# Patent

## Title
Method for measuring human megalin

## Abstract

This invention provides a method for measuring human megalin that can be performed in a simpler manner within a shorter period of time than is possible with conventional techniques, and that can also quantify human megalin. This invention also provides a method that enables diagnosis of functional diseases, which are specific to cells, tissues, or organs, in a site-directed manner at an early stage. Measurement of human megalin enables detection of a disease in an organ in which megalin expression is observed.

## Background
TECHNICAL FIELD
The present invention relates to a method for measuring human megalin. More particularly, the present invention relates to a method for detecting human megalin comprising quantitatively detecting megalin that is expressed topically and specifically in cells, tissues, and organs where megalin expression is observed in a rapid and simple manner, to thereby enable direct and early diagnosis of the degree to which cells, tissues, and organs are affected and improve and keep from worsening the conditions of disorders and the prognosis via treatment. The present invention can be applied to diagnosis of diseases of organs where megalin expression is observed, such as kidney or lung diseases.
BACKGROUND ART
1. Cloning of Megalin
As a result of a search for an etiologic antigen of Heymann nephritis, which is a model for experimental membranous nephropathy, Kerjaschki, D. and Farquhar, M. G. identified a cell membrane protein, gp330, in 1982 (Kerjaschki D., Farquhar M. G., 1982, Proc. Natl. Acad. Sci. U.S.A., 79, 5557-5561). In 1994, Saito, A. et al. determined the complete primary structure of a rat gp330 and designated it as megalin, because it was the largest cloned cell membrane protein of a vertebrate (Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729).
2. Megalin-Expressing Site
Megalin is also known as glycoprotein 330 (gp330) or low-density lipoprotein (LDL) receptor-related protein 2 (LRP-2). It is a glycoprotein having a molecular weight of about 600 kDa, which is expressed in kidney proximal tubule epithelial cells, other tissues and cells, such as type II alveolar cells, spermary, uterine endometrium, placenta, or inner ear epithelium, renal epithelium, germo-vitellarium, and neural ectoderm (see Christensen E. I., Willnow, T. E., 1999, J. Am. Soc. Nephrol. 10, 2224-2236; Juhlin C., Klareskog L. et al., 1990, J. Biol. Chem. 265, 8275-8279; and Zheng G, McCluskey R. T. et al., 1994, J. Histochem. Cytochem. 42, 531-542). In the kidney, megalin functions as an endocytosis receptor associated with endocytosis and reabsorption of proteins and the like in the proximal tubule prior to urinary excretion. The reabsorbed proteins and the like are then degraded by lysosomes (see Mausbach A. B., Christensen E. I., 1992, Handbook of Physiology: Renal Physiology, Windhager, editor, New York, Oxford University Press, 42-207).
3. Nucleotide Sequence of Megalin
Megalin is a glycoprotein that is the most frequently expressed on the kidney proximal tubule epithelial membrane of a mammalian animal. The cDNA-encoding sequence thereof has nucleotide identity with the human megalin cDNA sequence having gene accession number U04441 disclosed in Korenberg, J. R. et al. (1994) or the human megalin cDNA sequence having gene accession number U33837 disclosed in Hjaeln, G., et al. (1996) (see Korenberg J. R. et al., 1994, Genomics 22, 88-93; and Hjalm G. et al., 1996, Eur. J. Biochem. 239, 132-137).
Also, rat megalin having homology with human megalin has been discovered by Saito et al. (1994), and the cDNA sequence thereof having gene accession number L34049 has already been disclosed (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729).
4. Amino Acid Sequence and Protein Structure of Megalin
Megalin is a gigantic cell membrane protein consisting of 4,655 amino acids (in the case of human megalin) and 4,660 amino acids (in the case of rat megalin). The molecular weight deduced based on the amino acid sequence is about 520 kDa, and it can be as great as about 600 kDa, when including a sugar chain (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729). Megalin belongs to the LDL receptor gene family, a gigantic extracellular region thereof has four functional domains, and the extracellular region is connected to a thin intracellular region through a single transmembrane region. Megalin is mainly present in a clathrin-coated pit on the glomerulus (rat) or the epithelial luminal membrane (luminal and basal membrane in the glomerular epithelial cell) of the proximal tubule, type II alveolar cell, epididymal glands, thyroid glands, accessory thyroid glands, yolk sac membrane, inner ear, small intestine, or choroidea, and it is associated with intake of various ligands into the cells and metabolism thereof (see Farquhar M. G. et al., 1995, J. Am. Soc. Nephrol. 6, 35-47; and Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Low-molecular-weight proteinuria, bone metabolism disorders, respiratory failure, malformation of brain, and other disorders occur in megalin-knockout mice (see Willnow T. E. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8460-8464). A megalin homolog is also present in nematodes (C. elegans), and the biological importance thereof has been suggested (see Yochem J. et al., 1993, Proc. Natl. Acad. Sci. U.S.A., 90, 4572-4576).
5. Importance of Megalin as a Cause of Nephritis
Megalin, which is a major etiologic antigen of experimental membranous nephropathy (Heymann nephritis), is an epithelial scavenger receptor, and biological and pathological roles thereof have been elucidated. Animal models have been used for a long time in order to elucidate the mechanism of human membranous nephropathy development, and rat Heymann nephritis is a model of membranous nephropathy. The analysis of Heymann nephritis has been more advanced than that of any other model. Saito A. et al. disclosed the results of analysis of the pathological epitope and the ligand-binding domain of Heymann nephritis, and they have also demonstrated the major antigen region of megalin and a functional domain of megalin that mainly contribute to binding to a ligand (see Kerjaschki D. et al., 1992, Proc. Natl. Acad. Sci. U.S.A., 89, 11179-11183; Saito A., Farquhar M. G. et al., 1996, Proc. Natl. Acad. Sci. U.S.A., 93, 8601-8605; Yamazaki H., Farquhar M. G. et al., 1998, J. Am. Soc. Nephrol. 9, 1638-1644; and Orlando R. A., Farquhar M. G. et al., 1997, Proc. Natl. Acad. Sci. U.S.A., 94, 2368-2373).
6. Various Ligands of Megalin
Megalin is expressed most abundantly on the luminal side of the proximal tubule epithelial cells in vivo. In human kidney, megalin expression is not observed at sites other than the proximal tubule epithelial cells, including at glomeruli. Megalin incorporates various ligands (e.g., a low-molecular-weight protein or drugs) that are filtered by glomeruli into cells via endocytosis, megalin transports them to lysosomes, and they reappear on the cell surface via recycling (see Farquhar M. G. et al., 1995, J. Am. Soc. Nephrol. 6, 35-47; and Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Also, megalin is associated with transcytosis from the luminal side to the basal membrane side. Megalin is also associated with intake and metabolism of binding proteins, such as vitamins A, B12, and D (see Christensen E. I. et al., 2002, Nat. Rev. Mol. Cell. Biol. 3, 256-266). Christensen and Willnow demonstrated that megalin mediates reabsorption of three vitamin carrier proteins, vitamin D binding proteins (DBP), retinol binding protein (RBP), and transcobalamin (TC) and vitamins associated therewith; i.e., (OH) vitamin 25D3, vitamin A (retinol), and vitamin B12 (see Christensen E. I., Willnow T. E., 1999, J. Am. Soc. Nephrol. 10, 2224-2236). Saito A. et al. demonstrated that leptin, which is secreted from adipocytes and increase in the blood of obese patients, is incorporated into and metabolized by the proximal tubule epithelial cells as the megalin ligand (see Saito A., Gejyo F. et al., 2004, Endocrinology. 145, 3935-3940). The adipocytes, that is, accumulated visceral fats, result in combined pathological conditions, i.e., metabolic syndrome. Leptin, which is an adipocytokine secreted from adipocytes, increases in the blood of a metabolic syndrome patient. It is suggested that the kidney is the organ in which leptin in the blood is most likely to accumulate and that leptin plays a nephropathic role (see Tarzi R. M. Lord G. M. et al., 2004, Am. J. Pathol. 164, 385-390). A so-called leptin receptor is also found in a region between the proximal tubule and the collecting tubule located downstream of the megalin functioning region.
The term “metabolic syndrome” is defined as a disease complication of visceral obesity, elevated blood pressure, hyperlipidemia, impaired glucose tolerance, and other symptoms, the primary risk factor of which is insulin resistance. Such conditions are highly likely to lead to development of arteriosclerotic diseases and proteinuria, and may result in the development of nephropathy with glomerulus and renal tubular hypertrophy as histological features. When such a case is combined with apparent diabetes, the feature of hyperglycemia is further developed, diabetic nephropathy is manifested, and the disease conditions may further become serious. Type II diabetes is basically preceded by or simultaneously develops with metabolic syndrome. Accordingly, the feature of nephropathy could be included as nephropathy associated with metabolic syndrome.
