaxin 3	endothilin 1
	TNF RII	Eotaxin 3	NrCAM
	TNF RII	Eotaxin 3	Tenascin C
	TNF RII	Eotaxin 3	VCAM1
	TNF RII	Eotaxin 3	cortisol
	TNF RII	FABP	FGF basic
	TNF RII	FABP	myoglobin
	TNF RII	FABP	resistin
	TNF RII	FABP	TRAIL R3
	TNF RII	FABP	endothilin 1
	TNF RII	FABP	NrCAM
	TNF RII	FABP	Tenascin C
	TNF RII	FABP	VCAM1
	TNF RII	FABP	cortisol
	TNF RII	FGF basic	myoglobin
	TNF RII	FGF basic	resistin
	TNF RII	FGF basic	TRAIL R3
	TNF RII	FGF basic	endothilin 1
	TNF RII	FGF basic	NrCAM
	TNF RII	FGF basic	Tenascin C
	TNF RII	FGF basic	VCAM1
	TNF RII	FGF basic	cortisol
	TNF RII	myoglobin	resistin
	TNF RII	myoglobin	TRAIL R3
	TNF RII	myoglobin	endothilin 1
	TNF RII	myoglobin	NrCAM
	TNF RII	myoglobin	Tenascin C
	TNF RII	myoglobin	VCAM1
	TNF RII	myoglobin	cortisol
	TNF RII	resistin	TRAIL R3
	TNF RII	resistin	endothilin 1
	TNF RII	resistin	NrCAM
	TNF RII	resistin	Tenascin C
	TNF RII	resistin	VCAM1
	TNF RII	resistin	cortisol
	TNF RII	TRAIL R3	endothilin 1
	TNF RII	TRAIL R3	NrCAM
	TNF RII	TRAIL R3	Tenascin C
	TNF RII	TRAIL R3	VCAM1
	TNF RII	TRAIL R3	cortisol
	TNF RII	endothilin 1	NrCAM
	TNF RII	endothilin 1	Tenascin C
	TNF RII	endothilin 1	VCAM1
	TNF RII	endothilin 1	cortisol
	TNF RII	NrCAM	Tenascin C
	TNF RII	NrCAM	VCAM1
	TNF RII	NrCAM	cortisol
	TNF RII	Tenascin C	VCAM1
	TNF RII	Tenascin C	cortisol
	TNF RII	VCAM1	cortisol
	AXL	Eotaxin 3	FABP
	AXL	Eotaxin 3	FGF basic
	AXL	Eotaxin 3	myoglobin
	AXL	Eotaxin 3	resistin
	AXL	Eotaxin 3	TRAIL R3
	AXL	Eotaxin 3	endothilin 1
	AXL	Eotaxin 3	NrCAM
	AXL	Eotaxin 3	Tenascin C
	AXL	Eotaxin 3	VCAM1
	AXL	Eotaxin 3	cortisol
	AXL	FABP	FGF basic
	AXL	FABP	myoglobin
	AXL	FABP	resistin
	AXL	FABP	TRAIL R3
	AXL	FABP	endothilin 1
	AXL	FABP	NrCAM
	AXL	FABP	Tenascin C
	AXL	FABP	VCAM1
	AXL	FABP	cortisol
	AXL	FGF basic	myoglobin
	AXL	FGF basic	resistin
	AXL	FGF basic	TRAIL R3
	AXL	FGF basic	endothilin 1
	AXL	FGF basic	NrCAM
	AXL	FGF basic	Tenascin C
	AXL	FGF basic	VCAM1
	AXL	FGF basic	cortisol
	AXL	myoglobin	resistin
	AXL	myoglobin	TRAIL R3
	AXL	myoglobin	endothilin 1
	AXL	myoglobin	NrCAM
	AXL	myoglobin	Tenascin C
	AXL	myoglobin	VCAM1
	AXL	myoglobin	cortisol
	AXL	resistin	TRAIL R3
	AXL	resistin	endothilin 1
	AXL	resistin	NrCAM
	AXL	resistin	Tenascin C
	AXL	resistin	VCAM1
	AXL	resistin	cortisol
	AXL	TRAIL R3	endothilin 1
	AXL	TRAIL R3	NrCAM
	AXL	TRAIL R3	Tenascin C
	AXL	TRAIL R3	VCAM1
	AXL	TRAIL R3	cortisol
	AXL	endothilin 1	NrCAM
	AXL	endothilin 1	Tenascin C
	AXL	endothilin 1	VCAM1
	AXL	endothilin 1	cortisol
	AXL	NrCAM	Tenascin C
	AXL	NrCAM	VCAM1
	AXL	NrCAM	cortisol
	AXL	Tenascin C	VCAM1
	AXL	Tenascin C	cortisol
	AXL	VCAM1	cortisol
	Eotaxin 3	FABP	FGF basic
	Eotaxin 3	FABP	myoglobin
	Eotaxin 3	FABP	resistin
	Eotaxin 3	FABP	TRAIL R3
	Eotaxin 3	FABP	endothilin 1
	Eotaxin 3	FABP	NrCAM
	Eotaxin 3	FABP	Tenascin C
	Eotaxin 3	FABP	VCAM1
	Eotaxin 3	FABP	cortisol
	Eotaxin 3	FGF basic	myoglobin
	Eotaxin 3	FGF basic	resistin
	Eotaxin 3	FGF basic	TRAIL R3
	Eotaxin 3	FGF basic	endothilin 1
	Eotaxin 3	FGF basic	NrCAM
	Eotaxin 3	FGF basic	Tenascin C
	Eotaxin 3	FGF basic	VCAM1
	Eotaxin 3	FGF basic	cortisol
	Eotaxin 3	myoglobin	resistin
	Eotaxin 3	myoglobin	TRAIL R3
	Eotaxin 3	myoglobin	endothilin 1
	Eotaxin 3	myoglobin	NrCAM
	Eotaxin 3	myoglobin	Tenascin C
	Eotaxin 3	myoglobin	VCAM1
	Eotaxin 3	myoglobin	cortisol
	Eotaxin 3	resistin	TRAIL R3
	Eotaxin 3	resistin	endothilin 1
	Eotaxin 3	resistin	NrCAM
	Eotaxin 3	resistin	Tenascin C
	Eotaxin 3	resistin	VCAM1
	Eotaxin 3	resistin	cortisol
	Eotaxin 3	TRAIL R3	endothilin 1
	Eotaxin 3	TRAIL R3	NrCAM
	Eotaxin 3	TRAIL R3	Tenascin C
	Eotaxin 3	TRAIL R3	VCAM1
	Eotaxin 3	TRAIL R3	cortisol
	Eotaxin 3	endothilin 1	NrCAM
	Eotaxin 3	endothilin 1	Tenascin C
	Eotaxin 3	endothilin 1	VCAM1
	Eotaxin 3	endothilin 1	cortisol
	Eotaxin 3	NrCAM	Tenascin C
	Eotaxin 3	NrCAM	VCAM1
	Eotaxin 3	NrCAM	cortisol
	Eotaxin 3	Tenascin C	VCAM1
	Eotaxin 3	Tenascin C	cortisol
	Eotaxin 3	VCAM1	cortisol
	FABP	FGF basic	myoglobin
	FABP	FGF basic	resistin
	FABP	FGF basic	TRAIL R3
	FABP	FGF basic	endothilin 1
	FABP	FGF basic	NrCAM
	FABP	FGF basic	Tenascin C
	FABP	FGF basic	VCAM1
	FABP	FGF basic	cortisol
	FABP	myoglobin	resistin
	FABP	myoglobin	TRAIL R3
	FABP	myoglobin	endothilin 1
	FABP	myoglobin	NrCAM
	FABP	myoglobin	Tenascin C
	FABP	myoglobin	VCAM1
	FABP	myoglobin	cortisol
	FABP	resistin	TRAIL R3
	FABP	resistin	endothilin 1
	FABP	resistin	NrCAM
	FABP	resistin	Tenascin C
	FABP	resistin	VCAM1
	FABP	resistin	cortisol
	FABP	TRAIL R3	endothilin 1
	FABP	TRAIL R3	NrCAM
	FABP	TRAIL R3	Tenascin C
	FABP	TRAIL R3	VCAM1
	FABP	TRAIL R3	cortisol
	FABP	endothilin 1	NrCAM
	FABP	endothilin 1	Tenascin C
	FABP	endothilin 1	VCAM1
	FABP	endothilin 1	cortisol
	FABP	NrCAM	Tenascin C
	FABP	NrCAM	VCAM1
	FABP	NrCAM	cortisol
	FABP	Tenascin C	VCAM1
	FABP	Tenascin C	cortisol
	FABP	VCAM1	cortisol
	FGF basic	myoglobin	resistin
	FGF basic	myoglobin	TRAIL R3
	FGF basic	myoglobin	endothilin 1
	FGF basic	myoglobin	NrCAM
	FGF basic	myoglobin	Tenascin C
	FGF basic	myoglobin	VCAM1
	FGF basic	myoglobin	cortisol
	FGF basic	resistin	TRAIL R3
	FGF basic	resistin	endothilin 1
	FGF basic	resistin	NrCAM
	FGF basic	resistin	Tenascin C
	FGF basic	resistin	VCAM1
	FGF basic	resistin	cortisol
	FGF basic	TRAIL R3	endothilin 1
	FGF basic	TRAIL R3	NrCAM
	FGF basic	TRAIL R3	Tenascin C
	FGF basic	TRAIL R3	VCAM1
	FGF basic	TRAIL R3	cortisol
	FGF basic	endothilin 1	NrCAM
	FGF basic	endothilin 1	Tenascin C
	FGF basic	endothilin 1	VCAM1
	FGF basic	endothilin 1	cortisol
	FGF basic	NrCAM	Tenascin C
	FGF basic	NrCAM	VCAM1
	FGF basic	NrCAM	cortisol
	FGF basic	Tenascin C	VCAM1
	FGF basic	Tenascin C	cortisol
	FGF basic	VCAM1	cortisol
	myoglobin	resistin	TRAIL R3
	myoglobin	resistin	endothilin 1
	myoglobin	resistin	NrCAM
	myoglobin	resistin	Tenascin C
	myoglobin	resistin	VCAM1
	myoglobin	resistin	cortisol
	myoglobin	TRAIL R3	endothilin 1
	myoglobin	TRAIL R3	NrCAM
	myoglobin	TRAIL R3	Tenascin C
	myoglobin	TRAIL R3	VCAM1
	myoglobin	TRAIL R3	cortisol
	myoglobin	endothilin 1	NrCAM
	myoglobin	endothilin 1	Tenascin C
	myoglobin	endothilin 1	VCAM1
	myoglobin	endothilin 1	cortisol
	myoglobin	NrCAM	Tenascin C
	myoglobin	NrCAM	VCAM1
	myoglobin	NrCAM	cortisol
	myoglobin	Tenascin C	VCAM1
	myoglobin	Tenascin C	cortisol
	myoglobin	VCAM1	cortisol
	resistin	TRAIL R3	endothilin 1
	resistin	TRAIL R3	NrCAM
	resistin	TRAIL R3	Tenascin C
	resistin	TRAIL R3	VCAM1
	resistin	TRAIL R3	cortisol
	resistin	endothilin 1	NrCAM
	resistin	endothilin 1	Tenascin C
	resistin	endothilin 1	VCAM1
	resistin	endothilin 1	cortisol
	resistin	NrCAM	Tenascin C
	resistin	NrCAM	VCAM1
	resistin	NrCAM	cortisol
	resistin	Tenascin C	VCAM1
	resistin	Tenascin C	cortisol
	resistin	VCAM1	cortisol
	TRAIL R3	endothilin 1	NrCAM
	TRAIL R3	endothilin 1	Tenascin C
	TRAIL R3	endothilin 1	VCAM1
	TRAIL R3	endothilin 1	cortisol
	TRAIL R3	NrCAM	Tenascin C
	TRAIL R3	NrCAM	VCAM1
	TRAIL R3	NrCAM	cortisol
	TRAIL R3	Tenascin C	VCAM1
	TRAIL R3	Tenascin C	cortisol
	TRAIL R3	VCAM1	cortisol
	endothilin 1	NrCAM	Tenascin C
	endothilin 1	NrCAM	VCAM1
	endothilin 1	NrCAM	cortisol
	endothilin 1	Tenascin C	VCAM1
	endothilin 1	Tenascin C	cortisol
	endothilin 1	VCAM1	cortisol
	NrCAM	Tenascin C	VCAM1
	NrCAM	Tenascin C	cortisol
	NrCAM	VCAM1	cortisol
	Tenascin C	VCAM1	cortisol

