NZ736200B2 - Specific detection of clusterin isoforms - Google Patents

Specific detection of clusterin isoforms Download PDF

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Publication number
NZ736200B2
NZ736200B2 NZ736200A NZ73620016A NZ736200B2 NZ 736200 B2 NZ736200 B2 NZ 736200B2 NZ 736200 A NZ736200 A NZ 736200A NZ 73620016 A NZ73620016 A NZ 73620016A NZ 736200 B2 NZ736200 B2 NZ 736200B2
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New Zealand
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clusterin
wilkinson
sarah
kidney
specifically bind
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NZ736200A
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NZ736200A (en
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John Joseph Quinn
Murthy Vsn Yerramilli
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Idexx Laboratories Inc
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Priority claimed from PCT/US2016/030075 external-priority patent/WO2016176565A1/en
Publication of NZ736200A publication Critical patent/NZ736200A/en
Publication of NZ736200B2 publication Critical patent/NZ736200B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
    • G01N2333/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/775—Apolipopeptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G01N33/521—Single-layer analytical elements
    • G01N33/523—Single-layer analytical elements the element being adapted for a specific analyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere

Abstract

The invention provides methods and compositions for the detection of specific isoforms of clusterin.

Description

TITLE: Specific Detection of Clusterin lsoforms PRIORITY This application claims the benefit of US. Ser. No. 621 55,175, filed on April , 2015, which is incorporated by reference in its entirety herein.
BACKGROUND OF THE INVENTION Clusterin or Apolipoprotein J is a 75-80 kDa disulphide linked heterodimeric protein. Clusterin is part of many physiological processes including sperm tion, lipid transportation, complement inhibition, tissue remodeling, membrane recycling, stabilization of stressed proteins, and promotion of inhibition of apoptosis.
Clusterin is over-expressed during kidney al and distal tubular damage, has been noticed in various carcinomas, and is ulated in kidney injury.
There are several immunoassays that have been developed and marketed for measuring clusterin in various body fluids including plasma, serum, and urine.
Kidney specific clusterin can be used as a marker of kidney damage or disease.
However, contamination of urine samples with blood is a commonly observed occurrence due to infection, trauma, neoplasia, inflammation, and accidental ination during ization and cystocentisis. This is more profound m in veterinary medicine. In healthy tions serum concentrations of clusterin are 1000-fold higher (60-100 ug/ml) than the concentrations in urine (<1OO ng/ml). The blood contamination brings dney specific clusterin isoforms into the urine.
Hence, it is important to ensure that the quantification of kidney specific clusterin isoform is not impacted by contamination of serum clusterin from the blood. Failure to do so can result in false positive test s in urine clusterin assays. Methods are needed in the art to differentiate clusterin isoforms in bodily samples.
Y OF THE INVENTION The invention provides methods of specifically detecting a first clusterin isoform. The methods comprise contacting a sample with one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and one or more les that specifically bind to carbohydrate moieties of the first clusterin isoform and that do not specifically bind to carbohydrate moieties of other clusterin isoforms.
Complexes of the first clusterin, the one or more dies or n binding fragments thereof that specifically bind clusterin, and the one or more molecules that specifically bind to carbohydrate moieties of the first clusterin and that do not specifically bind to ydrate moieties of other clusterin isoforms are detected.
[Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson The invention also es methods of ing kidney specific clusterin.
The methods comprise contacting a sample with one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and one or more molecules that specifically bind to carbohydrate moieties of kidney ic clusterin and that do not specifically bind to carbohydrate moieties of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or orne, non-kidney ic clusterin).
Complexes of kidney specific rin, the one or more antibodies or antigen binding nts thereof that specifically bind clusterin, and the one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not 1O specifically bind to carbohydrate moieties of other clusterin isoforms are detected.
The one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not specifically bind to carbohydrate moieties of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin), can be one or more lectins or one or more molecules that specifically bind N-acetylglucosamine. The one or more s can be lectins that ically bind N-acetylglucosamine. Lectins can be Phaseo/us vulgaris leucoagglutanin (PHA-L), wheat germ agglutinin (WGA), WGA1, WGA2, WGAS, sWGA, Phaseo/us vulgaris agglutinin-E (PHA-E), Lycopersicon escu/entum lectin (LEL), Datura stramonium lectin (DSL), Phaseo/us vulgaris leucoagglutinin (PSA), or DoIiChos s lectin (DBA).
The one or more antibodies or n binding fragments thereof can be immobilized to a support. The sample and detectably labeled one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not specifically bind to carbohydrate moieties of other rin isoforms (which can be lectins) can be added to the support.
The one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not specifically bind to carbohydrate moieties of other clusterin isoforms (which can be lectins) can be immobilized to a support. The sample and detectably labeled one or more antibodies or n binding fragments thereof can be added to the support.
The one or more antibodies or antigen binding fragments thereof, the one or more molecules that specifically bind to carbohydrate moieties of kidney ic cluste'a and do not bind to carbohydrate moieties of other clusterin isoforms (e.g., [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson plasma clusterin, serum clusterin, or bloodborne, non—kidney specific clusterin), or both can be labeled with a detectable label.
The one or more lectins can be lectins that do not specifically bind serum and plasma clusterin. The sample can be a urine . The detection can be completed by a method selected from the group consisting of a lateral flow assay, a chemiluminescent d sandwich assay, and an enzyme-linked immunosorbent assay ), a competitive assay, an agglutination assay, a uminescent assay, a inescent assay, a gel electrophoresis immunoassay method, an immunohistochemistry assay, a radioimmunoassay (RIA), a label-free biosensor 1O assay, or an immunoradiometric assay. The antibodies can specifically bind plasma rin, serum clusterin, recombinant clusterin, kidney specific clusterin, or MDCK- derived clusterin. The kidney ic clusterin can be human, feline, or canine.
Other embodiments of the invention provide methods for detecting kidney e, kidney injury, or kidney damage in a mammal. The s comprise contacting a sample from a mammal with one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and one or more molecules that specifically bind to carbohydrate es of kidney specific clusterin and that do not specifically bind to carbohydrate moieties of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin). Complexes of kidney specific clusterin, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not specifically bind to carbohydrate moieties of other clusterin isoforms are detected. If the complexes are detected, then the mammal has kidney disease, kidney injury, or kidney damage. A kidney therapy or kidney therapeutic can be administered to the mammal if the mammal has kidney disease, kidney damage, or kidney injury. The kidney disease can be a urinary tract infection. The mammal can be a human, , or canine.
Other embodiments of the invention provide methods of distinguishing one more clusterin isoforms from other types of clusterin isoforms. The methods comprise contacting a sample with one or more antibodies or antigen binding fragments thereof that ically bind clusterin and one or more molecules that specially bind to carbohydrate moieties of the one or more rin isoforms anddo n ind to carbohydrate moieties of the other clusterin isoforms. Complexes of [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ed set by Sarah.Wilkinson the one or more isoforms of clusterin, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin, and the one or more molecules that ically bind to carbohydrate es of the one or more clusterin isoforms and that do not bind to carbohydrate moieties of the other rin isoforms are detected. The one or more clusterin isoforms can be kidney specific clusterin and the other clusterin ms can be, e.g., plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin. The one or more rin isoforms can be human, feline, or canine clusterin isoforms.
Other embodiments of the invention provide a complex comprising one or 1O more clusterin molecules, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin, and one or more s. The complex can comprise one or more kidney specific clusterin molecules, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin, and one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not bind to carbohydrate es of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or bloodborne, non-kidney ic clusterin). The complex can be lized to any type of solid support. The x can additionally comprise one or more detectable labels, which can be associated with one or more of the molecules of the complex.
Other embodiments of the invention e a kit comprising one or more antibodies or n binding fragments thereof that specifically bind clusterin and one or more the one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not bind to carbohydrate es of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or bloodborne, non- kidney specific clusterin). The one or more antibodies or antigen binding fragments thereof, the one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and that do not bind to carbohydrate moieties of other clusterin isoforms, or both are labeled with a detectable label. The detectable label can be an enzyme, an enzyme conjugate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a direct visual label, or a magnetic particle.
Other embodiments of the invention provide a method of improving detection of clufilin and clusterin ms. The methods comprise contacting a sample with one ore clusterin antibodies or specific binding fragments thereof and one or [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson more molecules that ically bind to one or more carbohydrate moieties of clusterin. Complexes of one or more clusterin antibodies or specific binding fragments thereof and one or more molecules that specifically bind to one or more ydrate moieties of clusterin are detected with improved sensitivity, icity, or both.
Therefore, the instant invention provides methods and compositions for the detection and/or quantification of a first specific clusterin isoform, optionally in the presence of one or more second clusterin isoforms, such that the one or more second clusterin isoforms do not significantly interfere with the detection and/or 1O quantification of the first specific clusterin isoform.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1A-B show clusterin levels in normal (i.e., healthy) canine urine that was spiked with varying dilutions of normal canine serum.
Figure 2 shows g of clusterin to a lectin solid phase.
Figure 3 shows a comparison of a commercial rin EIA and a Kidney Specific Clusterin Immunoassay in both whole blood and serum.
Figure 4 shows ement of kidney specific clusterin in urine from a canine gentamicin model.
Figure 5 shows measurement of kidney specific clusterin in urine of dogs with inflammatory or ischemic d active kidney injury.
Figure 6 shows measurement of kidney specific clusterin in patients with urinary tract infections .
Figure 7 shows a GE silver stain and western blot of feline clusterin.
Panel A. Silver stain of cell culture supernatants of MDCK and CRFK cell lines from ATCC. B. Western blots showing vity of anti-clusterin canine monoclonal antibody with Lanes 2 and 3 MDCK (canine) clusterin, 4 and 5 Plasma (canine) clusterin, and 6 and 7 CRFK (feline) clusterin.