Saito A. et al. have conducted an experiment using rat yolk sac epithelium-derived cells (L2 cells) in which megalin is expressed at high levels and found that incorporation of 125I-labelled AGE (advanced glycation end products) (derived from glucose) into L2 cells would be significantly inhibited by an anti-megalin antibody. Thus, they demonstrated that megalin is associated with a pathway for such incorporation (see Saito A. Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 1123-1131). As a mechanism of diabetic nephropathy development, association of advanced glycation end products (AGE) with glycated and modified proteins by the Maillard reaction has been pointed out. A low-molecular-weight AGE in the blood is filtered by glomeruli, and it is reabsorbed and metabolized by the proximal tubule epithelial cells. If nephropathy further advances, a higher-molecular weight AGE also is filtered by glomeruli, accumulates in the proximal tubule epithelial cells, and imposes excessive metabolic loads. Further, Saito A. et al. also demonstrate that megalin is also associated with incorporation of AGE derived from methylglyoxal, glyceraldehyde, or glycolaldehyde into cells, in addition to glucose. Also, metabolic syndrome is often complicated with hepatopathy, such as fatty liver. Liver type fatty acid binding proteins (L-FABP) that are abundantly present in the liver are released into the blood of a healthy person. In case of hepatopathy, more L-FABP is released into and increased in the blood. Saito A. et al. have also demonstrated that L-FABP in the blood is rapidly filtered by glomeruli and it is reabsorbed by the proximal tubule epithelial cells via megalin (see Takeda T., Gejyo F., Saito A. et al., 2005, Lab. Invest. 85, 522-531).
7. Functional Protein that Interacts with Megalin
In order to elucidate the mechanism of megalin transportation in cells, adaptor molecules that bind to megalin intracellular domains are searched for, and various proteins, such as Dab2, ANKRA, MAGI-1, GAIP, GIPC, Galphai3, MegBP, and ARH, have been identified (see Oleinikov A. V. et al., 2000, Biochem. J. 347, 613-621; Rader K., Farquhar M. G. et al., 2000, J. Am. Soc. Nephrol. 11, 2167-2178; Patrie K. M., Margolis B. et al., 2001, J. Am. Soc. Nephrol. 12, 667-677; Lou X., Farquhar M. G. et al., 2002, J. Am. Soc. Nephrol. 13, 918-927; Petersen H. H., Willnow T. E., 2003, J. Cell. Sci. 116, 453-461; and Takeda T., Farquhar M. G. et al., 2003, Mol. Biol. Cell. 14, 4984-4996). Through such molecules, megalin is associated with endocytosis or transcytosis, and megalin is also associated with signal transmission related thereto. Also, megalin functions conjugatively with a cell membrane receptor, i.e., cubilin, in the proximal tubule epithelial cells, so as to be further involved with incorporation of various ligands into cells (see Saito A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91, 9725-9729). For example, cubilin is a receptor that directly binds to transferrin, albumin, endogenous vitamin B12, or the like, and megalin is indirectly involved with endocytosis thereof. Also, megalin is known to interact with the Na+—H+ exchanger isoform 3 (NHE3) in the proximal tubule epithelial cells (see Biemesderfer D. et al., 1999, J. Biol. Chem. 274, 17518-17524). NHE3 is an antiporter that plays an important role in reabsorption of Na+, and NHE3 also influences incorporation of a ligand by megalin (see Hryciw D. H. et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379). Also, megalin may be involved with inactivation and metabolism of NHE3. At an early stage of diabetic nephropathy or metabolic syndrome-related nephropathy, glomerular filtration becomes excessive. Enhanced reabsorption of Na+ of the proximal tubule is deduced to be a primary cause (see Vallon V. et al., 2003, J. Am. Soc. Nephrol. 14, 530-537), NHE3 plays a key role in such a case, and inactivation and metabolism of MHE3 by megalin is considered to be involved therewith (see Hryciw D. H. et al., 2004, Clin. Exp. Pharmacol. Physiol. 31, 372-379).
8. Correlation of Urinary Excretion of Megalin and Urinary Excretion of Ligand by Megalin
Leheste et al. disclosed that megalin-knockout mice and Fanconi syndrome patients with weakened proximal tubule functions would experience increased excretion of proteins and retinol in the urine (see Leheste J. et al., 1999, Am. J. Pathol. 155, 1361-1370). Further, Moestrup S. K. et al. demonstrated that the amount of megalin excreted in urine of patients of Fanconi syndrome is significantly lower than that excreted by healthy individuals. This causes deterioration of megalin functions and expression in the proximal tubule and consequently increases the amount of glomerular-filtered proteins containing retinol-binding proteins excreted in urine (see Anthony G. W., Moestrup S. K. et al., 2002, J. Am. Soc. Nephrol. 13, 125-133).
9. Importance of Megalin Function Found by Experiments Using Models for Uremia and Models for Organ Regeneration
As described above, megalin is involved with intake of various low-molecular-weight proteins into the proximal tubule epithelial cells and metabolism thereof. When the pathological condition advances to kidney failure, the mechanism of the metabolism is disturbed, and low-molecular-weight proteins are consequently accumulated in the blood and tissues as uremic proteins. A representative example thereof is β2-microglobulin (β2-m), which may cause dialysis-related amyloidosis in a long-term dialysis patient (see Gejyo F., Schmid K. et al., 1985, Biochem. Biophys. Res. Commun. 129, 701-706). The aforementioned AGE is also suggested as a cause of arteriosclerosis or organ failure due to its accumulation in the blood of patients with kidney failure or dialysis, and AGE is considered as a type of uremic protein (see Henle T., Miyata T., 2003, Adv. Ren. Replace Ther. 10, 321-331). Further, leptin accumulates in the blood of a dialysis patient and thus is considered to be involved with malnutrition or immunity compromise. Tabata Y. and Gejyo F. et al. disclosed the effects and effectiveness of models for metabolizing uremic protein using megalin functions (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032 and WO 02/091955). That is, megalin-expressing cells are transplanted as scaffold proteins in vivo, and low-molecular-weight proteins leaked from peripheral blood vessels (newborn blood vessels) are incorporated into the cells with the aid of megalin for metabolization. The megalin-expressing cells used for transplantation (i.e., yolk sac epithelium-derived L2 cells) incorporate and metabolize β2-m with the aid of megalin (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032). Both kidneys of a nude mouse into which L2 cells had been subcutaneously transplanted were removed, the condition of kidney failure was induced, and cell incorporation in the tissue mass into which 125I-labeled β2-m had been transplanted and in organs via intraperitoneal injection was measured. As a result, the cell mass into which L2 cells had been transplanted was found to more significantly incorporate 125I-labeled β2-m compared with other organs, and the 125I-labeled β2-m clearance was found to significantly advance in a group to which L2 cells had been transplanted, compared with a control group into which L2 cells had not been transplanted (see Saito A., Tabata Y., Gejyo F. et al., 2003, J. Am. Soc. Nephrol. 14, 2025-2032).
10. Proteolysis and Urinary Excretion of Megalin
In recent years, the possibility of megalin being subjected to proteolysis in a Notch-like signaling pathway has been suggested (see Zou Z., Biemesderfer D. et al., 2004, J. Biol. Chem. 279, 34302-34310; and Grigorenko A. P. et al., 2004, Proc. Natl. Acad. Sci. U.S.A., 101, 14955-14960). This also includes a two-step cleavage system of shedding of an ectodomain mediated by metalloprotease and intramembrane proteolysis mediated by gamma-secretase.
Also, megalin is known to express in the type II alveolar cell.
Thus, megalin has been extensively studied in respect of its correlation with the metabolism in organs such as the kidney. However, the correlation between diseases of organs, including the kidney, and megalin has not yet been elucidated, and expression of megalin or excretion thereof to the body fluid in connection with a variety of organ diseases has not yet been studied.
To date, a method involving tissue staining or Western blotting using a polyclonal antibody obtained by immunizing an immune animal, such as a rabbit, has been known as a method for detecting megalin.
This technique, however, involves staining of a cell or a protein separated via electrophoresis, and this necessitates very complicated procedures and a long period of time for immobilizing tissues, preparing tissue slices, electrophoresis, and transfer onto the membrane. Thus, it is difficult to quantify megalin.
From the viewpoint of diagnosis of the degrees of functional disorders of tissues or organs, particularly in the case of kidney disorders, there is no effective means for diagnosing kidney tubule failure in a specific and simple manner. At present, many methods of diagnosis that detect albumin, creatinine, β2-microglobulin, L-FABP, or the like in urine or blood as a diagnosis marker for renal diseases have been employed. Such diagnosis markers, however, are not derived from kidney tissue, and they merely result from all phenomenon and functions during filtration in kidney glomeruli and reabsorption in kidney tubules. That is, it is difficult to identify glomerulus failure and failure of kidney tubule failure in the kidney even with the use of such marker. Also, such marker is an indirect marker derived from organs other than the kidney. Thus, effectiveness is poor for early diagnosis of a disease. The same applies to KL-6 (markers of acute inflammation), which is an existing diagnosis marker for lung diseases, and particularly for inflammation.
DISCLOSURE OF THE INVENTION
The present invention provides a method for measuring human megalin that can be performed in a simpler manner within a shorter period of time than is possible with conventional techniques, and that can also quantify human megalin. Further, this method enables diagnosis of functional diseases, which are specific to cells, tissues, or organs, in a site-directed manner at an early stage. In particular, the present invention provides a method of measuring megalin levels in urine to detect kidney disorders.