III. Test Sample

The method for diagnosing, monitoring, or determining a renal disorder involves determining the presence of sample analytes in a test sample. A test sample, as defined herein, is an amount of bodily fluid taken from a mammal. Non-limiting examples of bodily fluids include urine, blood, plasma, serum, saliva, semen, perspiration, tears, mucus, and tissue lysates. In an exemplary embodiment, the bodily fluid contained in the test sample is urine, plasma, or serum.

(a) Mammals

A mammal, as defined herein, is any organism that is a member of the class Mammalia. Non-limiting examples of mammals appropriate for the various embodiments may include humans, apes, monkeys, rats, mice, dogs, cats, pigs, and livestock including cattle and oxen. In an exemplary embodiment, the mammal is a human.

(b) Devices and Methods of Taking Bodily Fluids from Mammals

The bodily fluids of the test sample may be taken from the mammal using any known device or method so long as the analytes to be measured by the multiplexed assay are not rendered undetectable by the multiplexed assay. Non-limiting examples of devices or methods suitable for taking bodily fluid from a mammal include urine sample cups, urethral catheters, swabs, hypodermic needles, thin needle biopsies, hollow needle biopsies, punch biopsies, metabolic cages, and aspiration.

In order to adjust the expected concentrations of the sample analytes in the test sample to fall within the dynamic range of the multiplexed assay, the test sample may be diluted to reduce the concentration of the sample analytes prior to analysis. The degree of dilution may depend on a variety of factors including but not limited to the type of multiplexed assay used to measure the analytes, the reagents utilized in the multiplexed assay, and the type of bodily fluid contained in the test sample. In one embodiment, the test sample is diluted by adding a volume of diluent ranging from about ½ of the original test sample volume to about 50,000 times the original test sample volume.

In one exemplary embodiment, if the test sample is human urine and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 100 times the original test sample volume prior to analysis. In another exemplary embodiment, if the test sample is human serum and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 5 times the original test sample volume prior to analysis. In yet another exemplary embodiment, if the test sample is human plasma and the multiplexed assay is an antibody-based capture-sandwich assay, the test sample is diluted by adding a volume of diluent that is about 2,000 times the original test sample volume prior to analysis.

The diluent may be any fluid that does not interfere with the function of the multiplexed assay used to measure the concentration of the analytes in the test sample. Non-limiting examples of suitable diluents include deionized water, distilled water, saline solution, Ringer's solution, phosphate buffered saline solution, TRIS-buffered saline solution, standard saline citrate, and HEPES-buffered saline.

IV. Multiplexed Assay Device

In one embodiment, the concentration of a combination of sample analytes is measured using a multiplexed assay device capable of measuring up to 189 of the biomarker analytes. A multiplexed assay device, as defined herein, is an assay capable of simultaneously determining the concentration of three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, fifteen or more, sixteen or more, seventeen or more, eighteen or more, nineteen or more, or twenty or more of the biomarker analytes using a single device and/or method. Any known method of measuring the concentration of the biomarker analytes may be used for the multiplexed assay device. Non-limiting examples of measurement methods suitable for the multiplexed assay device include electrophoresis, mass spectrometry, protein microarrays, surface plasmon resonance, and immunoassays including, but not limited to western blot, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA) methods, vibrational detection using MicroElectroMagnetic Devices (MEMS), and particle-based capture-sandwich immunoassays.

(a) Multiplexed Immunoassay Device

In one embodiment, the concentrations of the analytes in the test sample are measured using a multiplexed immunoassay device that utilizes capture antibodies marked with indicators to determine the concentration of the sample analytes.