Figure 8 shows human clusterin expression in cells grown under various conditions of stress.
Figure 9 shows rabbit anti-beta chain clusterin binding to clusterin from MDCK (lane 2, 4), HEK 293 cell supernatants (lane 3), and the positive control recombinant canine clusterin beta chain n (lane 5).
DETAbED DESCRIPTION OF THE INVENTION [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson As used herein, the singular forms "a, an," and "the" include plural referents unless the context clearly dictates otherwise. The term “about” in association with a numerical value means that the cal value can vary plus or minus by 5% or less of the numerical value.
Kidney specific clusterin is an acute kidney injury (AKI) ker that increases during and, as a result of, kidney injury in mammals such as dogs, cats, and humans. A commercial EIA kit from Biovendor is available for the quantification of canine clusterin in both serum and urine. A recent study validated the biomarker using this kit in dogs with leishmaniasis. However, ination of urine samples with serum can provide false positive results due to the high concentration of clusterin in serum. The contamination of urine s with blood s in the lack of specificity in the detection of kidney specific clusterin due to the ination by serum clusterin.
To demonstrate the complications of false positives from general total clusterin measurements a negative canine urine sample was value assigned using a commercial kit (Biovendor) and then spiked with negative canine serum (0.002% to % v/v). The resulting mixtures were analyzed using the cial kit and the results obtained are shown in the table below: % Contamination Observed y Sample specific clusterin] ng/ml —__ 13 _—4869 _—2587 1142 ——E_ 113 Urine + 0.002% 0.001 Serum The commercial cut off is about 70 ng/ml. When the total clusterin is measured (all isoforms), even minute amounts of blood, which are not visible to the naked eye or detectable by conventional urinalysis (dipstick) can cause false positives. This means that the patient samples that have any hint of blood contamination have to be evaluated very carefully since the possibility of false positives leading to false clinical diagnoses is increased. The t invention provia methods of identifying specific isoforms of clusterin in bodily fluids, for [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson example, the determination of presence and/or ty of kidney specific clusterin with no erence by serum clusterin. That is, the instant invention can be used to detect and/or quantify specific clusterin isoforms, e.g. kidney ic clusterin in the presence of other clusterin isoforms.
The primary structures of all clusterin isoforms are highly homologous.
However, it was thought that there would be differences in the post-translational modification patterns between various clusterin isoforms. The specific oligosaccharide structures on clusterin isoforms are associated with tissue source, physiological status, disease state, and species. The methods of the t 1O invention take advantage of these differences in developing detection methods for specific clusterin isoforms (e.g., kidney injury specific clusterin isoforms) that are present in patient samples (e.g., urine samples).
Clusterin lsoforms “Clusterin isoforms” as used herein, are clusterin molecules that are a product of a gene splicing or duplication event, which are glycosylated (see, e.g., Rizzi et a/., Adv. Cancer Res. 104:9 (2009); Prochnow et a/., PLOS One, 03 (2013)).
Clusterin isoforms include nuclear, cytoplasmic, and secreted forms. A “clusterin isoform” also comprises clusterin orms, which are forms of clusterin that are differentially glycosylated due to, e.g., expression in a specific tissue type, expression in a specific physiological state, expression in a specific species type, expression in a specific disease state, or under conditions of cell damage.
“Kidney ic clusterin” or “kidney specific clusterin isoform” is rin produced in the renal system (i.e., kidneys, ureters, urethra, and the bladder) that can be present in the renal system, ing urine. Small amounts of kidney specific clusterin, however, can leak into blood, serum, or plasma. Increased levels of kidney specific rin can be present in the renal system, including urine, of animals and humans with kidney injury, kidney , and/or kidney disease as compared to animals and humans with no kidney injury, kidney damage, and/or kidney e.
“Serum clusterin” and It plasma clusterin” are clusterin isoforms that are synthesized in tissues such as heart, liver and lung that are released into circulation in blood, plasma, or fractions thereof. “Serum clusterin” and “plasma clusterin” do not include kidney specific clusterin that ated in the renal system or kidney specific in the renal system and then leaked into circulating blood, ustnefithat originated lasma, or fractions thereof. Non-kidney ic clusterin isoforms are those [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ed set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson ation] Sarah.Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson clusterin isoforms that are not produced in the renal system (e.g., serum or plasma clusterin). Bloodborne clusterin isoforms are those that are present in circulating blood, , serum or fractions thereof.
Secreted clusterin is produced from an initial protein precursor, presecretory psCLU (~60 kDa), heavily glycosylated, and then cleaved in the endoplasmic lum (ER). The resulting alpha- and beta-peptide chains are held together by 5 disulfide bonds in the mature secreted heterodimer protein form O kDa).
The glycosylation of clusterin can be different for different isoforms of clusterin. For example, kidney ic clusterin and serum or plasma clusterin can have different glycosylation patterns. This difference in glycosylation between isoforms of clusterin can be used to entiate one isoform of clusterin from other isoforms of clusterin.
Clusterin isoforms can be differentiated in, for example, mammals, humans, canines, felines, equines, bovines, ovines, simians, and other s using the methods of the invention. Differentiation includes, for example, determining the presence or absence of a first clusterin isoform in the presence of one or more second types of clusterin isoforms.
Antibodies Antibodies of the invention are antibody molecules or antigen binding fragments thereof that specifically bind to clusterin. The antibodies or antigen binding nts thereof can be specific for human, canine, , equine, bovine, ovine, or simian clusterin. The antibodies or antigen binding fragments thereof can be specific for any type of clusterin isoform (e.g., kidney specific clusterin, plasma rin, or serum clusterin). In embodiments of the invention, an antibody or antigen binding fragment thereof specifically binds kidney specific rin. In other embodiments an antibody or antigen binding fragment thereof specifically binds one or more isoforms of rin, all isoforms of clusterin, serum clusterin, or plasma clusterin.
An antibody of the invention can be a polyclonal antibody, a monoclonal dy, a single chain antibody (scFv), a bispecific antibody, a multispecific antibody, a chimeric dy, a monovalent antibody, a bivalent antibody, a multivalent antibody, an anti—idiotypic antibody, or an antigen or ic binding fragment of an antibody. An antigen binding fragments or specific binding fragment of an antibody is a porn of an intact antibody comprising the antigen binding site or variable region ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson of an intact antibody. Examples of antigen binding antibody fragments include Fab, Fab’, Fab’-SH, F(ab’)2, Fd, single chain Fvs (scFv), disulfide-linked Fvs (dev), fragments comprising a VL or a VH domain or a VL domain and a VH domain, and FV fragments.
An antibody of the invention can be any antibody class, including for example, lgG (lgG1, lgG2a, lgG2b, lgG3, lgG4), lgM, lgA (lgA1, lgA2), lgD and lgE. An antibody or antigen binding fragment thereof binds to one or more epitopes of a clusterin molecule, such as a kidney specific clusterin molecule, a plasma clusterin molecule, or a serum clusterin molecule. An antibody can be made in vivo in suitable 1O laboratory animals or in vitro using inant DNA techniques. Means for preparing and characterizing antibodies are well known in the art. See, e.g., Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol. Biol. 322361-79 (1994); Baileg, Methods Mol. Biol. 322381-88 (1994); Gullick, Methods Mol. Biol. 322389—99 (1994); Drenckhahn et al. Methods Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. l. 10:239-65 (1992); Wright et al. Crit. Rev. Immunol. 12:125-68 (1992). For example, polyclonal antibodies can be produced by administering a clusterin molecule or part of a clusterin molecule to an animal, such as a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, , or horse.
Serum from the zed animal is collected and the antibodies are ed from the plasma by, for e, itation with ammonium sulfate, ed by chromatography, such as affinity chromatography. Techniques for producing and sing polyclonal antibodies are known in the art.
“Specifically binds” or “specific for” means that a first antigen, e.g., a clusterin or a portion thereof, recognizes and binds to an dy or antigen binding fragment thereof with greater affinity than other ecific molecules. A non—specific molecule is an antigen that shares no common epitope with the first antigen. In embodiments of the invention a non-specific molecule is not a clusterin isoform and is not related to clusterin. For example, an antibody raised against a first antigen (e.g., a clusterin molecule) to which it binds more efficiently than to a non-specific antigen can be bed as specifically binding to the first antigen. In embodiments of the invention, an antibody or antigen-binding fragment thereof specifically binds to a rin molecule or portion thereof when it binds with a g affinity Ka of 107 l/mol flore. In the instant invention an antibody or antigen binding fragment can speci lly bind to 2 or more isoforms of clusterin or can specifically bind to only one [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson isoform of rin, e.g., kidney specific clusterin. Specific g can be tested using, for example, an enzyme-linked immunosorbant assay (ELISA), a radioimmunoassay (RIA), or a western blot assay using methodology well known in the art.
Antibodies of the invention can be chimeric (see, e.g., US. Patent No. ,482,856), humanized (see, e.g., Jones et al., Nature 321:522 (1986); ann et al., Nature 3 ; Presta, Curr. Op. Struct. Biol. 2:593 (1992)), caninized, , or human dies. Human antibodies can be made by, for example, direct immortilization, phage y, transgenic mice, or a Trimera 1O methodology, see e.g., Reisener eta/., Trends Biotechnol. 16:242-246 (1998).