As described above, many reports have been made regarding human megalin, and the correlation thereof with the metabolism of organs such as the kidney or the lung, has been suggested. When organ functions are impaired, however, the way that megalin expression varies and the way that prevalence of megalin changes are unknown.
The present inventors have conducted concentrated studies regarding a method for measuring human megalin with high sensitivity in a rapid manner. Consequently, they discovered a method for accurately measuring megalin in a body fluid sample, such as urine, using a ligand capable of binding to human megalin, and in particular, an anti-human megalin antibody.
Further, the present inventors measured human megalin in the body fluid of a patient with impaired organ functions and discovered that human megalin could be a marker for detecting and diagnosing organ diseases. This has led to the completion of the present invention.
Specifically, the present invention is as follows.
[1] A method for measuring human megalin in a sample using a first ligand capable of binding to human megalin that is bound to a solid support and a second ligand capable of binding to human megalin, the method comprising allowing the sample to react with the first ligand capable of binding to human megalin that is bound to a solid support, allowing the sample to react with the second ligand capable of binding to human megalin, and measuring the second ligand capable of binding to human megalin that is bound to a solid support resulting from formation of a complex of human megalin in the sample and the ligand capable of binding to human megalin.
[2] The method for measuring human megalin in a sample according to [1], which comprises the two steps of the reaction between the first ligand capable of binding to human megalin that is bound to a solid support and the sample and the following reaction between the second ligand capable of binding to human megalin and the sample.
[3] The method for measuring human megalin in a sample according to [1], wherein the reaction between the first ligand capable of binding to human megalin that is bound to a solid support and the sample and the reaction between the second ligand capable of binding to human megalin and the sample are carried out in a single step.
[4] The method for measuring human megalin in a sample according to any of [1] to [3], wherein the first ligand capable of binding to human megalin and the second ligand capable of binding to human megalin are both antibodies.
[5] The method for measuring human megalin in a sample according to any of [1] to [3], wherein the first ligand capable of binding to human megalin is lectin, which is specific to a sugar chain of human megalin, and the second ligand capable of binding to human megalin is an antibody.
[6] The method for measuring human megalin in a sample according to any of [1] to [3], wherein the first ligand capable of binding to human megalin is an antibody and the second ligand capable of binding to human megalin is lectin, which is specific to a sugar chain of human megalin.
[7] The method for measuring human megalin in a sample according to any of [1] to [6], wherein the first ligand capable of binding to human megalin and/or the second ligand capable of binding to human megalin are substances selected from the group consisting of: vitamin-binding protein, which is transcobalamin-vitamin B12, vitamin-D-binding protein, or retinol-binding protein; lipoprotein, which is apolipoprotein B, apolipoprotein E, apolipoprotein J/clusterin, or apolipoprotein H; hormone, which is parathyroid hormone (PTH), insulin, epithelial growth factor (EGF), prolactin, leptin, or thyroglobulin, a receptor of any thereof, or a receptor of such hormone; immune or stress response-associated protein, which is immunoglobulin light chain, PAP-1, or β2-microglobulin; enzyme, which is PAI-I, PAI-I-urokinase, PAI-I-tPA, prourokinase, lipoprotein lipase, plasminogen, α-amylase, β-amylase, α1-microglobulin, or lysozyme, an inhibitor of any thereof, or an inhibitor of such enzyme; drug or toxin, which is aminoglycoside, polymyxin B, aprotinin, or trichosantin; carrier protein, which is albumin, lactoferrin, hemoglobin, odorant-binding protein, transthyretin, or L-FABP; and receptor-associated protein (RAP), which is cytochrome-c, calcium (Ca2+), advanced glycation end products (AGE), cubilin, or Na+—H+ exchanger isoform 3 (NHE3) or binding fragment of such substance.
[8] A method for measuring human megalin in a sample using human megalin or a partial fragment of human megalin that is bound to a solid support and a ligand capable of binding to human megalin, the method comprising allowing the sample to react with the ligand capable of binding to human megalin, allowing the reaction product to react with the human megalin that is bound to a solid support, measuring the ligand capable of binding to human megalin that is bound to a solid support, and competitively quantifying human megalin in the sample based on a decrease in a percentage of the ligand capable of binding to human megalin that is bound to a solid support.
[9] The method for measuring human megalin in a sample according to [8], wherein the ligand capable of binding to human megalin is an anti-human megalin antibody.
[10] A method for measuring human megalin in a sample using a ligand capable of binding to human megalin, the method comprising allowing the sample to react with the ligand capable of binding to human megalin that is bound to a particle to induce agglutination and measuring human megalin based on the degree of resulting agglutination.
[11] The method for measuring human megalin according to [10], wherein the ligand capable of binding to human megalin is an anti-human megalin antibody and the agglutination is immune agglutination.
[12] A method of measuring human megalin by the method according to any of [1] to [11] to detect a disease in an organ in which megalin expression is observed.
[13] The method for detecting a disease in an organ according to [12], wherein the disease in an organ in which megalin expression is observed is a lung disease.
[14] The method for detecting a disease in an organ according to [12], wherein the disease in an organ in which megalin expression is observed is a renal disease.
[15] The method for detecting a renal disease according to [14], wherein the renal disease is renal tubular disorder.
[16] The method according to [14] or [15], wherein the sample is urine.
[17] A kit for detecting a disease in an organ in which megalin expression is observed comprising a ligand capable of binding to human megalin.
[18] The kit for detecting a disease in an organ in which megalin expression is observed according to [17], wherein the ligand capable of binding to human megalin is an anti-human megalin antibody.
[19] The kit for detecting a disease in an organ in which megalin expression is observed according to [18], wherein the disease in an organ in which megalin expression is observed is a lung disease.
[20] The kit for detecting a disease in an organ in which megalin expression is observed according to [18], wherein the disease in an organ in which megalin expression is observed is a renal disease.
[21] The kit for detecting a renal disease according to [20], wherein the renal disease is a renal tubular disorder.
[22] A disease-detecting marker for detecting a disease in an organ in which megalin expression is observed comprising human megalin.
[23] The disease-detecting marker according to [22], wherein the disease in an organ in which megalin expression is observed is a lung disease.
[24] The disease-detecting marker according to [22], wherein the disease in an organ in which megalin expression is observed is a renal disease.
[25] The disease-detecting marker according to [24], wherein the renal disease is a renal tubular disorder.
[26] Use of human megalin as a disease-detecting marker for detecting a disease in an organ in which megalin expression is observed.
[27] The use of human megalin as a disease-detecting marker according to [26], wherein the disease in an organ in which megalin expression is observed is a lung disease.
[28] The use of human megalin as a disease-detecting marker according to [26], wherein the disease in an organ in which megalin expression is observed is a renal disease.
[29] The use of human megalin as a disease-detecting marker according to [28], wherein the renal disease is a renal tubular disorder.
EFFECTS OF THE INVENTION
The method of the present invention enables measurement of human megalin in a sample, such as urine, with high sensitivity and accuracy. When functions of megalin-expressing cells, tissues, or organs are damaged, megalin escapes from the cells and accumulates in a sample. Specifically, measurement of human megalin in a sample enables direct detection and diagnosis of functional disorders of cells, tissues, or organs instead of indirect detection and diagnosis. Accordingly, measurement of human megalin in a sample via the method of the present invention enables detection of a disease in an organ in which megalin expression is observed, such as a renal or lung disease, at an early stage with high accuracy.
This description includes part or all of the contents as disclosed in the description and/or drawings of Japanese Patent Application No. 2006-089306, which is a priority document of the present application.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a gene locus and a general protein structure of human megalin.
FIG. 2 shows an ELISA calibration curve for detecting human megalin with the use of standard human megalin.
FIG. 3 shows the clinical results of human megalin in urine (part 1).
FIG. 4 shows the clinical results of human megalin in urine (part 2).