(i) Capture Antibodies

In the same embodiment, the multiplexed immunoassay device includes three or more capture antibodies. Capture antibodies, as defined herein, are antibodies in which the antigenic determinant is one of the Biomarker Analytes known in the art to have a documented association with early renal damage in humans. The biomarker analytes include, but are note limited to alpha-1-microglobulin, beta-2-microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF. Each of the at least three capture antibodies has a unique antigenic determinant that is one of the biomarker analytes. When contacted with the test sample, the capture antibodies form antigen-antibody complexes in which the analytes serve as antigens.

The term “antibody,” as used herein, encompasses a monoclonal ab, an antibody fragment, a chimeric antibody, and a single-chain antibody.

In some embodiments, the capture antibodies may be attached to a platform or other substrate having a contact surface in order to immobilize any analytes captured by the capture antibodies. The platform generally incorporates a porous material for immobilizing the analytes. Non-limiting examples of suitable substrates include paper, nitrocellulose, cellulose, glass, glass fiber mesh, silica gel, synthetic resins, or plastic strips, beads, or surfaces, such as the inner surface of the well of a microtitration tray. Suitable beads may include polystyrene or latex microspheres.

(ii) indicators

In one embodiment of the multiplexed immunoassay device, an indicator is attached to each of the three or more capture antibodies. The indicator, as defined herein, is any compound that registers a measurable change to indicate the presence of one of the sample analytes when bound to one of the capture antibodies. Non-limiting examples of indicators include visual indicators and electrochemical indicators.

Visual indicators, as defined herein, are compounds that register a change by reflecting a limited subset of the wavelengths of light illuminating the indicator, by fluorescing light after being illuminated, or by emitting light via chemiluminescence. The change registered by visual indicators may be in the visible light spectrum, in the infrared spectrum, or in the ultraviolet spectrum. Non-limiting examples of visual indicators suitable for the multiplexed immunoassay device include nanoparticulate gold, organic particles such as polyurethane or latex microspheres loaded with dye compounds, carbon black, fluorophores, phycoerythrin, radioactive isotopes, nanoparticles, quantum dots, and enzymes such as horseradish peroxidase or alkaline phosphatase that react with a chemical substrate to form a colored or chemiluminescent product.

Electrochemical indicators, as defined herein, are compounds that register a change by altering an electrical property. The changes registered by electrochemical indicators may be an alteration in conductivity, resistance, capacitance, current conducted in response to an applied voltage, or voltage required to achieve a desired current. Non-limiting examples of electrochemical indicators include redox species such as ascorbate (vitamin C), vitamin E, glutathione, polyphenols, catechols, quercetin, phytoestrogens, penicillin, carbazole, murranes, phenols, carbonyls, benzoates, and trace metal ions such as nickel, copper, cadmium, iron and mercury.

In this same embodiment, the test sample containing a combination of three or more sample analytes is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as the antigens. After removing any uncomplexed capture antibodies, the concentrations of the three or more analytes are determined by measuring the change registered by the indicators attached to the capture antibodies.

In one exemplary embodiment, the indicators are polyurethane or latex microspheres loaded with dye compounds and phycoerythrin.

(b) Multiplexed Sandwich Immunoassay Device

In another embodiment, the multiplexed immunoassay device has a sandwich assay format. In this embodiment, the multiplexed sandwich immunoassay device includes three or more capture antibodies as previously described. However, in this embodiment, each of the capture antibodies is attached to a capture agent that includes an antigenic moiety. The antigenic moiety serves as the antigenic determinant of a detection antibody, also included in the multiplexed immunoassay device of this embodiment. In addition, an indicator is attached to the detection antibody.

In this same embodiment, the test sample is contacted with the capture antibodies and allowed to form antigen-antibody complexes in which the sample analytes serve as antigens. The detection antibodies are then contacted with the test sample and allowed to form antigen-antibody complexes in which the capture agent serves as the antigen for the detection antibody. After removing any uncomplexed detection antibodies the concentration of the analytes are determined by measuring the changes registered by the indicators attached to the detection antibodies.

(c) Multiplexing Approaches

In the various embodiments of the multiplexed immunoassay devices, the concentrations of each of the sample analytes may be determined using any approach known in the art. In one embodiment, a single indicator compound is attached to each of the three or more antibodies. In addition, each of the capture antibodies having one of the sample analytes as an antigenic determinant is physically separated into a distinct region so that the concentration of each of the sample analytes may be determined by measuring the changes registered by the indicators in each physically separate region corresponding to each of the sample analytes.

In another embodiment, each antibody having one of the sample analytes as an antigenic determinant is marked with a unique indicator. In this manner, a unique indicator is attached to each antibody having a single sample analyte as its antigenic determinant. In this embodiment, all antibodies may occupy the same physical space. The concentration of each sample analyte is determined by measuring the change registered by the unique indicator attached to the antibody having the sample analyte as an antigenic determinant.

(d) Microsphere-Based Capture-Sandwich Immunoassay Device

In an exemplary embodiment, the multiplexed immunoassay device is a microsphere-based capture-sandwich immunoassay device. In this embodiment, the device includes a mixture of three or more capture-antibody microspheres, in which each capture-antibody microsphere corresponds to one of the biomarker analytes. Each capture-antibody microsphere includes a plurality of capture antibodies attached to the outer surface of the microsphere. In this same embodiment, the antigenic determinant of all of the capture antibodies attached to one microsphere is the same biomarker analyte.

In this embodiment of the device, the microsphere is a small polystyrene or latex sphere that is loaded with an indicator that is a dye compound. The microsphere may be between about 3 μm and about 5 μm in diameter. Each capture-antibody microsphere corresponding to one of the biomarker analytes is loaded with the same indicator. In this manner, each capture-antibody microsphere corresponding to a biomarker analyte is uniquely color-coded.

In this same exemplary embodiment, the multiplexed immunoassay device further includes three or more biotinylated detection antibodies in which the antigenic determinant of each biotinylated detection antibody is one of the biomarker analytes. The device further includes a plurality of streptaviden proteins complexed with a reporter compound. A reporter compound, as defined herein, is an indicator selected to register a change that is distinguishable from the indicators used to mark the capture-antibody microspheres.

The concentrations of the sample analytes may be determined by contacting the test sample with a mixture of capture-antigen microspheres corresponding to each sample analyte to be measured. The sample analytes are allowed to form antigen-antibody complexes in which a sample analyte serves as an antigen and a capture antibody attached to the microsphere serves as an antibody. In this manner, the sample analytes are immobilized onto the capture-antigen microspheres. The biotinylated detection antibodies are then added to the test sample and allowed to form antigen-antibody complexes in which the analyte serves as the antigen and the biotinylated detection antibody serves as the antibody. The streptaviden-reporter complex is then added to the test sample and allowed to bind to the biotin moieties of the biotinylated detection antibodies. The antigen-capture microspheres may then be rinsed and filtered.

In this embodiment, the concentration of each analyte is determined by first measuring the change registered by the indicator compound embedded in the capture-antigen microsphere in order to identify the particular analyte. For each microsphere corresponding to one of the biomarker analytes, the quantity of analyte immobilized on the microsphere is determined by measuring the change registered by the reporter compound attached to the microsphere.

For example, the indicator embedded in the microspheres associated with one sample analyte may register an emission of orange light, and the reporter may register an emission of green light. In this example, a detector device may measure the intensity of orange light and green light separately. The measured intensity of the green light would determine the concentration of the analyte captured on the microsphere, and the intensity of the orange light would determine the specific analyte captured on the microsphere.

Any sensor device may be used to detect the changes registered by the indicators embedded in the microspheres and the changes registered by the reporter compound, so long as the sensor device is sufficiently sensitive to the changes registered by both indicator and reporter compound. Non-limiting examples of suitable sensor devices include spectrophotometers, photosensors, colorimeters, cyclic coulometry devices, and flow cytometers. In an exemplary embodiment, the sensor device is a flow cytometer.