An assay for detection of a clusterin le can utilize one antibody or antigen binding fragment thereof or one or more antibodies or fragments (e.g., 1, 2, 3, 4, 5, 10 or more antibodies). An assay for clusterin can use, for example, a monoclonal antibody specific for a clusterin epitope, a combination of monoclonal antibodies specific for epitopes of one clusterin molecule, monoclonal antibodies specific for epitopes of different clusterins, polyclonal antibodies specific for the same clusterin epitope, polyclonal antibodies specific for ent clusterin epitopes, or a combination of onal and polyclonal dies. Assay protocols can be based upon, for example, competition, direct reaction, or sandwich type assays using, for example, labeled antibody.
Antibodies of the invention can be labeled with any type of label known in the art, including, for example, scent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope, and bioluminescent labels.
Antibodies that specifically bind clusterin include, for example, 9H7, 3A4, 2F2, antibodies specific for the alpha chain of clusterin, antibodies ic for the beta chain of clusterin, anti—clusterin urine isoform, Hs-3; 3R3-2; CLl-9; 1A11; 2F12; A4; 7D1; 3R3/2, clusterin C-Term antibody, clusterin m l antibody, CLU (AA 1- 333)(N-Term) antibody, CLU N-Term (AA 79—99) antibody, CLU (AA 312-325) antibody, CLU (AA 44-58) antibody, CLU (AA 402-501) antibody, CLU (AA 75-501) anfibody, CLLJ(A¢\ 312-325) anfibody; anfibody LS—B6759, anflbody LS—B3762, antibody LS-B2937, and LS-B2852, antibody 16B5. An antibody can specifically bind kidney specific clusterin or both kidney specific clusterin and other forms of clusterin (egg wnorpbsmacMswnm.
Lecti [Annotation] Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson ation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson Lectins are proteins that recognize and bind specific monosaccharide or oligosaccharide structures (carbohydrates). A lectin usually contains two or more binding sites for carbohydrate units. The carbohydrate-binding specificity of a certain lectin is determined by the amino acid residues that bind the carbohydrate. The binding strength of lectins to carbohydrates can increase with the number of molecular interactions. The dissociation constant for binding of lectins to carbohydrates is about Kd of 10'5 to 107. “Specifically binds” or “specific for” means that a first lectin, e.g., WGA, recognizes and binds to a specific type of carbohydrate (e.g., N-acetylglucosamine for WGA) with greater affinity than for other non-specific 1O types of carbohydrates. The specific type of carbohydrate is associated with a specific clusterin isoform (e.g., kidney specific clusterin or a species specific clusterin) and not significantly associated with one or more other clusterin ms (e.g., serum clusterin). For example, a lectin that binds more efficiently to a first specific type of carbohydrate than to a non-specific carbohydrate can be described as specifically binding to the first specific type of carbohydrate. ln embodiments of the ion, a lectin binds more efficiently to a first specific type of carbohydrate than to a non—specific carbohydrate when it binds to the first specific type of carbohydrate with a Kd that is lower by about 5, 10, 20, 30, 40, 50, 60% or more when compared to the binding of the non-specific carbohydrate. ln embodiments of the invention, a lectin specifically binds to a specific type of carbohydrate when it binds with a dissociation constant Kd of about 10'5 to 107. In the instant invention a lectin can specifically bind to 2 or more specific types of ydrates or can specifically bind to only one specific type of carbohydrate.
Lectins can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope and bioluminescent labels. ln embodiments of the invention lectins are used that specifically bind kidney specific clusterin and that do not ically bind plasma or serum clusterin. ln embodiments of the invention s that specifically bind N—acetylglucosamine are useful in the invention. Such s include, for example, WGA (wheat germ agglutinin), WGA1, WGA2, WGAS, sWGA, DSL lectin (Datura nium lectin), mannose binding lectin, PHA-L o/us is leucoagglutanin), PHA-E (Phafius vulgaris erythoagglutanin), and LEL (Lycopersicon escu/entum (Tomato)lectin. Other s that can be used e, for e jacalin, STL lectin [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson um tuberosum), LCA lectin (Lens culinaris), PSA lectin o/us vulgaris leucoagglutinin), ECL lectin (Erythina ga/li), RCA lectin (Ricin communis), DBA lectin (Do/ichos bif/orus), SBA lectin an), and CONA lectin (concanavlin).
Lectins are cially available from, e.g., Vector Laboratories.
Lectins can be used that specifically bind to carbohydrates on human, canine, feline, equine, bovine, ovine, or simian clusterin isoforms. Lectins can also be used that specifically bind one or more , serum, or kidney clusterin isoforms and that do not bind other clusterin isoforms.
Molecules that Specifically Bind to Carbohydrate Moieties of a First Clusterin lsoform 1O and That Do Not ically Bind to Carbohydrate Moieties of Other Clusterin lsoforms In embodiments of the invention one or more molecules that ically bind to carbohydrate moieties of a first rin isoform (e.g., kidney specific clusterin or a species specific clusterin, e.g., , feline, or human kidney specific clusterin) and that do not specifically bind to carbohydrate moieties of other clusterin isoforms can be used in assays of the invention. Other clusterin isoforms can be, for example, serum clusterin or plasma clusterin. In an example, the one or more molecules that specifically bind to carbohydrate moieties of a kidney specific clusterin isoform and that do not specifically bind to carbohydrate moieties of bloodborne, non-kidney specific clusterin isoforms can be used in assays of the invention. Examples of such molecules include the lectins discussed above and molecules that specifically bind ylglucosamine.
“Specifically binds” or “specific for” means that a first molecule specifically binds to carbohydrate moieties of a first clusterin isoform (e.g., kidney specific clusterin or a species specific clusterin) and does not specifically bind to carbohydrate moieties of one or more other clusterin isoforms. The first le recognizes and binds to a specific type of carbohydrate that occurs on a first clusterin isoform and does not significantly occur on one or more second clusterin isoforms (e.g., N-acetylglucosamine for bacterial -binding domain 3 protein, wherein N—acetylglucosamine is a carbohydrate that occurs on kidney specific clusterin isoforms and that does not significantly occur on serum clusterin isoforms) with r affinity than other non-specific carbohydrates. For example, a first molefi that binds more efficiently to a first ic type of carbohydrate than to a [Annotation] Sarah.Wilkinson None set by Wilkinson ation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson non—specific carbohydrate can be described as specifically binding to the first specific type of carbohydrate.
In embodiments of the invention, a first molecule that specifically binds to carbohydrate moieties of a first clusterin isoform and does not specifically bind to carbohydrate moieties of other clusterin isoforms, binds more efficiently to a first specific type of carbohydrate than to a non—specific carbohydrate when it binds to the first specific type of carbohydrate with a Kd that is lower by about 5, 10, 20, 30 , 40, 50, 60% or more when ed to the g of the to the non—specific carbohydrate. A first molecule that does not specifically bind to carbohydrate 1O moieties of other clusterin isoforms means that the le specifically binds via specific carbohydrate moieties of a first clusterin isoform and does not ically bind to carbohydrate moieties of a second clusterin isoform, such that g to the first clusterin isoform can detected and/or quantified in the presence of the second clusterin isoform, wherein the ce of the second clusterin isoform does not icantly interfere with the detection and/or quantification of the first clusterin isoform. In embodiments of the invention, a first molecule specifically binds to a specific type of carbohydrate when it binds with a dissociation constant Kd of about '5 to 10'7. In the instant invention a first molecule can specifically bind to 2 or more specific types of carbohydrates or can specifically bind to only one specific type of carbohydrate.
In embodiments of the invention one or more molecules that bind N- acetylglucosamine can be used to ically bind to kidney specific rin. One or more molecules that bind N-acetylglucosamine include, for example, a wild-type WGA (wheat germ agglutinin), mutated forms of WGA (e.g., WGA1, WGA2, WGAS, see Parasuraman et al. J. Mol. Recognit. (2014) —92), barley lectin (BL), rice lectin, Uritica dioica agglutinin (UDA), hevein, Phaseo/us vulgaris chitinase (PVC), potato wound—inducible protein 1 (WIN1), potato wound-inducible protein 2 (WIN2), Solanum tuberosum chitinase (STC), tobacco chitinase (TC), poplar wound—inducible protein (POP), bacterial N-acetylglucosamine-binding n A (Gpr) (from, e.g., Vibrio cholera, Shewane/la onedensis, Shewane/la baltica, Vibrio fascheri, Vibrio tapetis, Vibrio vulnificus, Yersinia mol/aretli, Yersinia a/dovae) CBP70, Plasmodium falciparum Pf120, Pf83, and Pf45 GlcNAc—binding proteins, Arsenophonus nasorfie n-acetylglucosamine-binding protein, bacterial chitin-binding domain 3 prote rom, e.g., Bacillus thuringiensis, us cereus, Burkho/derla amblfaria), N- [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson acetyl glucosamine chitinase like lectin from Tamarindus indica, phloem protein 2 (PP2, PP2-1A1) from Arabidopsis thaliana, Streptomyces o/ivaceoviridis Nch, urokinase plasminogen activation receptor—associated protein/ENDO180, amelogenin, and attenuated murine cytomegalovirus.
Assays The methods of the invention can be used to detect clusterin isoforms (e.g., kidney specific clusterin or species specific clusterin, e.g. canine, human or feline kidney specific clusterin) in a test sample, such as a biological sample or a laboratory sample. A test sample can potentially comprise (1) kidney specific 1O clusterin, (2) kidney specific clusterin and serum clusterin, (3) kidney ic rin and one or more types of other dney specific clusterin, (4) one or more types of other non-kidney specific clusterin; or (5) no clusterin. A biological sample can include, for example, tissue, urine, blood, serum, plasma, saliva, sputum, feces, cerebrospinal fluid, amniotic fluid, or wound exudate from a mammal such as a horse, bovine, ovine, cat, dog, mouse, rat, simian, or human. The test sample can be untreated, itated, fractionated, separated, diluted, concentrated, or purified. In embodiments of the invention kidney specific clusterin leaks into blood, plasma or serum and can be detected therein.