BEST MODES FOR CARRYING OUT THE INVENTION
The present invention concerns a method for measuring human megalin in a sample. SEQ ID NO: 1 shows the nucleotide sequence of human megalin, and SEQ ID NO: 2 shows the amino acid sequence of human megalin.
In the present invention, human megalin is measured using a first ligand capable of binding to human megalin that is bound to a solid support. Any solid support used in conventional immunoanalysis can be used. For example, wells of a plastic microtiter plate or magnetic particles can be preferably used.
An example of a ligand capable of binding to human megalin is an anti-human megalin antibody, and a monoclonal or polyclonal antibody can be used.
Also, lectin that is specific to a sugar chain of human megalin can be used as a ligand capable of binding to human megalin. Examples of lectin include, but are not limited to, concanavalin A, wheat germ lectin (WGA), Ricinus communis lectin (RCA), and lentil lectin (LCA).
Further, examples of a ligand capable of binding to human megalin include substances selected from the group consisting of: vitamin-binding proteins, such as transcobalamin-vitamin B12, vitamin-D-binding protein, or retinol-binding protein; lipoproteins, such as apolipoprotein B, apolipoprotein E, apolipoprotein J/clusterin, or apolipoprotein H; hormones, such as parathyroid hormone (PTH), insulin, epithelial growth factor (EGF), prolactin, leptin, or thyroglobulin, a receptor of any thereof, or a receptor of such hormone; immune or stress response-associated proteins, such as immunoglobulin light chain, PAP-1, or β2-microglobulin; enzymes, such as PAI-I, PAI-I-urokinase, PAI-I-tPA, prourokinase, lipoprotein lipase, plasminogen, α-amylase, β-amylase, α1-microglobulin, or lysozyme, an inhibitor of any thereof, or an inhibitor of such enzyme; drugs or toxins, such as aminoglycoside, polymyxin B, aprotinin, or trichosantin; carrier proteins, such as albumin, lactoferrin, hemoglobin, odorant-binding protein, transthyretin, or L-FABP; and receptor-associated proteins (RAP), such as cytochrome-c, calcium (Ca2+), advanced glycation end products (AGE), cubilin, or Na+—H+ exchanger isoform 3 (NHE3) or binding fragments of such substances. The term “binding fragment” used herein refers to a fragment of the aforementioned substance that includes a site binding to human megalin.
A ligand capable of binding to human megalin, such as an anti-human megalin antibody, can be bound to a solid support by a technique well-known in the art. When a ligand is to be bound to microtiter plate wells, for example, about 3 to 10 μg/ml (preferably about 5 μg/ml) of a solution of a ligand capable of binding to human megalin, such as an antibody, is applied to a solid support and the resultant is then allowed to stand at 4° C. overnight (preferably 12 hours or longer). The recommended density of a solid support mentioned above was theoretically determined when immobilizing a full-length antibody.
The density is determined by theoretical formulae:

Q=(2/√{square root over ( )}3)·(MW/N)·(2r)−2·109(ng/cm2)

Q: molecular weight density (ng/cm2)
MW: molecular weight (dalton: Da)
N: Avogadro's number=6·1023 (mole−1)
r: Stokes radius of molecular=(R·T20)/(6·π·η20·D20·N) (cm)
R: gas constant=8.3·107 (g·cm2·sec−2·° K−1·mole−1)
T20: room temperature (20° C.)=293° K
η20: viscosity of water at 20° C.=1·10−2 (g·cm−1·sec−1)
D20: diff. coeff. of molecular ref. to water at 20° C. (cm2·sec−1)
Such value is applied when immobilizing via physical adsorption. When a ligand capable of binding to human megalin is immobilized, the theoretical density of the relevant solid support is determined, and such density is affected by the aforementioned variation factors, such as individual molecular weight. Thus, the density varies depending on type of individual solid-support molecule, configuration of solid phase, or other conditions. Accordingly, the density is not limited to the aforementioned values. When absorbed on a solid support via covalent binding, also, such density is utilized in the present invention. In such a case, the number of functional groups that are present on the adsorption surface and used for covalent binding is also taken into consideration. The density of a solid support is not limited. After binding, blocking is carried out using bovine serum albumin (hereafter abbreviated to “BSA”) or casein, for the purpose of blocking non-specific adsorption sites of a protein, based on a conventional technique. When a solid support is a magnetic bead, the solid support is treated in the same manner as in the case of a microtiter plate.
A ligand capable of binding to human megalin, such as an anti-human megalin antibody, bound to a solid support is allowed to react with a sample, and human megalin in a sample is bound to a solid support with the aid of the ligand capable of binding to human megalin bound to the solid support by a ligand-receptor binding reaction, such as an antigen-antibody reaction. Specifically, a complex of a first ligand capable of binding to human megalin, such as an anti-human megalin antibody, bound to a solid support and human megalin is formed. Any sample may be used, provided that it contains human megalin. Examples of a sample include urine, an alveolar wash, blood, blood serum, blood plasma, and an exhaled air condensate. Such antigen-antibody reaction can be carried out at 4° C. to 45° C., more preferably 20° C. to 40° C., and further preferably 25° C. to 38° C. The duration of the reaction is approximately 10 minutes to 18 hours, more preferably 10 minutes to 1 hour, and further preferably 30 minutes to 1 hour.
After washing, the second ligand capable of binding to human megalin is then allowed to react with the human megalin in a sample bound to a solid support. Specifically, a complex of a first ligand capable of binding to human megalin, such as an anti-human megalin antibody, bound to a solid support, human megalin, and a second ligand capable of binding to human megalin is formed. As the second ligand capable of binding to human megalin, the same substance used as the first ligand capable of binding to human megalin, such as an anti-human megalin antibody, can be used. However, when both the first ligand capable of binding to human megalin and the second ligand capable of binding to human megalin are anti-human megalin monoclonal antibodies, an epitope that is recognized and bound by the first anti-human megalin antibody needs to be different from an epitope that is recognized and bound by the second anti-human megalin antibody. A combination of the first anti-human megalin antibody and the second anti-human megalin antibody can be any combination of a monoclonal antibody and a monoclonal antibody, a monoclonal antibody and a polyclonal antibody, a polyclonal antibody and a monoclonal antibody, and a polyclonal antibody and polyclonal antibody. The reaction can be carried out at 4° C. to 45° C., more preferably 20° C. to 40° C., and further preferably 25° C. to 38° C. The duration of the reaction is about 10 minutes to 18 hours, more preferably 10 minutes to 1 hour, and further preferably 30 minutes to 1 hour. Thus, the second ligand capable of binding to human megalin is bound to a solid support with the aid of human megalin and the first ligand capable of binding to human megalin.
After washing, the second ligand capable of binding to human megalin, such as the second anti-human megalin antibody, bound to a solid support is then measured. This can be carried out via a variety of techniques that are commonly employed in the immunoanalysis field. For example, the second ligand capable of binding to human megalin is labeled with an enzyme, fluorescence, biotin, or radiation label to prepare an enzyme-labeled substance. By assaying such label, the second ligand capable of binding to human megalin bound to a solid support can be measured. Labeling with an enzyme or fluorescence is particularly preferable. Examples of enzyme include peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase, and an example of fluorescence is fluorescein isothiocyanate (FITC), although labels are not limited thereto. A label can be detected by allowing a relevant substrate to react with an enzyme-labeled substance and then measuring the resulting dye, fluorescence, emission, or the like. When the second ligand capable of binding to human megalin is not labeled, a labeled third antibody is allowed to react with the second ligand capable of binding to human megalin, and the third antibody can be measured based on such labeling. Thus, the second ligand capable of binding to human megalin can be measured.
A solid support or an anti-human megalin antibody used for labeling may be an immunoglobulin fragment, such as Fab or F(ab′)2, specific to human megalin or a recombinant antibody, such as scFv, dsFv, diabody, or minibody, expressed as a recombinant. In the present invention, the term “antibody” also refers to a fragment specific to human megalin. A method for preparing such fragment is well-known in the art.
The aforementioned method comprises the two steps of the reaction between a first ligand capable of binding to human megalin, such as an anti-human megalin antibody, bound to a solid support and a sample, followed by washing, and the reaction of a second ligand capable of binding to human megalin with a sample. Alternatively, a method comprising a single step in which the reaction between a first ligand capable of binding to human megalin, such as an anti-human megalin antibody, bound to a solid support and a sample is carried out simultaneously with the reaction between a second ligand capable of binding to human megalin and a sample.
The present invention further comprises a method for measuring human megalin in a sample using human megalin or a partial fragment of human megalin that is bound to a solid support and a ligand capable of binding to human megalin, the method comprising allowing a sample to react with a ligand capable of binding to human megalin, allowing the reaction product to react with the human megalin that is bound to a solid support, measuring the ligand capable of binding to human megalin that is bound to a solid support, and competitively quantifying human megalin in a sample based on a decrease in a percentage of the ligand capable of binding to human megalin that is bound to a solid support. This method requires binding of human megalin to a solid support, and such binding can be carried out in accordance with the method for binding a substance to a solid support. Also, a partial fragment of human megalin is not limited, and a partial fragment of human megalin to which a ligand capable of binding to human megalin is bound may be used. As a partial fragment of human megalin, a partial sequence of the amino acid sequence of human megalin as shown in SEQ ID NO: 2 can be prepared via chemical synthesis or genetic engineering. The aforementioned ligand capable of binding to human megalin can be used, and an anti-human megalin antibody is particularly preferable. In a competitive method, the amount of human megalin or a partial fragment of human megalin that is bound to a solid support and a ligand capable of binding to human megalin to be used is important. A competitive method is a known technique, and such amount can be adequately determined based on a known technique.
Further, the present invention comprises a method for measuring human megalin using a ligand capable of binding to human megalin, the method comprising allowing a sample to react with a ligand capable of binding to human megalin that is bound to a particle to induce agglutination and measuring human megalin based on the degree of resulting agglutination.
Examples of particles that are used in such method include particles having diameters of 0.05 to 10 μm, preferably latex particles having diameters of 0.1 to 0.4 μm, gelatin particles having diameters of 0.5 to 10 μm, and animal erythrocytes. An antibody can be bound to a particle by a method well-known in the art, such as physical adsorption or covalent binding.
In this method, particles comprising anti-human megalin antibodies bound thereto are mixed with a sample on, for example, a black glass slide, and particles precipitated as a result of agglutination are observed. Thus, human megalin in a sample can be detected. Also, the absorption of the agglutinate may be measured to quantify human megalin. Further, human megalin can also be detected via pulse immunoassay.
The method for measuring human megalin of the present invention enables measurement of not only intact human megalin but also fragments of human megalin.