(e) Vibrational Detection Device

In another exemplary embodiment, the multiplexed immunoassay device has a vibrational detection format using a MEMS. In this embodiment, the immunoassay device uses capture antibodies as previously described. However, in this embodiment, the capture antibodies are attached to a microscopic silicon microcantilever beam structure. The microcantilevers are micromechanical beams that are anchored at one end, such as diving spring boards that can be readily fabricated on silicon wafers and other materials. The microcantilever sensors are physical sensors that respond to surface stress changes due to chemical or biological processes. When fabricated with very small force constants, they can measure forces and stresses with extremely high sensitivity. The very small force constant of a cantilever allows detection not surface stress variation due to the binding of an analyte to the capture antibody on the microcantilever. Binding of the analyte results in a differential surface stress due to adsorption-induced forces, which manifests as a deflection which can be measured. The vibrational detection may be multiplexed. For more details, see Datar et al., MRS Bulletin (2009) 34:449-459 and Gaster et al., Nature Medicine (2009) 15:1327-1332, both of which are hereby incorporated by reference in their entireties.

It will be understood by one skilled in the art that the devices described herein, as well as all those embodiments within the scope of the current invention may be incorporated into a kit. Generally, the kit may include any of the devices described herein in addition to a collection apparatus suitable for collecting a sample of bodily fluid from the mammal. The collection apparatus may include, but it not limited to urine sample cups, urethral catheters, swabs, hypodermic needles, thin needles, hollow needles, metabolic cages, aspiration needles, and combinations thereof.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1

Least Detectable Dose and Lower Limit of Quantitation of Assay for Analytes Associated with Renal Disorders

To assess the least detectable doses (LDD) and lower limits of quantitation (LLOQ) of a variety of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.

The concentrations of the analytes were measured using a capture-sandwich assay using antigen-specific antibodies. For each analyte, a range of standard sample dilutions ranging over about four orders of magnitude of analyte concentration were measured using the assay in order to obtain data used to construct a standard dose response curve. The dynamic range for each of the analytes, defined herein as the range of analyte concentrations measured to determine its dose response curve, is presented below.

To perform the assay, 5 μL of a diluted mixture of capture-antibody microspheres were mixed with 5 μL of blocker and 10 μL of pre-diluted standard sample in each of the wells of a hard-bottom microtiter plate. After incubating the hard-bottom plate for 1 hour, 10 μL of biotinylated detection antibody was added to each well, and then the hard-bottom plate was incubated for an additional hour. 10 μL of diluted streptavidin-phycoerythrin was added to each well and then the hard-bottom plate was incubated for another 60 minutes.

A filter-membrane microtiter plate was pre-wetted by adding 100 μL wash buffer, and then aspirated using a vacuum manifold device. The contents of the wells of the hard-bottom plate were then transferred to the corresponding wells of the filter-membrane plate. All wells of the hard-bottom plate were vacuum-aspirated and the contents were washed twice with 100 μL of wash buffer. After the second wash, 100 μL of wash buffer was added to each well, and then the washed microspheres were resuspended with thorough mixing. The plate was then analyzed using a Luminex 100 Analyzer (Luminex Corporation, Austin, Tex., USA). Dose response curves were constructed for each analyte by curve-fitting the median fluorescence intensity (MFI) measured from the assays of diluted standard samples containing a range of analyte concentrations.

The least detectable dose (LDD) was determined by adding three standard deviations to the average of the MFI signal measured for 20 replicate samples of blank standard solution (i.e. standard solution containing no analyte). The MFI signal was converted to an LDD concentration using the dose response curve and multiplied by a dilution factor of 2.

The lower limit of quantification (LLOQ), defined herein as the point at which the coefficient of variation (CV) for the analyte measured in the standard samples was 30%, was determined by the analysis of the measurements of increasingly diluted standard samples. For each analyte, the standard solution was diluted by 2 fold for 8 dilutions. At each stage of dilution, samples were assayed in triplicate, and the CV of the analyte concentration at each dilution was calculated and plotted as a function of analyte concentration. The LLOQ was interpolated from this plot and multiplied by a dilution factor of 2.

The LDD and LLOQ results for each analyte are summarized in Table 2:

TABLE 2
LDD, LLOQ, and Dynamic Range of Analyte Assay

Dynamic Range

Analyte	Units	LDD	LLOQ	minimum	maximum

Calbindin	ng/mL	1.1	3.1	0.516	2580
Clusterin	ng/mL	2.4	2.3	0.676	3378
CTGF	ng/mL	1.3	3.8	0.0794	400
GST-alpha	ng/mL	1.4	3.6	0.24	1,200
KIM-1	ng/mL	0.016	0.028	0.00478	24
VEGF	pg/mL	4.4	20	8.76	44,000
β-2M	μg/mL	0.012	0.018	0.0030	15
Cystatin C	ng/mL	2.8	3.7	0.60	3,000
NGAL	ng/mL	4.1	7.8	1.2	6,000
Osteopontin	ng/mL	29	52	3.9	19,500
TIMP-1	ng/mL	0.71	1.1	0.073	365
A-1M	μg/mL	0.059	0.29	0.042	210
THP	μg/mL	0.46	0.30	0.16	800
TFF-3	μg/mL	0.06	0.097	0.060	300

The results of this experiment characterized the least detectible dose and the lower limit of quantification for fourteen analytes associated with various renal disorders using a capture-sandwich assay.

Example 2

Precision of Assay for Analytes Associated with Renal Disorders

To assess the precision of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. The percent errors for each run at each concentration are presented in Table 3 for all of the analytes tested:

TABLE 3
Precision of Analyte Assay

	Average		Run 2		Interrun
	concentration	Run 1	Error	Run 2	Error
Analyte	(ng/mL)	Error (%)	(%)	Error (%)	(%)

Calbindin	4.0	6	2	6	13
	36	5	3	2	7
	281	1	6	0	3
Clusterin	4.4	4	9	2	6
	39	5	1	6	8
	229	1	3	0	2
CTGF	1.2	10	17	4	14
	2.5	19	19	14	14
	18	7	5	13	9
GST-alpha	3.9	14	7	5	10
	16	13	7	10	11
	42	1	16	6	8
KIM-1	0.035	2	0	5	13
	0.32	4	5	2	8
	2.9	0	5	7	4
VEGF	65	10	1	6	14
	534	9	2	12	7
	5,397	1	13	14	9
β-2M	0.040	6	1	8	5
	0.43	2	2	0	10
	6.7	6	5	11	6
Cystatin C	10.5	4	1	7	13
	49	0	0	3	9
	424	2	6	2	5
NGAL	18.1	11	3	6	13
	147	0	0	6	5
	1,070	5	1	2	5
Osteopontin	44	1	10	2	11
	523	9	9	9	7
	8,930	4	10	1	10
TIMP-1	2.2	13	6	3	13
	26	1	1	4	14
	130	1	3	1	4
A-1M	1.7	11	7	7	14
	19	4	1	8	9
	45	3	5	2	4
THP	9.4	3	10	11	11
	15	3	7	8	6
	37	4	5	0	5
TFF-3	0.3	13	3	11	12
	4.2	5	8	5	7
	1.2	3	7	0	13

The results of this experiment characterized the precision of a capture-sandwich assay for fourteen analytes associated with various renal disorders over a wide range of analyte concentrations. The precision of the assay varied between about 1% and about 15% error within a given run, and between about 5% and about 15% error between different runs. The percent errors summarized in Table 2 provide information concerning random error to be expected in an assay measurement caused by variations in technicians, measuring instruments, and times of measurement.

Example 3

Linearity of Assay for Analytes Associated with Renal Disorders

To assess the linearity of an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were measured in triplicate during three runs using the methods described in Example 1. Linearity of the assay used to measure each analyte was determined by measuring the concentrations of standard samples that were serially-diluted throughout the assay range. The % recovery was calculated as observed vs. expected concentration based on the dose-response curve. The results of the linearity analysis are summarized in Table 4.