The methods of the invention can be used to improve detection of clusterin and clusterin isoforms by providing assays that use both a rin dy or specific binding fragment thereof combined with a molecule (e.g., a lectin) that specifically binds to one or more carbohydrate moieties of clusterin. The methods comprise contacting a sample with one or more clusterin antibodies or ic binding fragments thereof and one or more molecules that specifically bind to one or more carbohydrate moieties of rin. Complexes of one or more clusterin antibodies or specific binding fragments thereof and one or more molecules that specifically bind to one or more carbohydrate moieties of clusterin are detected with improved sensitivity, specificity, or both. The sensitivity or icity can be improved by about 2, 5, 10, 20, 30, 40, 50% or more.
In certain embodiments, methods of the invention can be used to detect specific clusterin isoforms (e.g., a kidney specific clusterin or species specific clusterin). The methods comprise ting one or more antibodies or antigen bindifiragments thereof that specifically bind clusterin and one or more other mole s that specifically bind kidney ic clusterin (e.g., molecules that [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson specifically bind to carbohydrate moieties of kidney specific clusterin and do not bind to carbohydrate moieties of other clusterin isoforms) with a test sample under conditions that allow complexes of kidney ic clusterin, antibody or antigen binding fragment thereof, and one or more other molecules that specifically bind kidney specific clusterin to form. The complexes are then detected. The presence of complexes indicates the presence of kidney specific clusterin. The absence of complexes indicates the absence of kidney ic clusterin. One of skill in the art is familiar with assays and conditions that are used to detect x binding.
Complexes can se, for example, one or more kidney specific clusterin 1O molecules, one or more antibodies that ically bind clusterin, and one or more other molecules that specifically bind to kidney specific clusterin and that do not specifically bind other isoforms of clusterin. The other forms of clusterin can be, for e, bloodborne, non-kidney specific clusterin isoforms. The amount of the complexes can be determined and can be used to establish the severity of disease.
Assays of the invention can be used to, e.g., distinguish kidney specific clusterin from other types of clusterin isoforms, to detect kidney specific clusterin in a , to quantify kidney specific clusterin in a sample, to distinguish one or more rin isoforms (e.g., kidney ic clusterin, serum clusterin, plasma rin, species specific clusterin isoforms) from other clusterin isoforms, to quantify clusterin isoforms in a sample, or to detect specific rin isoforms in a sample.
Embodiments of the invention provide methods of distinguishing one more clusterin isoforms from other types of clusterin ms. The methods comprise ting a sample with one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and one or more molecules that specifically bind to carbohydrate moieties of the one or more clusterin isoforms (e.g. kidney specific clusterin) and do not bind to carbohydrate moieties of the other clusterin isoforms (e.g., plasma rin, serum clusterin, or bloodborne, non-kidney specific clusterin isoforms). Complexes comprising the one or more isoforms of clusterin, one or more antibodies or antigen g fragments thereof that specifically bind clusterin, and the one or more molecules that specifically bind to ydrate moieties of the one or more rin isoforms and that do not bind to carbohydrate moieties of the other clusterin isoforms are detected. The one or more clusterin clustisofoifi can be mammalian, human, , feline, equine, bovine, ovine, or simianisoforms.
[Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson ation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson ation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson Competitive assays can be used in s of the invention. For example, one or more antibodies or antigen binding nts thereof that specifically bind clusterin can be immobilized to a support. Kidney specific clusterin bound to a detectably labeled lectin and a sample treated with an unlabeled lectin that ically binds kidney specific clusterin are added to the support. The amount of detectably labeled -kidney specific clusterin that is not bound to the one or more antibodies or antigen binding fragments thereof is detected. The amount of detectably d lectin-kidney specific clusterin that is not bound to the one or more antibodies or antigen binding fragments is proportional to the amount of kidney 1O specific clusterin in the sample. Alternatively, the detectably labeled lectin-kidney specific clusterin that is not bound to the one or more antibodies or antigen binding fragments is washed away and the remaining detectably d lectin-kidney specific clusterin is detected. Alternatively, the assay can begin with one or more lectins that specifically bind a clusterin isoform are immobilized to the support.
Kidney specific clusterin bound to one or more detectably labeled antibodies or antigen binding fragments thereof that specifically bind clusterin along with a sample treated with unlabeled antibodies that specifically bind kidney specific clusterin are added to the support. Detection is ted as bed above.
Methods of the invention can be used in the diagnosis or detection of kidney disease, kidney injury, or kidney damage by obtaining a test sample from, e.g., a human or mammal suspected of having kidney disease or kidney damage. The methods comprise contacting a sample from a mammal with one or more antibodies that specifically bind clusterin and one or more molecules that ically bind to ydrate moieties of one or more clusterin isoforms (e.g., kidney specific clusterin) and that do not specifically bind other clusterin isoforms (e.g., plasma clusterin, serum rin, or orne, non-kidney specific rin ms).
One of skill in the art is aware of conditions that enable and are appropriate for formation of complexes. The complexes of kidney specific clusterin, one or more antibodies that specifically bind clusterin and one or more one or more les that specifically bind to carbohydrate moieties of clusterin and that do not specifically bind other clusterin isoforms that specifically bind kidney specific clusterin are detected. If the complexes are detected, then the mammal is diagnosed with kidney diseafi kidney injury, or kidney damage. The amount of complexes can be deter ed by any methodology known in the art. A level that is higher than that [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ed set by Sarah.Wilkinson formed in a control sample indicates kidney damage, kidney , or kidney disease. A control sample is a sample that contains either no kidney specific clusterin or kidney specific clusterin at a level observed in humans or mammals with no kidney disease, kidney injury, or kidney damage. Both types of control samples can be used in an assay. A kidney therapy or kidney therapeutic can be administered to the mammal if the mammal has kidney disease or kidney damage.
In embodiments canine kidney specific clusterin can be ed with one or more clusterin antibodies or n g fragments thereof and one or more of PHA-L, WGA, sWGA, STL, LEL, PHA-E, or DSL lectins. In embodiments feline 1O kidney specific clusterin can be detected with one or more clusterin antibodies or antigen binding fragments thereof and one or more ofjacalin, ECL, LCA, RCA, PHA- E, WGA, PSA, DSL, DBA, PHA-L, SBA, or CONA s. In embodiments feline and canine kidney specific clusterin can be detected with one or more clusterin antibodies or antigen binding nts thereof and one or more of WGA, sWGA, DSL, PHA-L, or PHA—E lectins. In embodiments human and feline kidney specific clusterin can be detected with one or more clusterin antibodies or antigen binding fragments thereof and one or more of PSA or DBA lectins.
Kidney damage, kidney injury, and kidney e include, for example, acute kidney injury (AKI; functional and structural disorder or signs of renal damage including any defect from blood and urine test, or tissue imaging that is less than 3 months), a progressive or worsening acute kidney injury, an early AKI, a mild AKI, a moderate AKI, a severe AKI, chronic renal/kidney e, diabetic nephropathy, acute tubular is, acute interstitial nephritis, a glomerulonephropathy, a ulonephritis, proximal and distal tubular damage, a renal vasculitis, an obstruction of the renal artery, a renal ischemic injury, a tumor Iysis syndrome, rhandomyolysis, a urinary tract obstruction, a prerenal azotemia, a renal vein thrombosis, a cardiorenal syndrome, a hepatorenal syndrome, a pulmonary-renal syndrome, an abdominal compartment syndrome, urinary tract ion, upper urinary tract infection, lower urinary tract infection, an injury from a toxic agent, bladder cancer, kidney cancer, urological cancer, or a contrast nephropathy.
Methods of the ion can detect kidney disease, kidney injury, and kidney damage earlier than known methods (e.g., serum creatinine assays). Methods of the inven'fi can detect kidney disease, kidney injury, and kidney damage within about [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson ation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson , 4, 3, 2, 1, or less days of onset of the detect kidney disease, kidney injury, and kidney damage.
In embodiments of the invention, the complexes are detected when an able label, such as an enzyme conjugate or other able label, which is bound to the one or more antibodies, the one or more other molecules that specifically bind carbohydrate moieties of kidney specific clusterin and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g., serum rin, plasma clusterin, or bloodborne, non-kidney specific clusterin ms), or both, catalyzes or es a detectable reaction. Optionally, one or more detectable 1O labels comprising a signal generating compound can be applied to the complex under conditions that allow formation of a detectable label complex. A detectable label x comprises clusterin, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin, one or more other molecules that specifically bind carbohydrate moieties of clusterin and that do not specifically bind carbohydrate moieties of other isoforms of rin, and one or more detectable label molecules. The detectable label complex is detected. Optionally, the one or more antibodies or one or more other molecules that specifically bind carbohydrate moieties of clusterin and that do not specifically bind ydrate moieties of other isoforms of clusterin can be labeled with a detectable label prior to the formation of a detectable label x. The method can optionally comprise a positive or negative control.
A complex comprising clusterin, one or more antibodies that specifically bind rin, one or more other molecules that specifically bind carbohydrate moieties of kidney specific clusterin and that do not specifically bind ydrate moieties of other isoforms of clusterin (e.g. plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin isoforms) can also be detected using methods that do not require labels or detectable label regents. For example, e plasmon resonance biosensors, Corning EP|C® biosensors, or colorimetric resonant reflectance biosensors can be used to detect complexes of the ion in a label- free manner.