By measuring human megalin in a sample, whether or not a subject from which a sample has been obtained has disorders in human megalin-expressing cells, tissues, organs, or the like can be evaluated. Specifically, an organ disease or the like can be detected or diagnosed.
Any cells, tissues, or organs may be targets, provided that megalin expression is observed therein. Lung and kidney are preferable, and kidney is further preferable. In case of renal diseases, nephritis or renal tubular disorder can be particularly detected. Also, diabetic nephropathy can be adequately detected. Further, such cells, tissues, or organs can also be used for detecting metabolic syndrome or metabolic syndrome-associated nephropathy.
In the aforementioned subject having functional disorders of cells, tissues, or organs, human megalin escapes from cells and the amount of human megalin in a sample is increased. When human megalin in a sample obtained from the subject is measured in vitro and the concentration of human megalin in a sample is significantly enhanced compared with the concentration of human megalin obtained from a healthy individual, the subject can be diagnosed as having a functional disorder of cells, tissues, or organs.
As described above, urine, an alveolar wash, blood, blood serum, blood plasma, an exhaled air condensate, and the like can be used as samples. When a lung disease is to be detected, use of an alveolar wash is particularly preferable. When a renal disease is to be detected, use of urine is preferable.
Further, measurement of human megalin in a sample obtained from a subject enables evaluation of the risk of being afflicted with functional disorders of cells, tissues, or organs, such as a renal disease. When human megalin in a sample obtained from the subject is measured in vitro and the concentration of human megalin in a sample is significantly enhanced compared with the concentration of human megalin obtained from a healthy individual, the subject can be evaluated as being highly likely to be afflicted with functional disorders of cells, tissues, or organs. That is, measurement of human megalin in a sample enables screening for of subjects who are highly likely to be afflicted with a disease, such as patients-to-be of renal disease, and provision of adequate treatment.
Further, periodical measurement of human megalin in a sample obtained from the subject and monitoring of human megalin concentration enable management of organ functions.
Human megalin can be used as a marker for detecting or diagnosing functional disorders in cells, tissues, or organs where human megalin expression is observed. The present invention comprises use of human megalin as a marker for detecting a functional disorder, i.e., a disease in an organ in which megalin expression is observed. The present invention further comprises a disease-detecting/diagnosing marker for detecting and diagnosing a functional disorder, i.e., a disease in a cell, tissue, or an organ in which megalin expression is observed.
Also, when detecting or diagnosing a functional disorder, i.e., a disease in a cell, tissue, or an organ in which megalin expression is observed, fragments of human megalin may be measured, as well as intact human megalin.
Examples
Hereafter, the present invention is described in greater detail with reference to the examples, although the present invention is not limited to these examples. It should be noted that the method of enzyme-linked immunosorbent assay (ELISA) used in the examples has been heretofore reported by many researchers, since Engvall E. and Perlmann P. made the first report in 1971. There are solid grounds for using this technique (Engvall E, Perlmann P., 1971, Immunochemistry, 8, 871-874).
Hereafter, the present invention is described in greater detail with reference to the examples, although the present invention is not limited to these examples. Detection of human megalin in urine via enzyme-linked immunosorbent assay (ELISA)
(1) Preparation of Mouse Anti-Human Megalin Monoclonal Antibody
A mouse was immunized intraperitoneally with 50 μg of human megalin with an adjuvant several times, and the elevated blood serum titer was confirmed. The spleen was removed 3 days after a booster shot (intravenous immunization), and spleen cells were obtained. The spleen cells were fused with mouse myeloma cells (10:1) in the presence of polyethylene glycol 3500 to prepare hybridoma cells. The resulting cells were cultured for a week in the presence of CO2 at 37° C., and the presence or absence of an anti-human megalin antibody in the culture supernatant was inspected. The cells in the positive wells in which antibody production was observed were diluted via limiting dilution, the resultants were cultured for 2 weeks, and the presence or absence of an anti-human megalin antibody in the culture supernatant was inspected in the same manner. Thereafter, the cells in the positive wells in which antibody production was observed were diluted via limiting dilution, and the resultants were cultured in the same manner. At this stage, cells in which anti-human megalin antibodies have been produced are cultured in a flask, part thereof is suspended in fetal calf serum (FCS) containing 10% dimethyl sulfoxide (DMSO) (5×106 cells/ml), and the resultant was stored in liquid nitrogen.
Subsequently, supernatants in the wells were used to inspect reactivity of antibodies against human megalin produced in culture supernatants. Human megalin was dissolved in 140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4 (pH 7.3; hereafter abbreviated as “PBS, pH 7.3”). To wells of a plastic microtiter plate (Nunc-ImmunoTMModule F8 MaxisorpTM Surface plate, Nalge Nunc International), 100 μl of the solution of human megalin in PBS (pH 7.3) was added per well, and human megalin was then immobilized on the microtiter plate at 3 pmol/well at 4° C. for 12 hours. Thereafter, the solution of human megalin in PBS (pH 7.3) that had been added to the wells was removed via decantation, 145 mM NaCl, 3.6 mM Na2HPO4, 1.4 mM KH2PO4, and 0.05% (v./v.) Tween 20 (hereafter abbreviated as “PBS-T”) were applied to wells of the microtiter plate at 200 μl/well, PBS-T was removed via decantation, and the excessively adsorbed human megalin in the wells was washed. This process of washing was carried out twice in total. Thereafter, 145 mM NaCl, 7.2 mM Na2HPO4, 2.8 mM KH2PO4, 1% (wt./v.) BSA, and 5% (wt./v.) lactose (hereafter abbreviated as a “blocking solution for an antigen-immobilized plate) were applied at 200 μl/well, and insides of the wells of the microtiter plate on which human megalin had been immobilized were blocked at 4° C. for 12 hours. Thereafter, the resultant was stored at 4° C. In order to inspect the reactivity of antibodies in the culture supernatant, the microtiter plate on which human megalin had been immobilized after blocking treatment was used. To wells of the microtiter plate on which human megalin had been immobilized, a hybridoma culture supernatant was added at 100 μl/well, and the plate was heated at 37° C. for 1 hour. Thereafter, the culture supernatant that had been applied to the wells was removed via decantation, PBS-T was applied to the wells of the microtiter plate at 200 μl/well, PBS-T was removed via decantation, and insides of the wells were then washed. This process of washing was carried out three times in total. Thereafter, peroxidase-conjugated goat anti-mouse immunoglobulins (DAKO) were applied to the wells at 100 μl/well (2,000-fold diluted, 0.55 μg/ml), and the resultant was heated at 37° C. for 1 hour. The enzyme-labeled antibody was diluted using 145 mM NaCl, 3.6 mM Na2HPO4, 1.4 mM KH2PO4, 0.05% (v./v.) Tween 20, and 0.5% (wt./v.) BSA (hereafter referred to as a “diluent of enzyme-labeled antibody”). Thereafter, the enzyme-labeled antibodies that had been applied to the wells were removed via decantation, PBS-T was applied to the wells of the microtiter plate at 200 μl/well, PBS-T was removed via decantation, and insides of the wells were then washed. This process of washing was carried out three times in total. Thereafter, a 3,3′,5,5′-tetramethylbenzidine (hereafter abbreviated as “TMB”) solution (TMB One-Step Substrate System: DAKO) was applied to the wells at 100 μl/well as a substrate solution for peroxidase enzyme reaction, and the resultant was allowed to stand at 25° C. for 30 minutes. Immediately thereafter, a 313 mM H2SO4 solution (hereafter referred to as a “reaction terminator”) was applied at 100 μl/well to the substrate solution for reaction in the wells to terminate the enzyme reaction in the wells. Thereafter, the absorption of the wells was measured, and the value obtained by subtracting the absorption at 630 nm from that at 450 nm was designated as an indicator for evaluation of reactivity (Josephy P. D., Mason R. P. et al., 1982, J. Biol. Chem. 257, 3669-3675). Many reports have been heretofore made regarding TMB-based colorimetry since Bos E. S. et al made the first report in 1981, and there are solid grounds for using this technique (Bos E. S. et al., 1981, J. Immunoassay, 2, 187-204).
As a result, monoclonalized hybridoma cells exhibiting strong reactivity of an anti-human megalin antibody to immobilized human megalin were selected, and the class and the subclass of immunoglobulin in the culture supernatant were examined regarding each clone from 100 μl of the stock culture supernatant solution using the mouse immunoglobulin typing kit (Wako Pure Chemical Industries, Ltd.). Based on the results, clones of the IgG class were selected from the resulting monoclone cell library and then the process of ascites preparation was carried out as described below.
Subsequently, these cells were cultured in a 25-ml flask and then in a 75-ml flask. The cells were injected intraperitoneally into a pristane-treated mouse, and ascites was sampled.