TABLE 4
Linearity of Analyte Assay

		Expected	Observed	Recovery
Analyte	Dilution	concentration	concentration	(%)

Calbindin	1:2	61	61	100
(ng/mL)	1:4	30	32	106
	1:8	15	17	110
Clusterin	1:2	41	41	100
(ng/mL)	1:4	21	24	116
	1:8	10	11	111
CTGF	1:2	1.7	1.7	100
(ng/mL)	1:4	0.84	1.0	124
	1:8	0.42	0.51	122
GST-alpha	1:2	25	25	100
(ng/mL)	1:4	12	14	115
	1:8	6.2	8.0	129
KIM-1	1:2	0.87	0.87	100
(ng/mL)	1:4	0.41	0.41	101
	1:8	0.21	0.19	93
VEGF	1:2	2,525	2,525	100
(pg/mL)	1:4	1,263	1,340	106
	1:8	631	686	109
β-2M	1:100	0.63	0.63	100
(μg/mL)	1:200	0.31	0.34	106
	1:400	0.16	0.17	107
Cystatin C	1:100	249	249	100
(ng/mL)	1:200	125	122	102
	1:400	62	56	110
NGAL	1:100	1,435	1,435	100
(ng/mL)	1:200	718	775	108
	1:400	359	369	103
Osteopontin	1:100	6,415	6,415	100
(ng/mL)	1:200	3,208	3,275	102
	1:400	1,604	1,525	95
TIMP-1	1:100	35	35	100
(ng/mL)	1:200	18	18	100
	1:400	8.8	8.8	100
A-1M	1:2000	37	37	100
(μg/mL)	1:4000	18	18	99
	1:8000	9.1	9.2	99
THP	1:2000	28	28	100
(μg/mL)	1:4000	14	14	96
	1:8000	6.7	7.1	94
TFF-3	1:2000	8.8	8.8	100
(μg/mL)	1:4000	3.8	4.4	86
	1:8000	1.9	2.2	86

The results of this experiment demonstrated reasonably linear responses of the sandwich-capture assay to variations in the concentrations of the analytes in the tested samples.

Example 4

Spike Recovery of Analytes Associated with Renal Disorders

To assess the recovery of analytes spiked into urine, serum, and plasma samples by an assay used to measure the concentration of analytes associated with renal disorders, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Prior to analysis, all urine samples were diluted 1:2000 (sample: diluent), all plasma samples were diluted 1:5 (sample: diluent), and all serum samples were diluted 1:2000 (sample: diluent).

The concentrations of the analytes in the samples were measured using the methods described in Example 1. The average % recovery was calculated as the proportion of the measurement of analyte spiked into the urine, serum, or plasma sample (observed) to the measurement of analyte spiked into the standard solution (expected). The results of the spike recovery analysis are summarized in Table 5.

TABLE 5
Spike Recovery of Analyte Assay in Urine, Serum, and Plasma Samples

		Recovery in	Recovery in	Recovery in
	Spike	Urine	Serum	Plasma
Analyte	Concentration	Sample (%)	Sample (%)	Sample (%)

Calbindin	66	76	82	83
(ng/mL)	35	91	77	71
	18	80	82	73
	average	82	80	76
Clusterin	80	72	73	75
(ng/mL)	37	70	66	72
	20	90	73	70
	average	77	70	72
CTGF	8.4	91	80	79
(ng/mL)	4.6	114	69	78
	2.4	76	80	69
	average	94	77	75
GST-alpha	27	75	84	80
(ng/mL)	15	90	75	81
	7.1	82	84	72
	average	83	81	78
KIM-1	0.63	87	80	83
(ng/mL)	.029	119	74	80
	0.14	117	80	78
	average	107	78	80
VEGF	584	88	84	82
(pg/mL)	287	101	77	86
	123	107	84	77
	average	99	82	82
β-2M	0.97	117	98	98
(μg/mL)	0.50	124	119	119
	0.24	104	107	107
	average	115	108	105
Cystatin C	183	138	80	103
(ng/mL)	90	136	97	103
	40	120	97	118
	average	131	91	108
NGAL	426	120	105	111
(ng/mL)	213	124	114	112
	103	90	99	113
	average	111	106	112
Osteopontin	1,245	204	124	68
(ng/mL)	636	153	112	69
	302	66	103	67
	average	108	113	68
TIMP-1	25	98	97	113
(ng/mL)	12	114	89	103
	5.7	94	99	113
	average	102	95	110
A-1M	0.0028	100	101	79
(μg/mL)	0.0012	125	80	81
	0.00060	118	101	82
	average	114	94	81
THP	0.0096	126	108	90
(μg/mL)	0.0047	131	93	91
	0.0026	112	114	83
	average	123	105	88
TFF-3	0.0038	105	114	97
(μg/mL)	0.0019	109	104	95
	0.0010	102	118	93
	average	105	112	95

The results of this experiment demonstrated that the sandwich-type assay is reasonably sensitive to the presence of all analytes measured, whether the analytes were measured in standard samples, urine samples, plasma samples, or serum samples.

Example 5

Matrix Interferences of Analytes Associated with Renal Disorders

To assess the matrix interference of hemoglobin, bilirubin, and triglycerides spiked into standard samples, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. For each analyte, three concentration levels of standard solution were spiked into known urine, serum, and plasma samples. Matrix interference was assessed by spiking hemoglobin, bilirubin, and triglyceride into standard analyte samples and measuring analyte concentrations using the methods described in Example 1. A % recovery was determined by calculating the ratio of the analyte concentration measured from the spiked sample (observed) divided by the analyte concentration measured form the standard sample (expected). The results of the matrix interference analysis are summarized in Table 6.

TABLE 6
Matrix Interference of Hemoglobin, Bilirubin, and Triglyceride on
the Measurement of Analytes

		Matrix
		Compound	Maximum	Overall
		Spiked into	Spike	Recovery
	Analyte	Sample	Concentration	(%)

	Calbindin	Hemoglobin	500	110
	(mg/mL)	Bilirubin	20	98
		Triglyceride	500	117
	Clusterin	Hemoglobin	500	125
	(mg/mL)	Bilirubin	20	110
		Triglyceride	500	85
	CTGF	Hemoglobin	500	91
	(mg/mL)	Bilirubin	20	88
		Triglyceride	500	84
	GST-alpha	Hemoglobin	500	100
	(mg/mL)	Bilirubin	20	96
		Triglyceride	500	96
	KIM-1	Hemoglobin	500	108
	(mg/mL)	Bilirubin	20	117
		Triglyceride	500	84
	VEGF	Hemoglobin	500	112
	(mg/mL)	Bilirubin	20	85
		Triglyceride	500	114
	β-2M	Hemoglobin	500	84
	(μg/mL)	Bilirubin	20	75
		Triglyceride	500	104
	Cystatin C	Hemoglobin	500	91
	(ng/mL)	Bilirubin	20	102
		Triglyceride	500	124
	NGAL	Hemoglobin	500	99
	(ng/mL)	Bilirubin	20	92
		Triglyceride	500	106
	Osteopontin	Hemoglobin	500	83
	(ng/mL)	Bilirubin	20	86
		Triglyceride	500	106
	TIMP-1	Hemoglobin	500	87
	(ng/mL)	Bilirubin	20	86
		Triglyceride	500	93
	A-1M	Hemoglobin	500	103
	(μg/mL)	Bilirubin	20	110
		Triglyceride	500	112
	THP	Hemoglobin	500	108
	(μg/mL)	Bilirubin	20	101
		Triglyceride	500	121
	TFF-3	Hemoglobin	500	101
	(μg/mL)	Bilirubin	20	101
		Triglyceride	500	110

The results of this experiment demonstrated that hemoglobin, bilirubin, and triglycerides, three common compounds found in urine, plasma, and serum samples, did not significantly degrade the ability of the sandwich-capture assay to detect any of the analytes tested.

Example 6

Sample Stability of Analytes Associated with Renal Disorders

To assess the ability of analytes spiked into urine, serum, and plasma samples to tolerate freeze-thaw cycles, the following experiment was conducted. The analytes measured were alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF. Each analyte was spiked into known urine, serum, and plasma samples at a known analyte concentration. The concentrations of the analytes in the samples were measured using the methods described in Example 1 after the initial addition of the analyte, and after one, two and three cycles of freezing and thawing. In addition, analyte concentrations in urine, serum and plasma samples were measured immediately after the addition of the analyte to the samples as well as after storage at room temperature for two hours and four hours, and after storage at 4° C. for 2 hours, four hours, and 24 hours.

The results of the freeze-thaw stability analysis are summarized in Table 7. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any freeze-thaw cycles.