One embodiment of the invention comprises a complex comprising one or more clusterin molecules, one or more antibodies or antigen binding fragments therfiat specifically bind clusterin, and one or more lectins. The x can com one or more kidney specific clusterin molecules, one or more antibodies or [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson antigen binding nts that specifically bind clusterin and one or more molecules that specifically bind to carbohydrate moieties of kidney specific clusterin and do not bind to carbohydrate moieties of other clusterin isoforms (e.g. plasma rin, serum clusterin, or orne, non-kidney specific clusterin isoforms). The complex can optionally comprise one or more detectable labels that are covalently or non- covalently bound to any component of the complex. The complex can be immobilized to a solid support.
In embodiments of the invention, one or more antibodies that specifically bind clusterin are immobilized to a solid phase or substrate. A test sample is added to the 1O substrate. One or more other molecules that specifically bind carbohydrate moieties of kidney ic clusterin and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g. plasma clusterin, serum clusterin, or bloodborne, non—kidney specific clusterin isoforms) are added to the substrate before the test sample, with the test sample, or after the test sample is added to the substrate. The one or more other molecules that ically bind carbohydrate moieties of kidney specific clusterin and that do not ically bind carbohydrate es of other isoforms of clusterin can be ably labeled. Wash steps can be performed prior to each addition to the substrate. The detectable label can be directly detected or indirectly detected via, for example, a chromophore or enzyme substrate that is added to react with the detectable label. A detectable reaction (e.g., development of color) is allowed to develop. The reaction is stopped and the detectable reaction can be quantified using, for example, a spectrophotometer. This type of assay can quantitate the amount of kidney ic clusterin in a test sample.
In embodiments of the invention, one or more other les that specifically bind carbohydrate moieties of kidney specific clusterin and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g. plasma clusterin, serum clusterin, or bloodborne, dney specific clusterin ms) are ed to a solid phase or ate. A test sample is added to the substrate. One or more antibodies that specifically bind kidney specific clusterin are added to the substrate before the test sample, with the test sample, or after the test sample is added to the substrate. The one or more antibodies or antigen binding fragments f can be detectably labeled. Wash steps can be performed prior to each addition to the substfi. The dy label can be directly detected or indirectly detected via, forexam a chromophore or enzyme substrate that is added to the substrate to react ation] Sarah.Wilkinson None set by Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson ation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson with the detectable label. A detectable reaction (e.g., color) is allowed to develop.
The detectable reaction is stopped and the reaction can be quantified using, for example, a spectrophotometer. This type of assay can quantitate the amount kidney specific clusterin in a test sample.
In embodiments of the invention, a sample is depleted of a first rin isoform (or multiple clusterin isoforms) in order to better detect a second clusterin isoform (or multiple other clusterin isoforms). The sample is contacted with one or more lectins that specifically bind the first clusterin isoform so that a complex of one or more lectins and one or more first clusterin isoforms are formed. In one example, 1O N lectins specifically bind carbohydrate moieties of semen clusterin, but do not bind carbohydrate moieties of serum clusterin. Alternatively, a sample can be contacted with one or more les that specifically bind to carbohydrate moieties of the first clusterin isoforms and that do not specifically bind to carbohydrate moieties of the second clusterin isoforms so that a complex of one or more molecules that specifically bind to ydrate moieties of the first clusterin isoform and that do not specifically bind to carbohydrate moieties of the second clusterin isoforms and one or more first clusterin isoforms are formed. The complexes can then optionally be removed from the sample by, for example precipitation. An assay for the second clusterin can be performed using, e.g., any assay of the invention.
Alternatively, any assay for the second clusterin m can be performed once the first rin isoform are depleted from the sample (e.g., contacting the sample with one or more dies specific for clusterin and detection of clusterin/antibody xes). Sandwich assays using tow antibodies or direct assays using one antibody can be used.
In embodiments of the ion, a sample is ed of non—kidney specific clusterin in order to better detect kidney specific clusterin. A sample is contacted with one or more lectins that specifically bind one or more non-kidney specific clusterin isoforms (e.g., serum or plasma clusterin isoforms) so that a complex of one or more lectins and one or more non-kidney specific clusterin isoforms are formed. WGA does not bind plasma clusterin and binds to kidney specific rin.
Alternatively, a sample can be contacted with one or more molecules that specifically bind to carbohydrate moieties of non—kidney specific clusterin and that do not spec’fily bind to carbohydrate moieties of kidney specific clusterin isoforms so thata co x of one or more molecules that specifically bind to carbohydrate moieties [Annotation] Sarah.Wilkinson None set by Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson of non-kidney specific clusterin isoforms and that do not specifically bind to ydrate moieties of kidney specific clusterin isoforms and one or more non- kidney specific clusterin isoforms are formed. The complexes can then be removed from the sample. An assay for kidney ic clusterin can be performed, e.g., any assay of the invention. Alternatively, any assay for kidney specific clusterin can be performed once the non-kidney specific clusterin isoforms are depleted from the sample (e.g., contacting the sample with one or more dies specific for clusterin and detection of clusterin/antibody xes). ch assays using two antibodies or direct assays using one antibody can be used. 1O Assays of the invention include, but are not limited to those based on ition, direct reaction or sandwich-type assays, including, but not limited to enzyme linked immunosorbent assay (ELISA), competitive assay, western blot, lFA, radioimmunoassay (RIA), hemagglutination assay (HA), agglutination assay, fluorescence polarization immunoassay (FPIA), and iter plate assays (any assay done in one or more wells of a microtiter plate). One assay of the invention comprises a reversible flow chromatographic binding assay, for example a SNAP® assay. See US. Pat. No. 5,726,010.
Assays can use solid phases, ates, or supports or can be performed by immunoprecipitation or any other methods that do not utilize supports. Where a solid phase, substrate, or support is used, one or more antibodies, one or more other les that specifically bind carbohydrate moieties of kidney specific rin and that do not specifically bind carbohydrate moieties of other isoforms of clusterin, or combinations thereof, are directly or indirectly attached to a t or a substrate such as a iter well, magnetic bead, non-magnetic bead, column, matrix, membrane, glass, polystyrene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, magletite, fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic rs, such as, polyethylene, polypropylene, or polyester), sintered structure composed of particulate materials (e.g., glass or various plastic polymers), or cast ne film composed of nitrocellulose, nylon, polysulfone or the like (generally synthetic in nature). In embodiments of the invention a substrate is sintered, fine particles of polyethylene, commonly known as porous polyethylene, for example, 10- meifin porous polyethylene from Chromex Corporation (Albuquerque, NM). All of thes bstrate materials can be used in suitable shapes, such as films, sheets, or [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or s. Suitable methods for immobilizing antibodies, ns, and lectins on solid phases include ionic, hydrophobic, covalent interactions and the like.
The antibodies, lectins, or les that specifically bind to carbohydrate moieties of one or more clusterin isoforms (e.g., kidney specific clusterin) and that do not specifically bind to carbohydrate moieties of other clusterin isoforms (e.g. plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin isoforms) can be affixed to a solid support by, for example, adsorption or by covalent linkage so 1O that the molecules retain their selective binding activity. Optionally, spacer groups can be included so that the binding site of the molecule remains accessible. The immobilized molecules can then be used to bind clusterin molecules from a sample, such as a biological sample including , serum, sputum, blood, urine, feces, cerebrospinal fluid, amniotic fluid, wound e, or tissue.
The formation of a complex (e.g., a complex of one or more of the following: (1) clusterin, antibody or antigen binding fragment thereof, les that specifically bind carbohydrate moieties of one or more rin isoforms (e.g., kidney specific isoforms) and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g. plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin isoforms); (2) able label, clusterin, antibody or antigen binding fragments thereof, one or more other molecules that specifically bind carbohydrate moieties of one or more clusterin isoforms (e.g., kidney specific clusterin) and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g. plasma clusterin, serum clusterin, or bloodborne, non-kidney specific clusterin isoforms) can be detected by e.g., radiometric, colorimetric, fluorometric, size-separation, biosensor methods, precipitation methods, or label—free methods. ally, ion of a complex can be by the addition of a ary dy that is coupled to a detectable label. Detectable labels comprising signal generating compounds associated with a complex can be ed using the methods described above and include chromogenic agents, catalysts such as enzyme conjugates, scent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridiniums, phenanthridiniums, ruthenium, and direct visual labels, as well as cofactors, inhibitors, maglumirhradioactive ts, les, and the like. Examples of enzyme conjugates include alkaline [Annotation] Sarah.Wilkinson None set by Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The ion of a particular label is not critical, but it will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
Formation of the complex is tive of the presence of one or more clusterin isoforms (e.g., kidney specific clusterin) in a test sample. The methods of the invention can te the amount or quantity of one or more clusterin isoforms (e.g. kidney specific clusterin) in a test sample. With many detectable labels, such as enzyme conjugates, the amount of clusterin present is proportional to the signal generated. Depending upon the type of test sample, it can be diluted with a suitable 1O buffer reagent, concentrated, or ted with a solid phase without any manipulation. For example, test samples can be diluted or concentrated in order to determine the presence and/or amount of clusterin.
Assays of the invention can be also used to monitor the course of amelioration of a kidney e, kidney injury, or kidney damage. By ing the increase or decrease of kidney ic rin in a test sample from a subject, it can be determined whether a particular eutic regiment aimed at ameliorating the disease or damage is effective.