(2) Purification of Mouse Anti-Human Megalin Monoclonal (IgG) Antibody
The obtained ascites (10 ml) was mixed with an opacified blood serum-treating agent (FRIGEN (registered trademark) II: Kyowa Pure Chemical Co., Ltd.) at a ratio of 1:1.5 by volume, and the resultant was shaken and stirred for 1 to 2 minutes to delipidize the ascites. The ascites was centrifuged using a centrifuger at 3,000 rpm (1930×g) for 10 minutes, and the centrifuged supernatant of clarified ascites (10 ml) was fractionated. The centrifuged supernatant of ascites (10 ml) was subjected to ammonium sulfate fractionation (final concentration: 50% saturated ammonium sulfate) in an ice bath for 1 hour, and the precipitated immunoglobulin fraction was suspended and dissolved in PBS. This process of ammonium sulfate fractionation was carried out twice in total to obtain a crude immunoglobulin fraction from ascites. The resulting crude immunoglobulin fraction (10 ml) was mixed with an equivalent amount of 20 mM sodium phosphate (pH 7.0; hereafter referred to as “20 mM NaPB (pH 7.0)” and then subjected to affinity purification using a protein G column (HiTrap Protein G HP, 5 ml; Amersham BioSciences)). The sample was adsorbed on a protein G column, 20 mM NaPB (pH 7.0, 50 ml) was flushed through the protein G column, and contaminants other than IgG in the sample were removed by washing. Thereafter, affinity-adsorbed IgG on the protein G column was eluted with 0.1 M glycine-HCl (pH 2.7), and the elution fraction immediately after elution from the column was neutralized with 1M tris(hydroxymethyl)aminomethane-HCl (pH 9.0) and then recovered (hereafter “tris(hydroxymethyl)aminomethane” is abbreviated as “Tris”). After neutralization, the affinity-purified product was dialyzed against PBS in an amount 500 times greater than the purified product by volume at 4° C. for 6 hours, and this process of dialysis was carried out twice in total. The dialysis membrane used for dialysis was a cellulose tube for dialysis (Viskase Companies). The resulting IgG elution fraction was designated as a purified anti-human megalin monoclonal antibody and subjected to storage at 4° C. and procedures described below. The process of purification was performed by connecting the aforementioned protein G column to the BioLogic LP System (Bio Rad Laboratories) at a constant flow rate of 1 ml/min.
(3) Preparation of Microtiter Plate on which Anti-Human Megalin Monoclonal Antibody has been Immobilized
The purified anti-human megalin monoclonal antibody was dissolved in PBS (pH 7.3) to result in a final concentration of 5 μg/ml therein. To wells of a plastic microtiter plate (Nunc-ImmunoTMModule F8 MaxisorpTM Surface plate, Nalge Nunc International), 100 μl of the solution of the anti-human megalin monoclonal antibody in PBS (pH 7.3) was added per well, and the anti-human megalin monoclonal antibody was immobilized on the microtiter plate at 4° C. for 12 hours. Thereafter, the solution of the anti-human megalin monoclonal antibody in PBS (pH 7.3) that had been added to the wells was removed via decantation, PBS-T was added to the wells of the microtiter plate at 200 μl/well, PBS-T was removed via decantation, and the excessively adsorbed anti-human megalin monoclonal antibody in the wells was washed. This process of washing was carried out twice in total. Thereafter, 86 mM NaCl, 100 mM Tris, 0.5% (wt./v.) BSA, and 0.05% (v./v.) Tween 20 (hereafter referred to as a blocking solution for an antibody-immobilized plate) were added at 200 μl/well, and the insides of the wells of the human megalin-immobilized microtiter plate were subjected to blocking at 4° C. for 12 hours. Thereafter, the resultant was stored at 4° C.
(4) Preparation of Peroxidase-Labeled Anti-Human Megalin Monoclonal Antibody
Horseradish peroxidase (hereafter abbreviated as “HRP”) (peroxidase from horseradish, Type VI, Sigma) was dissolved in pure water at a concentration of 4 mg/ml, 100 μl of a 100 mM sodium metaperiodate solution was added to 500 μl of the HRP solution (2 mg), and the mixture was agitated at room temperature for 20 minutes. The resultant was dialyzed against a 1 mM sodium acetate (pH 4.0) solution (hereafter referred to as a “1 mM acetate buffer”) in an amount 500 times greater than that of the HRP solution by volume at 4° C. for 6 hours, and this procedure was performed twice. The dialysis membrane used for dialysis was a cellulose tube for dialysis (Viskase Companies). Subsequently, the anti-human megalin monoclonal antibody was dissolved in a solution of 2.4 mM Na2CO3 and 7.6 mM NaHCO3 (pH 9.6) (hereafter referred to as a “10 mM carbonate buffer”) at a concentration of 8 mg/ml. A solution of 120 mM Na2CO3 and 380 mM NaHCO3 (pH 9.6) (hereafter referred to as a “0.5 M carbonate buffer”) was added to 500 μl of the HRP solution (2 mg) in an amount one-third thereof by volume, 500 μl of the aforementioned anti-human megalin monoclonal antibody (4 mg) was added thereto, and the resultant was agitated at room temperature for 2 hours. Thereafter, 50 μl of a solution of sodium borohydride (4 mg/ml) was added, and the resultant was agitated in an ice bath for 2 hours. The resultant was subjected to ammonium sulfate fractionation (final concentration: 50% saturated ammonium sulfate) in an ice bath for 1 hour, and the precipitated fraction was suspended and dissolved in 1 ml of a solution of 100 mM Tris, 145 mM NaCl, and 1% (v./v.) BSA (pH 7.6) (hereafter referred to as a “suspension of labeled antibody”). This ammonium sulfate fractionation was carried out twice in total, and a solution of 2.8 mM KH2PO4, 7.2 mM Na2HPO4, 145 mM NaCl, 1% (wt./v.) BSA, 0.02% (v./v.) phenol, and 40% (wt./v.) D-sorbitol (hereafter referred to as a labeled-antibody stock solution) was added to the solution of the labeled antibody in an amount three-fourths of the solution of the labeled antibody (hereafter referred to as a labeled-antibody stock solution). The HRP-labeled anti-human megalin monoclonal antibody was obtained. Many reports have been heretofore made regarding the method of HRP labeling, ever since Nakane, P. K. and Kawaoi, A. made the first report in 1974. Thus, there are solid grounds for using this technique (Nakane, P. K., Kawaoi, A., 1974, J. Histochem. Cytochem. 22, 1084).
(5) Measurement of Human Megalin in Urine
The aforementioned anti-human megalin monoclonal antibody-immobilized microtiter plate and the HRP-labeled anti-human megalin monoclonal antibody were used to measure human megalin in urine. At the outset, 90 μl of glomerular filtrate was mixed with 10 μl of a solution of 2 M Tris and 0.2 M ethylenediamine-N,N,N′,N′-tetraacetic acid (hereafter “ethylenediamine-N,N,N′,N′-tetraacetic acid” is abbreviated as EDTA, pH 8.0), and 100 μl of the resulting solution was applied to wells of the microtiter plate to which the anti-human megalin monoclonal antibody has been immobilized. The resultant was allowed to stand at 37° C. for 1 hour, the urine sample solution that had been applied to wells was removed via decantation, PBS-T was applied to wells of the microtiter plate at 200 μl/well, and PBS-T was removed via decantation, followed by washing. The process of washing was carried out three times. Thereafter, the solution of HRP-labeled anti-human megalin monoclonal antibody (the above stock solution was diluted to 10,000-fold with the solution of diluted labeled antibody) was added at 100 μl/well. The resultant was allowed to stand at 37° C. for 1 hour, the solution of HRP-labeled antibody that had been applied to the wells was removed via decantation, PBS-T was added to wells of the microtiter plate at 200 μl/well, and PBS-T was removed via decantation, followed by washing. The process of washing was carried out three times. Subsequently, a TMB solution (TMB One-Step Substrate System; DAKO) was applied to wells as a substrate solution for peroxidase enzyme reaction at 100 μl/well, and the resultant was allowed to stand at 25° C. for 30 minutes. Immediately thereafter, the reaction terminator was added to the substrate solution in the wells at 100 μl/well to terminate the enzyme reaction in the wells. Thereafter, the absorbance of the wells was measured, and the value obtained by subtracting the absorbance at 630 nm from that at 450 nm was designated as an indicator for evaluation of measurement of human megalin in the urine. As the reference sample for the calibration curve, human megalin that was used as an immunological antigen at the time of preparation of an anti-human megalin monoclonal antibody was used, and the results of analysis are shown in Table 1 and FIG. 2. The results of actual clinical measurement of human megalin in the urine are shown in Table 2, FIG. 3, and FIG. 4. As a result, the amount of human megalin excreted to the urine was found to be significantly greater in patients with renal diseases and patient-to-be of renal diseases, compared with healthy individuals (FIGS. 3 and 4). The results of the creatinine clearance tests regarding the amount of megalin excreted to the urine were also similar. This indicates that the concentration at the time of urinary excretion would not matter (FIGS. 3 and 4). The present invention provides a method for measuring human megalin that can be performed in a simpler manner within a shorter period of time than is possible with conventional techniques, and that can also quantify human megalin. Further, this method enables diagnosis of functional diseases that are specific to cells, tissues, or organs, in a site-directed manner at an early stage. The clinical results shown above apparently support such feature of the present invention.