TABLE 7
Freeze-Thaw Stability of the Analytes in Urine, Serum, and Plasma

Period

Urine Sample

Serum Sample

Plasma Sample

	and		Recovery		Recovery		Recovery
Analyte	Temp	Concentration	(%)	Concentration	(%)	Concentration	(%)

Calbindin	Control	212	100	31	100	43	100
(ng/mL)	1X	221	104	30	96	41	94
	2X	203	96	30	99	39	92
	3X	234	110	30	97	40	93
Clusterin	0	315	100	232	100	187	100
(ng/mL)	1X	329	104	227	98	177	95
	2X	341	108	240	103	175	94
	3X	379	120	248	107	183	98
CTGF	0	6.7	100	1.5	100	1.2	100
(ng/mL)	1X	7.5	112	1.3	82	1.2	94
	2X	6.8	101	1.4	90	1.2	100
	3X	7.7	115	1.2	73	1.3	107
GST-	0	12	100	23	100	11	100
alpha	1X	13	104	24	105	11	101
(ng/mL)	2X	14	116	21	92	11	97
	3X	14	111	23	100	12	108
KIM-1	0	1.7	100	0.24	100	0.24	100
(ng/mL)	1X	1.7	99	0.24	102	0.22	91
	2X	1.7	99	0.22	94	0.19	78
	3X	1.8	107	0.23	97	0.22	93
VEGF	0	1,530	100	1,245	100	674	100
(pg/mL)	1X	1,575	103	1,205	97	652	97
	2X	1,570	103	1,140	92	612	91
	3X	1,700	111	1,185	95	670	99
β-2M	0	0.0070	100	1.2	100	15	100
(μg/mL)	1X	0.0073	104	1.1	93	14	109
	2X	0.0076	108	1.2	103	15	104
	3X	0.0076	108	1.1	97	13	116
Cystatin C	0	1,240	100	1,330	100	519	100
(ng/mL)	1X	1,280	103	1,470	111	584	113
	2X	1,410	114	1,370	103	730	141
	3X	1,420	115	1,380	104	589	113
NGAL	0	45	100	245	100	84	100
(ng/mL)	1X	46	102	179	114	94	112
	2X	47	104	276	113	91	108
	3X	47	104	278	113	91	109
Osteopontin	0	38	100	1.7	100	5.0	100
(ng/mL)	1X	42	110	1.8	102	5.5	110
	2X	42	108	1.5	87	5.5	109
	3X	42	110	1.3	77	5.4	107
TIMP-1	0	266	100	220	100	70	100
(ng/mL)	1X	265	100	220	10	75	108
	2X	255	96	215	98	77	110
	3X	295	111	228	104	76	109
A-1M	0	14	100	26	100	4.5	100
(μg/mL)	1X	13	92	25	96	4.2	94
	2X	15	107	25	96	4.3	97
	3X	16	116	23	88	4.0	90
THP	0	4.6	100	31	100	9.2	100
(μg/mL)	1X	4.4	96	31	98	8.8	95
	2X	5.0	110	31	100	9.2	100
	3X	5.2	114	27	85	9.1	99
TFF-3	0	4.6	100	24	100	22	100
(μg/mL)	1X	4.4	96	23	98	22	103
	2X	5.0	110	24	103	22	101
	3X	5.2	114	19	82	22	102

The results of the short-term stability assessment are summarized in Table 8. The % recovery of each analyte was calculated as a percentage of the analyte measured in the sample prior to any short-term storage.

TABLE 8
Short-Term Stability of Analytes in Urine, Serum, and Plasma

Storage

Urine Sample

Serum Sample

Plasma Sample

	Time/	Sample	Recovery	Sample	Recovery	Sample	Recovery
Analyte	Temp	Conc.	(%)	Conc.	(%)	Conc.	(%)

Calbindin	Control	226	100	33	100	7	100
(ng/mL)	2 hr/	242	107	30	90	6.3	90
	room
	temp
	2 hr. @	228	101	29	89	6.5	93
	4° C.
	4 hr @	240	106	28	84	5.6	79
	room
	temp
	4 hr. @	202	89	29	86	5.5	79
	4° C.
	24 hr. @	199	88	26	78	7.1	101
	4° C.
Clusterin	Control	185	100	224	100	171	100
(ng/mL)	2 hr @	173	94	237	106	180	105
	room
	temp
	2 hr. @	146	79	225	100	171	100
	4° C.
	4 hr @	166	89	214	96	160	94
	room
	temp
	4 hr. @	157	85	198	88	143	84
	4° C.
	24 hr. @	185	100	207	92	162	94
	4° C.
CTGF	Control	1.9	100	8.8	100	1.2	100
(ng/mL)	2 hr @	1.9	99	6.7	76	1	83
	room
	temp
	2 hr. @	1.8	96	8.1	92	1.1	89
	4° C.
	4 hr @	2.1	113	5.6	64	1	84
	room
	temp
	4 hr. @	1.7	91	6.4	74	0.9	78
	4° C.
	24 hr. @	2.2	116	5.9	68	1.1	89
	4° C.
GST-	Control	14	100	21	100	11	100
alpha	2 hr @	11	75	23	107	11	103
(ng/mL)	room
	temp
	2 hr. @	13	93	22	104	9.4	90
	4° C.
	4 hr @	11	79	21	100	11	109
	room
	temp
	4 hr. @	12	89	21	98	11	100
	4° C.
	24 hr. @	13	90	22	103	14	129
	4° C.
KIM-1	Control	1.5	100	0.23	100	0.24	100
(ng/mL)	2 hr @	1.2	78	0.2	86	0.22	90
	room
	temp
	2 hr. @	1.6	106	0.23	98	0.21	85
	4° C.
	4 hr @	1.3	84	0.19	82	0.2	81
	room
	temp
	4 hr. @	1.4	90	0.22	93	0.19	80
	4° C.
	24 hr. @	1.1	76	0.18	76	0.23	94
	4° C.
VEGF	Control	851	100	1215	100	670	100
(pg/mL)	2 hr @	793	93	1055	87	622	93
	room
	temp
	2 hr. @	700	82	1065	88	629	94
	4° C.
	4 hr @	704	83	1007	83	566	84
	room
	temp
	4 hr. @	618	73	1135	93	544	81
	4° C.
	24 hr. @	653	77	1130	93	589	88
	4° C.
β-2M	Control	0.064	100	2.6	100	1.2	100
(μg/mL)	2 hr @	0.062	97	2.4	92	1.1	93
	room
	temp
	2 hr. @	0.058	91	2.2	85	1.2	94
	4° C.
	4 hr @	0.064	101	2.2	83	1.2	94
	room
	temp
	4 hr. @	0.057	90	2.2	85	1.2	98
	4° C.
	24 hr. @	0.06	94	2.5	97	1.3	103
	4° C.
Cystatin C	Control	52	100	819	100	476	100
(ng/mL)	2 hr @	50	96	837	102	466	98
	room
	temp
	2 hr. @	44	84	884	108	547	115
	4° C.
	4 hr @	49	93	829	101	498	105
	room
	temp
	4 hr. @	46	88	883	108	513	108
	4° C.
	24 hr. @	51	97	767	94	471	99
	4° C.
NGAL	Control	857	100	302	100	93	100
(ng/mL)	2 hr @	888	104	287	95	96	104
	room
	temp
	2 hr. @	923	108	275	91	92	100
	4° C.
	4 hr @	861	101	269	89	88	95
	room
	temp
	4 hr. @	842	98	283	94	94	101
	4° C.
	24 hr. @	960	112	245	81	88	95
	4° C.
Osteopontin	Control	2243	100	6.4	100	5.2	100
(ng/mL)	2 hr @	2240	100	6.8	107	5.9	114
	room
	temp
	2 hr. @	2140	95	6.4	101	6.2	120
	4° C.
	4 hr @	2227	99	6.9	108	5.8	111
	room
	temp
	4 hr. @	2120	95	7.7	120	5.2	101
	4° C.
	24 hr. @	2253	100	6.5	101	6	116
	4° C.
TIMP-1	Control	17	100	349	100	72	100
(ng/mL)	2 hr @	17	98	311	89	70	98
	room
	temp
	2 hr. @	16	94	311	89	68	95
	4° C.
	4 hr @	17	97	306	88	68	95
	room
	temp
	4 hr. @	16	93	329	94	74	103
	4° C.
	24 hr. @	18	105	349	100	72	100
	4° C.
A-1M	Control	3.6	100	2.2	100	1	100
(μg/mL)	2 hr @	3.5	95	2	92	1	105
	room
	temp
	2 hr. @	3.4	92	2.1	97	0.99	99
	4° C.
	4 hr @	3.2	88	2.2	101	0.99	96
	room
	temp
	4 hr. @	3	82	2.2	99	0.97	98
	4° C.
	24 hr. @	3	83	2.2	100	1	101
	4° C.
THP	Control	1.2	100	34	100	2.1	100
(μg/mL)	2 hr @	1.2	99	34	99	2	99
	room
	temp
	2 hr. @	1.1	90	34	100	2	98
	4° C.
	4 hr @	1.1	88	27	80	2	99
	room
	temp
	4 hr. @	0.95	79	33	97	2	95
	4° C.
	24 hr. @	0.91	76	33	98	2.4	116
	4° C.
TFF-3	Control	1230	100	188	100	2240	100
(μg/mL)	2 hr @	1215	99	179	95	2200	98
	room
	temp
	2 hr. @	1200	98	195	104	2263	101
	4° C.
	4 hr @	1160	94	224	119	2097	94
	room
	temp
	4 hr. @	1020	83	199	106	2317	103
	4° C.
	24 hr. @	1030	84	229	122	1940	87
	4° C.