Kits The invention further comprises assay kits (e.g., articles of manufacture) for detecting kidney specific clusterin. A kit can comprise one or more antibodies or antigen binding fragments thereof of the invention and one or more other molecules that specifically bind carbohydrate moieties of one or more clusterin isoforms (e.g., kidney ic rin) and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g., plasma clusterin, serum clusterin, or bloodborne, dney specific clusterin isoforms) and compositions for determining specific binding of the antibodies, the one or more other molecules, and clusterin in the sample. These components can comprise one or more detectable labels (i.e., the detectable labels can be immobilized to one or more of the components) or detectable labels can be provided separately. A kit can comprise a device containing one or more antibodies or antigen g fragments thereof of the invention and one or more other molecules that specifically bind carbohydrate moieties of one or more clusterin ms (e.g., kidney specific ms) and that do n’fipecifically bind carbohydrate moieties of other isoforms of clusterin (e.g., seru plasma clusterin) and instructions for use of the molecules for, e.g., the [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson identification of kidney disease, kidney injury, or kidney damage in a mammal. A kit can comprise a support with one or more dies or antigen binding fragments thereof or one or more other molecules that specifically bind carbohydrate moieties of one or more isoforms of clusterin (e.g. kidney specific clusterin) and that do not specifically bind carbohydrate moieties of other isoforms of clusterin (e.g., plasma or serum clusterin) or both immobilized on the support. The kit can also comprise ing material comprising a label that indicates that the one or more one or more other molecules that specifically bind carbohydrate moieties of kidney specific clusterin and that do not specifically bind carbohydrate moieties of other isoforms of 1O clusterin and antibodies of the kit can be used for the identification kidney disease, kidney injury, or kidney damage. Other components such as buffers, controls (e.g., positive controls like kidney specific clusterin; negative controls like plasma clusterin, serum clusterin or buffers), and the like, known to those of ry skill in art, can be included in such test kits. The one or more other molecules that specifically bind carbohydrate moieties of kidney specific clusterin and that do not specifically bind carbohydrate moieties of other isoforms of clusterin, antibodies, assays, and kits of the invention are useful, for example, in the diagnosis of individual cases of kidney disease, kidney injury, or kidney damage in a patient, as well as epidemiological studies of kidney disease, kidney injury, or kidney damage.
A kit can also comprise one or more lectins that specifically bind one or more non-kidney ic clusterin isoforms (e.g., serum or plasma rin isoforms) for formation of a complex of one or more lectins and one or more non-kidney specific clusterin isoforms. A kit can also comprise one or more molecules that specifically bind to ydrate moieties of non-kidney specific clusterin and that do not specifically bind to ydrate moieties of kidney specific rin isoforms, for x formation between one or more non-kidney specific clusterin isoforms and the one or more les.
All patents, patent applications, and other scientific or technical writings referred to anywhere herein are orated by nce herein in their entirety.
The invention illustratively described herein suitably can be practiced in the e of any element or elements, limitation or tions that are not specifically disclosed herein. Thus, for example, in each ce herein any of the terms "comprising", "consflwg essentially of", and sting of" may be replaced with either of the other terms, while retaining their ordinary meanings. The terms and expressions [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson which have been employed are used as terms of description and not of tion, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by embodiments, al features, cation and variation of the concepts herein disclosed may be resorted to by those d in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the ption and the appended claims. 1O In addition, where features or aspects of the invention are described in terms of Markush groups or other ng of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of s of the Markush group or other group.
The following are provided for exemplification purposes only and are not intended to limit the scope of the invention described in broad terms above.
EXAMPLES Blood Contamination Normal canine serum was spiked into negative urine (i.e., urine from y canines) and the amount of clusterin measured using the cial Clusterin EIA (Biovendor). As shown in Figure 1A-B significant clusterin levels are measured even at 1:1000 dilution (1 ul per ml). Therefore, it is ant to be able to detect kidney specific clusterin isoform while excluding any detection of serum or plasma clusterin isoform.
Example 2: Materials Isolation of Clusterin Molecules The sequence of canine clusterin was used to design and synthesize a vector to express a recombinant his tagged canine clusterin molecule (Life logies).
After expression and purification of the protein, the sequence was confirmed by LC- MS. This molecule is referred to as recombinant rin or his-tagged recombinant clusterin herein.
Plasma clusterin was purified from pooled plasma of 30 canines by affinity chroragraphy. Madin-Darby canine kidney (MDCK) cell-derived clusterin (which is [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson a kidney specific clusterin) was obtained by growing MDCK cells to confluence in 125 ml T flasks at 37°C, 7.5% 002 in 1X MEM supplemented medium with antibiotics. Supernatants were ted and the clusterin was affinity purified over an anti-clusterin column using a AKTA chromatography system (GE Healthcare).
Kidney specific clusterin was purified by affinity chromatography from pooled urine of canines suspected of having an acute injury to the kidney.
Antibody Preparation Polyclonal antiserum against plasma—derived clusterin was raised in rabbits.
Monoclonal antibodies were ted in mice using multiple forms of rin as 1O an immunogen (lmmunoprecise, Inc. ver, BC). The s forms included recombinant whole molecule clusterin, alpha—chain of clusterin, beta—chain of clusterin, plasma-derived clusterin, erived clusterin, and urine-derived clusterin (which is a kidney specific clusterin).
Immunoaffinity Chromatography Recombinant clusterin was used to immunize rabbits. The lusterin lgG was purified by protein A chromatography. The anti-recombinant clusterin lgG antibodies were used to purify native plasma clusterin from a pool of canine plasma by ty chromatography. Monoclonal antibodies were made by immunizing mice with plasma clusterin and the resulting anti-clusterin lgG antibodies were purified by protein A chromatography. ion Antibodies The anti-clusterin a-native) monoclonal or polyclonal antibodies were labeled with horseradish peroxide (HRP) by standard SMCC chemistry (Thermo- Pierce).
Clusterin Standard rin was purified by affinity chromatography from the culture supernatants of MDCK cell line (ATCC) or pooled canine plasma. The resulting clusterin was quantitated by LCMS. Values (mg/ml) were assigned and standard curves and controls were made.
Exam Ie 3: General Clusterin Assa Protocol A standard curve of clusterin was prepared in assay buffer (1x PBS containing 1% BSA and 0.5 % Tween® orbate) 20) by serial dilution of a 500 ng/ml standa(.j Urine samples were diluted 1:100 in assay buffer and 100 pl was incub for 1 hour at ambient temperature in duplicate on the plate. After 3 washes [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson ionNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson with PetChek® buffer (IDEXX tories), 100 pl of anti-Clusterin antibody d with horseradish peroxidase was incubated for 30 minutes at ambient temperature.
Following 3 washes as above, 50 ul of TMB substrate (IDEXX Laboratories) was added and color was allowed to develop for 5 minutes. The colorimetric reaction was stopped by the 100 pl addition of acid (1N HCL). The plates were ately read at 450 nm. rin Coated Plates Microtiter plates were coated with 5 ug/ml of plasma clusterin, MDCK—derived clusterin, recombinant gged clusterin, and BSA overnight at 4°C in 0.05M carbonate buffer, pH 9.5. ing 3 washes with PBS-Tween® (polysorbate) 20 (0.1%), plates were blocked with 1% bovine serum albumin (BSA) in PBST for 2 hours. Plates were dried under vacuum for 4 hours after 3 additional washes with PBST.
Lectin Coated Plates Biotinylated lectins (Vector Labs, Burlingame, CA) were diluted to 5 ug/ml in PBS, pH 7.4 and 100 pi and added to wells of a streptavidin coated plated (IDEXX Laboratories). After overnight binding at 4°C, plates were washed 3 times with PBST. All plates were stored, desiccated, at 4°C until use.
Example 4: Clusterin Lectin Specificity Clusterin coated microtiter plates were incubated for 1 hour with 1ug/ml of biotinylated lectins in PBST. Following 3 washes with PBST, 100 pl of HRP-labeled streptavidin was incubated for 30 minutes at ambient temperature on a plate shaker.
After 3 additional washes with PBST, 100 pl TMB substrate was added and ted for 5 minutes and the reaction was stopped with 100 pl of 1N HCL. The plates were read at 450.
Table 2 Carbohydrate specificity of Clusterin preparations — Clusterin ation Lectin lawn-Ea: MDCK/Plasma [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson ation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson Reactivity of clusterin preparations to specific s is shown in OD. 450 units in Table 2. An OD > 0.5 was used as a positive response to a lectin. This CD. was chosen since binding of non-glycosylated proteins, His-tagged clusterin and BSA resulted in values 5 0.4 0D. units. A ratio of MDCK/plasma binding was taken and ratios > 2.0 were chosen for further characterization. Four (4) lectins met this criteria, PHA-E, PHA-L, WGA, and LEL. Wheat germ lectin (WGA) was selected for r characterization.
Example 5: Feasibility of Detection of Kidney Specific Clusterin s forms of clusterin (MDCK—derived clusterin, native plasma rin, and recombinant his-tagged clusterin) were serial diluted in assay buffer and detected with anti-clusterin HRP-labeled monoclonal antibody. Figure 2 shows binding of only the MDCK-derived clusterin preparation in a dose dependent manner.
The his-tagged recombinant clusterin, which has no carbohydrate, and the native plasma clusterin, which contains carbohydrate, do not bind to the lectin solid phase at any tration tested.
Specificity of Lectin Towards Kidney Specific Clusterin Native plasma clusterin, erived clusterin, and derived clusterin samples, were diluted to 1 ug/ml in assay buffer and detected with anti-clusterin HRP moncutal antibodies on different lectin solid phases. Table 3 below, shows ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson binding of only the MDCK—derived clusterin and clusterin purified from urine to the WGA solid phase. There was reduced binding to succinylated WGA (sWGA) suggesting sialic acid residues are not playing a role in binding.
Table 3. Solid Phase Clusterin Antigen Both polyclonal and monoclonal anti-clusterin antibodies are able to bind MDCK—derived clusterin bound to multiple lectin solid phases and do not bind clusterin from plasma sources because plasma-derived rin was not able to bind to the lectin solid phases. WGA is specific for kidney specific rin (MDCK- derived and urine).