The results of this experiment demonstrated that the analytes associated with renal disorders tested were suitably stable over several freeze/thaw cycles, and up to 24 hrs. of storage at a temperature of 4° C.

Example 8

Diagnosis of Renal Damage Using Detection of Analytes in Human Urine Samples

To assess the effectiveness of a human kidney toxicity panel to detect renal damage due to disease states, the following experiment was conducted. Urine samples were obtained from healthy control patients (n=5), renal cancer patients (n=4) and “other” cancer patients (n=8) afflicted with lung cancer, pancreatic cancer, liver cancer, or colon cancer. All urine samples were diluted as described in Example 4 and subjected to a sandwich-capture assay as described in Example 1. Urine concentrations of analytes included in a human kidney toxicity panel were measured by the assay, including alpha-1 microglobulin (A1M), beta-2 microglobulin (B2M), calbindin, clusterin, CTGF, cystatin C, GST-alpha, KIM-1, NGAL, osteopontin (OPN), THP, TIMP-1, TFF-3, and VEGF.

FIG. 1 summarizes the urine concentrations of those analytes that differed significantly from control urine concentrations. The urine concentrations of A1M, NGAL, and THP were slightly elevated for the renal cancer patient group and more significantly elevated for the “other” cancer patient group. Urine B2M concentrations appeared to be elevated for both the renal cancer and “other” cancer patient groups, although the BRM concentrations exhibited more variability than the other analyte concentrations shown in FIG. 1.

The results of this experiment demonstrated that panels of analytes detected in urine samples were capable of identifying patients having renal damage resulting from renal cancer and other cancers.

Example 9

Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury

A screen for potential protein biomarkers in relation to kidney toxicity/damage was performed using a panel of biomarkers, in a set of urine and plasma samples from patients with documented renal damage. The investigated patient groups included diabetic nephropathy (DN), obstructive uropathy (OU), analgesic abuse (AA) and glomerulonephritis (GN) along with age, gender and BMI matched control groups. Multiplexed immunoassays were applied in order to quantify the following protein analytes: Alpha-1 Microglobulin (α1M), KIM-1, Microalbumin, Beta-2-Microglobulin (β32M), Calbindin, Clusterin, CystatinC, TreFoilFactor-3 (TFF-3), CTGF, GST-alpha, VEGF, Calbindin, Osteopontin, Tamm-HorsfallProtein (THP), TIMP-1 and NGAL.

Li-Heparin plasma and mid-stream spot urine samples were collected from four different patient groups. Samples were also collected from age, gender and BMI matched control subjects. 20 subjects were included in each group resulting in a total number of 160 urine and plasma samples. All samples were stored at −80° C. before use. Glomerular filtration rate for all samples was estimated using two different estimations (Modification of Diet in Renal Disease or MDRD, and the Chronic Kidney Disease Epidemiology Collaboration or CKD-EPI) to outline the eGFR (estimated glomerular filtration rate) distribution within each patient group (FIG. 2). Protein analytes were quantified in human plasma and urine using multiplexed immunoassays in the Luminex xMAP™ platform. The microsphere-based multiplex immunoassays consist of antigen-specific antibodies and optimized reagents in a capture-sandwich format. Output data was given as g/ml calculated from internal standard curves. Because urine creatinine (uCr) correlates with renal filtration rate, data analysis was performed without correction for uCr. Univariate and multivariate data analysis was performed comparing all case vs. control samples as well as cases vs. control samples for the various disease groups.

The majority of the measured proteins showed a correlation to eGFR. Measured variables were correlated to eGFR using Pearson's correlations coefficient, and samples from healthy controls and all disease groups were included in the analysis. 11 and 7 proteins displayed P-values below 0.05 for plasma and urine (Table 9) respectively.

TABLE 9
Correlation analysis of eGFR and variables for all case samples

URINE

PLASMA

Variable	Pearson's r	P-Value	Variable	Pearson's r	P-Value

Alpha-1-	−0.08	0.3	Alpha-1-	−0.33
Microglobulin			Microglobulin
Beta-2-	−0.23	0.003	Beta-2-	−0.39
Microglobulin			Microglobulin
Calbindin	−0.16	0.04	Calbindin	−0.18	<0.02
Clusterin	−0.07	0.4	Clusterin	−0.51
CTGF	−0.08	0.3	CTGF	−0.05	0.5
Creatinine	−0.32		Cystatin-C	−0.42	<0.0001
Cystatin-C	−0.24	0.002	GST-alpha	−0.12	0.1
GST-alpha	−0.11	0.2	KIM-1	−0.17	0.03
KIM-1	−0.08	0.3	NGAL	−0.28	<0.001
Microalbumin_UR	−0.17	0.03	Osteopontin	−0.33
NGAL	−0.15	0.07	THP	−0.31
Osteopontin	−0.19	0.02	TIMP-1	−0.28	<0.001
THP	−0.05	0.6	TFF3	−0.38
TIMP-1	−0.19	0.01	VEGF	−0.14	0.08
TFF2	−0.09	0.3
VEGF	−0.07	0.4
P values <0.0001 are shown in bold italics
P values <0.005 are shown in bold
P values <0.05 are shown in italics

For the various disease groups, univariate statistical analysis revealed that in a direct comparison (T-test) between cases and controls, a number of proteins were differentially expressed in both urine and plasma (Table 10 and FIG. 3). In particular, clusterin showed a marked differential pattern in plasma.

TABLE 10
Differentially regulated proteins by
univariate statistical analysis

	Group	Matrix	Protein	p-value

	AA	Urine	Calbindin	0.016
	AA	Urine	NGAL	0.04
	AA	Urine	Osteopontin	0.005
	AA	Urine	Creatinine	0.001
	AA	Plasma	Calbindin	0.05
	AA	Plasma	Clusterin	0.003
	AA	Plasma	KIM-1	0.03
	AA	Plasma	THP	0.001
	AA	Plasma	TIMP-1	0.02
	DN	Urine	Creatinine	0.04
	DN	Plasma	Clusterin	0.006
	DN	Plasma	KIM-1	0.01
	GN	Urine	Creatinine	0.004
	GN	Urine	Microalbumin	0.0003
	GN	Urine	NGAL	0.05
	GN	Urine	Osteopontin	0.05
	GN	Urine	TFF3	0.03
	GN	Plasma	Alpha 1 Microglobulin	0.002
	GN	Plasma	Beta 2 Microglobulin	0.03
	GN	Plasma	Clusterin	0.00
	GN	Plasma	Cystatin C	0.01
	GN	Plasma	KIM-1	0.003
	GN	Plasma	NGAL	0.03
	GN	Plasma	THP	0.001
	GN	Plasma	TIMP-1	0.003
	GN	Plasma	TFF3	0.01
	GN	Plasma	VEGF	0.02
	OU	Urine	Clusterin	0.02
	OU	Urine	Microalbumin	0.007
	OU	Plasma	Clusterin	0.00

Application of multivariate analysis yielded statistical models that predicted disease from control samples (plasma results are shown in FIG. 4).