Lectins were then screened for clusterin antigens that were captured on the solid phase by monoclonal or polyclonal antibodies. 3A4 monoclonal antibodies, 9H7 monoclonal antibodies, 2E2 monoclonal antibodies, 2F2 monoclonal antibodies, lpha chain clusterin polyclonal antibodies, eta chain clusterin polyclonal dies, or rine clusterin polyclonal antibodies were immobilized to a solid phase. MDCK—derived or plasma-derived clusterin (1 ug/ml) was added to the solid phase along with biotinylated WGA, sWGA, Pha—L, Pha-E or buffer control. The results are show in Table 4.
Table 4.
Biotinylated s or Controls Clusterin Solid PhaseAb WGA sWGA Pha—L Pha-E Buffer Antigen anti2-al2pha anti-urine ation] Wilkinson None set by Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson ation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson (1 rig/ml) 9H7 0.2 0.1 0.1 0.2 0.0 2F2 0.2 0.1 0.1 0.1 0.0 anti-alpha anti-beta anti-urine Monoclonal antibody 9H7 and polyclonal clusterin anti—beta chain, and polyclonal clusterin anti-urine exhibit the best sensitivity. WGA, Pha-L, Pha-E ically bound the MDCK-derived clusterin and did not specifically bind the plasma-derived clusterin.
WGA (5ug/ml), sWGA (5ug/ml), polyclonal anti—plasma clusterin antibody, or buffer were bound to a solid phase. Plasma—derived clusterin (1ug/ml), MDKC- derived clusterin (1 ug/ml), urine-derived clusterin (1 ug/ml) or buffer was added. The results are shown in Table 5. Monoclonal antibody 9H7 (100 ng/ml) conjugated to the horseradish peroxidase was then added. Specific binding was detected. The MDCK—derived clusterin and urine-derived clusterin specifically bound to the immobilized WGA and was detected by the 9H7 dy. The plasma-derived clusterin did not specifically bind to the immobilized WGA and was not ed by the 9H7 antibody. The results are shown in Table 5.
Table 5.
Solid Phase rin Preparation (1 ug/ml) sug/ml MDKC —EE_——— —IE_—II_IWE- Freshly prepared serum was spiked into urine and the formation of sandwich immune complex was tested with a solid phase sing immobilized WGA lectin and sWGA lectin. Lectin and kidney specific clusterin xes were detected with HRP-conjugated 9H7 monoclonal antibody. The results are shown in Table 6. The results suggest that the complex (WGA, kidney specific rin, and antibody) is formed only when MDCK—derived clusterin was spiked into the urine with no significant reactivity when serum was spiked into urine between 0.1 — 10%.
Therefore, serum clusterin is not detected by the assay.
Table 6.
—Normal Serum Spikes mg._ 0.10% 1.00% 5.00% 10.00% MDKC [Annotation] Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson ation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson ______(50009) His-tagged recombinant clusterin, plasma-derived clusterin and MDCK- derived clusterin samples were reduced with DDT to te alpha and beta chains of clusterin or remained non—reduced. In Western blots, monoclonal antibody 9H7 was trated to bind to both MDCK-derived clusterin and plasma-derived clusterin. WGA lectin, however binds only to the non—reduced or reduced MDCK— derived clusterin. See Table 7. WGA did not bind to non-reduced or reduced his- tagged recombinant clusterin or non-reduced or reduced plasma-derived rin.
Table 7. -tagged Plasma— MDCK— His-tagged Plasma— MDCK- recombinant derived derived recombinant derived derived clusterin clusterin clusterin rin clusterin clusterin ___—___ ___—___ Exam le 6 Clusterin Levels in Field Do 5 with Hematuria The urine from healthy canines was examined by UA dipstick (IDEXX Laboratories, Inc.) for the presence of blood. Kidney ic clusterin levels were measured using the Commercial Clusterin EIA according to the manufacturers’ instructions (Biovendor Research and Diagnostic Products). As shown below (Table 8), healthy canines with no detectable blood in their urine had levels of clusterin within the reference range (70 ng/ml) while those having blood contamination (samples 5 to 8) had clusterin levels 10-100 times above the normal reference range. This result indicates that the presence of blood in urine may result in high rin measurements, leading to false positives.
Table 8 rin EIA Blood —Negative —Negative _Neative 1045 1015 —3 65000 [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson ation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson Example 7 Specificity of the Kidney ic Clusterin Immunoassay A Kidney Specific rin Immunoassay (KSCI) was designed using a monoclonal antibody (lgGZa, kappa) raised against canine Clusterin purified from plasma and Wheat Germ Lectin (WGA). The WGA was coated onto wells of a microtiter plate. The monoclonal antibody was labeled with HRP. To illustrate the specificity of the KSCI, fresh whole blood or plasma from a healthy dog was spiked into buffer and analyzed using both the KSCI and the Commercial Clusterin EIA ndor) assay.
As shown in Figure 3, Clusterin was detected at high concentrations in both whole blood and serum by the Commercial Clusterin EIA but not the KSCI. Taking into consideration the fact that a high tage of urine samples from healthy dogs and cats have blood contamination, the only way to accurately measure Clusterin is to use the Kidney Specific Clusterin Immunoassay.
Example 8: Kidney Specific Clusterin in a Canine Gentamicin Model Kidney specific Clusterin was ed in urine from a canine icin model (Figure 4). In the model system, dogs were given 40mg/kg gentamicin daily for 5 days. In this dog model, serum creatinine was essentially ged hout the study while kidney specific Clusterin in urine increased rapidly, ng approximately 5 times baseline when closing was stopped and peaking at approximately 10 times baseline at day 11. This shows that kidney specific Clusterin is an earlier and more sensitive marker than serum nine for active kidney injury.
Example 9: Kidney Specific Clusterin in Patients with Active Kidney lnjum Kidney ic Clusterin was ed in the urine of dogs presenting to a clinic with inflammatory or ischemic induced active kidney injury (Figure 5). The data shows a clear separation in the concentration of kidney specific Clusterin between healthy patients and those diagnosed with active kidney injury. In conclusion, kidney specific Clusterin is a sensitive and ic marker for active kidney injury.
Exam le 10: Kidne S ecific Clusterin in Patients with Urina Tract Infections Kidney specific Clusterin was measured in cats and dogs with urinary tract infections (UTIs) (Figure 6). Kidney specific Clusterin levels were ically increased in a subset of the UTI patients. Kidney specific Clusterin is a marker for UTI.
Exam le11: Kidne S ecific Clusterin in Cats.
Feline Clusterin was isolated from feline renal CRFK cells (ATCC, Manassas, VA). Analysis of the soluble feline Clusterin was done using SDS—PAGE western blottiand a lectin screening array.
[Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson Supernatants from the canine and feline renal cell lines (MDCK and CRFK, respectively) and a clusterin preparation purified from canine plasma were run in SDS—PAGE and blotted onto nitrocellulose. The blot was probed with an anti— clusterin monoclonal antibody raised against canine clusterin. The results (Figure 7) show that the monoclonal antibody was cross reactive with the feline clusterin produced by the CRFK. Thus, the monoclonal antibody can be used for the ion of feline renal clusterin in the two site immunoassay (ELISA) .
Screening of s to Feline Clinical Samples ylated lectins (Vector Labs) were coated at 1 ug/ml in PBST (Tween 1O 20® (polysorbate) at 0.01%) to streptavidin coated plates overnight at 4°C. Plates were washed 3 times and feline clusterin affinity-purified from plasma (1 ug/ml) or 1:10 diluted feline clinical urine incubated for 1 hour at ambient temperature. After 3 washes, 100 pl of HRP labeled monoclonal antibody raised against canine clusterin (250 ng/ml) was added and incubated 30 minutes as above. After another three washes, 100 pl TMB was added and color developed for 5 minutes after which 100 pl 1N HCL was added to stop the reaction. Absorbance was read at 450 nm.
Results are shown in Table 9.
Table 9 Feline Sample Purified Plasma Lectin Abbreviation Lectin Source Clusterin Urine. 1:10 dilution (1 rig/ml) jacalin Jacalin 0.00 0.51 GSL-l Griffonia(Bandeiraea) simplicifolia l 0.00 0.14 LCA Lens culinaris 0.33 2.04 ECL Erythina cristagalli 0.30 1.36 LEL rsicon esculentum -0.01 -0.23 STL Solanum tuberosum 0.00 0.19 RCA Ricin communis 0.44 2.39 VVA Vicia a -0.01 -0.45 GSL-ll Griffonia(Bandeiraea) cifolia || 0.00 0.01 SJA Sophora japonica -0.01 0.12 PHA-E Phaseolus vulgaris Erythroagglutinin 0.10 2.53 sWGA Succinylated wheat germ 0.01 0.72 WGA Wheat Pisum sativum germ 0.05 2.1 p5A Pisum sativum 1.04 2.62 DSL Datura stratonium 0.36 2.90 DBA Dolichos us 0.08 1.59 a-L Phaseolus vulgaris Leucoagglutinin 0.07 1.93 Ulex europaeus | -0.01 0.18 [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson ation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Sarah.Wilkinson SBA Soybean 0.04 2.06 CONA Concanavlin A 0.65 3.12 PNA Peanut 0.02 0.60 Twelve lectins (bold) were able to form a sandwich with feline clusterin and the anti- clusterin monoclonal antibody. As shown, WGA binds to feline clusterin. Thus, the KSCI assay can be used to detect clusterin in both dogs and cats.
Detection of urinary clusterin in clinical samples using lectin format Urine was collected from felines visiting a local nary hospital, diluted 1:100, and subjected to the KSCI assay. As shown, animals ented the range of the assay demonstrating that the KSCI assay developed for canines is cross- reactive with feline clinical samples. (<LOD = below limit of detection; >ULOQ = above upper limit of quantification). See Table 10.
Table 10.
Renal (ng/mls) <LOD <LOD >ULOQ <LOD <LOD Exam le 12: Kidne S ecific Clusterin in Humans Adherent human embryonic epithelial kidney cell line HEK293, canine kidney cell line MDCK, and green monkey kidney epithelial cell line Vero (ATCC, Manassas, VA) were grown per the supplier’s instructions. When cells were confluent, the cells were fissed using a nephrotoxic drug, Gentamicin 0.2 mg/ml, heated at 40°C, or [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson Unmarked set by Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson treated with a ation of heat and drug. Supernatants were harvested and tested for their reactivity in a commercially ble human rin ELISA (Biovendor). The results (Table 11) shown that the ELISA is reactive with clusterin sed by HEK293 cells.
Table 11: Specificity of Human Cell lines used for Clusterin expression Commercial Cell Line Species/Tissue Organ Human Clusterin Assay Reactivity can'ne MDCK Kidney epithelial Green Monkey Vero kidney epithelial Human Kidney HEK293 epithelial onic) Kidney cell lines were stressed with a nephrotoxic drug gentamicin 0.2 mg/ml, heat 40°C for 24 hours, or a combination of drug (0.2mg/ml) and heat (40°C for 24 hrs.). The supernatants were diluted 1:100 and run in the human clusterin ELISA (Biovendor). As shown below in Figure 8, no reactivity was seen with canine kidney cell control (MDCK). Slight reactivity was seen with the Green Monkey kidney Vero line. The human line, HEK 293 showed the strongest reactivity. This confirms that the HEK2993 cell line secreted human clusterin when grown under a variety of conditions. dies reactive with human renal-expressed clusterin In order to develop a two site ELISA (sandwich ELISA), a y of monoclonal and polyclonal anti-canine-clusterin antibodies raised against the recombinant canine rin were screened to determine their binding to human clusterin. The results indicated that multiple anti-clusterin antibodies against recombinant canine clusterin, were able to bind to human clusterin. Western blot confirmation, Figure 9, shows rabbit anti-beta chain clusterin binding to clusterin from MDCK (lane 2, 4), HEK 293 cell supernatants (lane 3), and the positive control recombinant canine clusterin beta chain antigen (lane 5).
Human Clusterin ELISA Plates were coated with 10 ug/ml of purified anti-beta chain rin polchal antibody overnight at 4°C. The plates were washed 3 times and blocked 0443 with 0.1% BSA overnight followed by 3 final washes. The plates were dried for 2 hours under a vacuum and stored at 4°C until use. The supernatants for the human kidney cell line and the MDCK (canine) control were diluted 1:10 with PBS and 100 μl placed in wells in duplicate. The supernatants were incubated for 1 hour at ambient temperature with shaking. After 3 washes, 100 μl of ylated lectins (1μg/ml) in PBS was added and incubated for 1 hour as above. Following three additional washes, the plates were incubated for 30 minutes with streptavidin-HRP (1:5000) in PBS. After a final 3 washes the plates were developed with 100 μl TMB substrate for 5 minutes and the reaction was stopped with 100 μl 1M HCL. ance was read at 450nm.
See Table 12. Two lectins (PSA, DBA) were shown to form a sandwich with human clusterin and the canine anti-beta chain polyclonal antibody.
Table 12: Lectin Specificity PSA DBA WGA MDCK 0.43 2.95 2.66 HEK 293 0.38 1.06 0.12 VERO 0.50 1.02 0.06 nce to any prior art in the specification is not an ledgement or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be combined with any other piece of prior art by a skilled person in the art. 1003664763

Claims (22)

1. A method of detecting kidney specific clusterin comprising contacting a sample with one or more antibodies or n binding fragments thereof that specifically bind clusterin and one or more lectins that ically bind to N- acetylglucosamine carbohydrate moieties of kidney specific clusterin and 10 detecting complexes of kidney ic clusterin, the one or more dies or antigen binding fragments thereof that specifically bind clusterin, and the one or more lectins that specifically bind to N-acetylglucosamine carbohydrate moieties of kidney specific clusterin. 15
2. The method of claim 1, wherein the one or more antibodies or antigen binding fragments thereof are immobilized to a support.
3. The method of claim 2, wherein the sample and detectably labeled one or more lectins that specifically bind to N-acetylglucosamine carbohydrate moieties of 20 kidney specific clusterin are added to the t.
4. The method of claim 1, wherein the one or more lectins are immobilized to a support. 25
5. The method of claim 4, wherein the sample and detectably labeled one or more antibodies or antigen g nts thereof are added to the support.
6. The method of any one of claims 1 to 5, wherein the one or more antibodies or antigen binding fragments thereof, the one or more lectins, or both are labeled 30 with a detectable label.
7. The method of any one of claims 1 to 6, wherein the one or more s do not specifically bind serum and plasma clusterin. 35
8. The method of any one of claims 1 to 7, wherein the sample is a urine sample. 1003664763
9. The method of any one of claims 1 to 8, wherein the detecting is completed by a method selected from the group consisting of a lateral flow assay, a chemiluminescent labeled sandwich assay, and an enzyme-linked immunosorbent assay (ELISA), a competitive assay, an agglutination assay, a 5 chemiluminescent assay, a bioluminescent assay, a gel electrophoresis assay method, an immunohistochemistry assay, a radioimmunoassay (RIA), a label-free biosensor assay, or an immunoradiometric assay.
10. The method of any one of claims 1 to 9, wherein the antibodies specifically bind 10 plasma clusterin, serum clusterin, recombinant clusterin, kidney specific clusterin, or MDCK-derived rin.
11. The method of any one of claims 1 to 10, wherein the kidney specific clusterin is human, feline, or canine.
12. A method for detecting kidney disease, kidney injury, or kidney damage in a mammal comprising contacting a sample from a mammal with one or more antibodies or n binding fragments thereof that specifically bind clusterin and one or more lectins that specifically bind to N-acetylglucosamine 20 carbohydrate moieties of kidney specific clusterin and detecting complexes of kidney specific rin, one or more antibodies or antigen binding nts thereof that ically bind clusterin and one or more s that specifically bind to N-acetylglucosamine ydrate moieties of kidney specific clusterin, wherein if the complexes are detected, then the mammal has kidney disease, 25 kidney injury, or kidney damage.
13. The method of claim 12, wherein the kidney disease is a urinary tract infection.
14. The method of claim 12 or 13, wherein the mammal is a human, feline, or 30 canine.
15. A method of distinguishing one or more kidney specific clusterin ms from non-kidney specific, bloodborne clusterin isoforms sing contacting a sample with one or more antibodies or antigen binding fragments thereof that 35 specifically bind clusterin and one or more lectins that specifically bind to N- 1003664763 acetylglucosamine carbohydrate moieties of the kidney specific clusterin isoforms and ing complexes of the kidney specific isoforms of clusterin, one or more antibodies or antigen binding fragments thereof that specifically bind clusterin, and the one or more lectins that specifically bind to N- 5 acetylglucosasmine carbohydrate moieties of the kidney specific rin isoforms.
16. The method of claim 15, wherein the kidney specific clusterin isoforms are 10 human, feline, or canine kidney specific clusterin isoforms.
17. The method of any one of claims 1 to 16, wherein the one or more lectins are Phaseolus vulgaris leucoagglutinin (PHA-L), wheat germ agglutinin (WGA), WGA1, WGA2, WGA3, sWGA, Phaseolus vulgaris agglutinin-E (PHA-E), 15 Lycopersicon esculentum lectin (LEL), Datura stramonium lectin (DSL), jacalin lectin, Solanum tuberosum lectin (STL).
18. A complex comprising one or more kidney ic clusterin molecules, one or more antibodies or antigen binding fragments f that specifically bind 20 clusterin, and one or more lectins that specifically bind to N-acetylglucosamine carbohydrate moieties of kidney specific clusterin.
19. The complex of claim 18, wherein the complex is immobilized to a solid support. 25
20. A kit when used in a method of any one of claims 1 to 17, comprising one or more antibodies or antigen binding fragments thereof that specifically bind clusterin and the one or more lectins that specifically bind to N- glucosamine carbohydrate moieties of kidney specific clusterin, wherein the lectins comprise Phaseolus vulgaris leucoagglutinin (PHA-L), wheat germ 30 agglutinin (WGA), WGA1, WGA2, WGA3, sWGA, Phaseolus vulgaris inin-E (PHA-E), Lycopersicon esculentum lectin (LEL), Datura stramonium lectin (DSL), n lectin, or Solarium tuberosum lectin (STL).
21. The kit of claim 20, n the one or more antibodies or antigen g 35 nts f, the one or more lectins that specifically bind to N- 1003664763 acetylglucosamine ydrate moieties of kidney specific clusterin, or both are labeled with a able label.
22. The kit of claim 21, wherein the detectable label is an enzyme, an enzyme 5 conjugate, a fluorescent compound, a chemiluminescent compound, a radioactive element, a direct visual label, or a magnetic particle. [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson ed set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson None set by Sarah.Wilkinson [Annotation] Sarah.Wilkinson MigrationNone set by Sarah.Wilkinson [Annotation] Wilkinson Unmarked set by Sarah.Wilkinson a mam 5g......&m. , . .g. . , “N9,” ”WEEMM “fig...“ HQ 1:20 1:53 "£1130 1:500 $1300 @5603 Q
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