In conclusion, these results form a valuable base for further studies on these biomarkers in urine and plasma both regarding baseline levels in normal populations and regarding the differential expression of the analytes in various disease groups. Using this panel of analytes, error rates from adaboosting and/or random forest were low enough (<10%) to allow a prediction model to differentiate between control and disease patient samples. Several of the analytes showed a greater correlation to eGFR in plasma than in urine.

Example 10

Statistical Analysis of Kidney Biomarkers in Plasma and Urine from Patients with Renal Injury

Urine and plasma samples were taken from 80 normal control group subjects and 20 subjects from each of four disorders: analgesic abuse, diabetic nephropathy, glomerulonephritis, and obstructive uropathy. The samples were analyzed for the quantity and presence of 16 different proteins (alpha-1 microglobulin (α1M), beta-2 microglobulin (β2M), calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) as described in Example 1 above. The goal was to determine the analytes that distinguish between a normal sample and a diseased sample, a normal sample and an obstructive uropathy (OU) sample, and finally, an glomerulonephritis sample from the other disease samples (diabetic nephropathy (DN), analgesic abuse (AA), and glomerulonephritis (GN)).

From the above protein analysis data, bootstrap analysis was used to estimate the future performance of several classification algorithms. For each bootstrap run, training data and testing data was randomly generated. Then, the following algorithms were applied on the training data to generate models and then apply the models to the testing data to make predictions: automated Matthew's classification algorithm, classification and regression tree (CART), conditional inference tree, bagging, random forest, boosting, logistic regression, SVM, and Lasso. The accuracy rate and ROC areas were recorded for each method on the prediction of the testing data. The above was repeated 100 times. The mean and the standard deviation of the accuracy rates and of the ROC areas were calculated.

The mean error rates and AUROC were calculated from urine and AUROC was calculated from plasma for 100 runs of the above method for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 5, Table 11), AA vs. normal (FIG. 7, Table 13), DN vs. AA (FIG. 9, Table 15, AA vs. GN (FIG. 11, Table 17), and AA vs. OU (FIG. 13, Table 19).

The average relative importance of 16 different analytes (alpha-1 microglobulin, beta-2 microglobulin, calbindin, clusterin, CTGF, creatinine, cystatin C, GST-alpha, KIM-1, microalbumin, NGAL, osteopontin, THP, TIMP-1, TFF-3, and VEGF) and 4 different clinical variables (weight, BMI, age, and gender) from 100 runs were analyzed with two different statistical methods—random forest (plasma and urine samples) and boosting (urine samples)—for each of the following comparisons: disease (AA+GN+OU+DN) vs. normal (FIG. 6, Table 12), AA vs. normal (FIG. 8, Table 14), DN vs. AA (FIG. 10, Table 16), AA vs. GN (FIG. 12, Table 18), and AA vs. OU (FIG. 14, Table 20).

TABLE 11
Disease v. Normal

			Standard
		Mean	deviation
	method	AUROC	AUROC

	random	0.931	0.039
	forest
	bagging	0.919	0.045
	svm	0.915	0.032
	boosting	0.911	0.06
	lasso	0.897	0.044
	logistic	0.891	0.041
	regression
	ctree	0.847	0.046
	cart	0.842	0.032
	matt	0.83	0.023

TABLE 12
Disease v. Normal

		relative
	analyte	importance

	Creatinine	11.606
	Kidney_Injury_M	8.486
	Tamm_Horsfall_P	8.191
	Total_Protein	6.928
	Osteopontin	6.798
	Neutrophil_Gela	6.784
	Tissue_Inhibito	6.765
	Vascular_Endoth	6.716
	Trefoil_Factor—	6.703
	Cystatin_C	6.482
	Alpha_1_Microgl	6.418
	Beta_2_Microglo	6.228
	Glutathione_S_T	6.053
	clusterin	5.842

TABLE 13
AA v. NL

			Standard
			deviation
		Mean	of
	method	AUROC	AUROC

	cart	1	0
	bagging	1	0
	boosting	1	0
	lasso	0.998	0.008
	ctree	0.998	0.015
	random	0.997	0.012
	forest
	svm	0.977	0.033
	logistic	0.933	0.092
	regression
	matt	0.873	0.112

TABLE 14
AA v. NL

		Relative
	analyte	importance

	Creatinine	17.800
	Tissue_Inhibito	9.953
	Total_Protein	8.837
	Tamm_Horsfall_P	7.379
	Cystatin_C	6.237
	Kidney_Injury_M	6.174
	Beta_2_Microglo	5.915
	Neutrophil_Gela	5.761
	Alpha_1_Microgl	5.742
	Trefoil_Factor—	5.736
	Osteopontin	5.561
	Vascular_Endoth	5.338
	clusterin	4.892
	Glutathione_S_T	4.675

TABLE 15
AA v. DN

			Standard
		Mean	deviation
	method	AUROC	AUROC

	lasso	0.999	0.008
	random	0.989	0.021
	forest
	svm	0.988	0.039
	boosting	0.988	0.022
	bagging	0.972	0.036
	logistic	0.969	0.057
	regression
	cart	0.93	0.055
	ctree	0.929	0.063
	matt	0.862	0.12

TABLE 16
AA v. DN

		Relative
	analyte	importance

	Creatinine	17.57
	Total_Protein	10.90
	Tissue_Inhibito	8.77
	clusterin	6.89
	Glutathione_S_T	6.24
	Alpha_1_Microgl	6.15
	Beta_2_Microglo	6.06
	Cystatin_C	5.99
	Trefoil_Factor—	5.88
	Kidney_Injury_M	5.49
	Vascular_Endoth	5.38
	Tamm_Horsfall_P	5.33
	Osteopontin	4.86
	Neutrophil_Gela	4.47

TABLE 17
AA v. GN

			Standard
			deviation
		Mean	of
	method	AUROC	AUROC

	svm	0.689	0.11
	boosting	0.675	0.102
	bagging	0.674	0.106
	random	0.66	0.096
	forest
	matt	0.631	0.085
	cart	0.626	0.089
	logistic	0.614	0.091
	regression
	lasso	0.606	0.102
	ctree	0.53	0.061

TABLE 18
AA v. GN

		Relative
	analyte	importance

	Creatinine	10.780
	Alpha_1_Microgl	8.847
	Kidney_Injury_M	8.604
	clusterin	8.109
	Total_Protein	7.679
	Glutathione_S_T	7.493
	Neutrophil_Gela	6.721
	Vascular_Endoth	6.461
	Cystatin_C	6.444
	Beta_2_Microglo	6.261
	Trefoil_Factor—	6.184
	Tamm_Horsfall_P	5.872
	Tissue_Inhibito	5.690
	Osteopontin	4.855

TABLE 19
AA v. OU

			Standard
			deviation
		Mean	of
	method	AUROC	AUROC

	random	0.814	0.11
	forest
	bagging	0.792	0.115
	svm	0.788	0.112
	lasso	0.786	0.118
	boosting	0.757	0.117
	matt	0.687	0.111
	logistic	0.683	0.116
	regression
	cart	0.665	0.097
	ctree	0.659	0.118

TABLE 20
AA v. OU

		Relative
	analyte	importance

	Total_Protein	11.502
	Tissue_Inhibito	9.736
	Cystatin_C	9.161
	Alpha_1_Microgl	8.637
	Trefoil_Factor—	7.329
	Osteopontin	7.326
	Beta_2_Microglo	6.978
	Neutrophil_Gela	6.577
	Glutathione_S_T	6.100
	Tamm_Horsfall_P	6.066
	Kidney_Injury_M	6.038
	Vascular_Endoth	5.946
	clusterin	4.751
	Creatinine	3.854

It should be appreciated by those of skill in the art that the techniques disclosed in the examples above represent techniques discovered by the inventors to function well in the practice of the invention. Those of skill in the art should, however, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.