TABLE 1







[h-megalin]
ELISA calibration curve for detecting human megalin












(nM)
n = 1
n = 2
n = 3
AVR.
S.D.





6.250
2.4356
2.4416
2.3576
2.4116
0.0469


3.125
1.2551
1.2596
1.2261
1.2469
0.0182


1.563
0.6288
0.6576
0.6358
0.6407
0.0150


0.781
0.3282
0.3296
0.3282
0.3287
0.0008


0.313
0.1341
0.1359
0.1370
0.1357
0.0015


0.156
0.0788
0.0917
0.0858
0.0854
0.0065


0.078
0.0582
0.0638
0.0727
0.0649
0.0073


0.031
0.0390
0.0503
0.0468
0.0454
0.0058


0.000
0.0465
0.0409
0.0431
0.0435
0.0028


















TABLE 2








Creatinine in urine
Megalin in urine


Item
Enzyme method:
ELISA










Method of measurement
Color method

Creatinine clearance












Background
Sample
[u-Cre]
O.D. (450 nm) −
[Megalin]
(nmol megalin/g


of samples
No.
(mg/dl)
O.D. (630 nm)
(nM)
Cre)















Diabetes
D-1
117.96
0.241
0.513
0.435



D-2
68.16
0.110
0.168
0.246



D-3
102.53
0.102
0.146
0.142


Nephropathy
N-1
178.52
2.472
6.398
3.584



N-2
33.55
0.459
1.088
3.243



N-3
41.24
0.161
0.302
0.732



N-4
78.29
0.110
0.168
0.215



N-5
36.97
0.129
0.218
0.590


Metabolic
M-1
302.32
0.169
0.323
0.107


syndrome
M-2
59.56
0.086
0.104
0.175


Healthy
H-1
44.11
0.061
0.038
0.086


individuals
H-2
92.72
0.082
0.094
0.101



H-3
134.72
0.056
0.025
0.019



H-4
123.59
0.063
0.044
0.036



H-5
104.31
0.052
0.015
0.014



H-6
96.64
0.050
0.009
0.009









All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

## Claims

1. A method for detecting a renal disease in which megalin expression is observed by measuring human megalin in a urine sample, comprising:
(i) allowing the sample to react with a first ligand that is capable of binding to human megalin and that is bound to a solid support,
(ii) allowing the sample to react with the second ligand, wherein the second ligand is capable of binding to human megalin, and then
(iii) measuring the level of human megalin in the sample by measuring the amount of the complex formed between the first ligand, the human megalin and the second ligand, wherein an increased level of human megalin indicates a renal disease that is selected from the group consisting of nephritis, tubular renal disorder, and nephropathy.

2. The method according to claim wherein step (i) and step (ii) are carried out sequentially.

3. The method according to claim 1, wherein step (i) and step (ii) are carried out concurrently.

4. The method according to claim 1, wherein the first ligand and the second ligand are both antibodies.

5. The method according to claim 1, wherein the first ligand is lectin, which is specific to a sugar chain of human megalin, and the second ligand is an antibody.

6. The method according to claim 1, wherein the first ligand is an antibody and the second ligand is lectin, which is specific to a sugar chain of human megalin.

7. The method according to claim 1, wherein the first ligand is selected from the group consisting of: vitamin-binding protein, which is transcobalamin-vitamin B12, vitamin-D-binding protein, or retinol-binding protein; lipoprotein, which is apolipoprotein B, apolipoprotein E, apolipoprotein J/clusterin, or apolipoprotein H; hormone, which is parathyroid hormone (PTH), insulin, epithelial growth factor (EGF), prolactin, leptin, or thyroglobulin, a receptor of any thereof, or a receptor of such hormone; immune or stress response-associated protein, which is immunoglobulin light chain, purple acid phosphatase 1 (PAP-1), or β2-microglobulin; enzyme, which is plasminogen activator inhibitor-I (PAI-I), plasminogen activator inhibitor-I-urokinase (PAI-I-urokinase), plasminogen activator inhibitor-I-tissue plasminogen activator (PAI-I-tPA), prourokinase, lipoprotein lipase, plasminogen, α-amylase, β-amylase, α1-microglobulin, or lysozyme, an inhibitor of any thereof, or an inhibitor of such enzyme; drug or toxin, which is aminoglycoside, polymyxin aprotinin, or trichosantin; carrier protein, which is albumin, lactoferrin, hemoglobin, odorant-binding protein, transthyretin, or liver type fatty acid binding proteins (L-FABP); and receptor-associated protein (RAP), which is cytochrome-c, calcium (Ca2+), advanced glycation end products (AGE), cubilin, or Na+—H+ exchanger isoform 3 (NHE3) or binding fragment of such substance, and the second ligand is an antibody.

8. The method according to claim 1, wherein the first ligand is an antibody and the second ligand is selected from the group consisting of vitamin-binding protein, which is transcobalamin-vitamin B12, vitamin-D-binding protein, or retinol-binding protein; lipoprotein, which is apolipoprotein B, apolipoprotein E, apolipoprotein J/clusterin, or apolipoprotein H; hormone, which is parathyroid hormone (PTH), insulin, epithelial growth factor (EGF), prolactin, leptin, or thyroglobulin, a receptor of any thereof, or a receptor of such hormone; immune or stress response-associated protein, which is immunoglobulin light chain, purple acid phosphatase 1 (PAP-1), or β2-microglobulin; enzyme, which is plasminogen activator inhibitor-I (PAI-I), plasminogen activator inhibitor-I-urokinase (PAI-I-urokinase), plasminogen activator inhibitor-I-tissue plasminogen activator (PAI-I-tPA), prourokinase, lipoprotein lipase, plasminogen, α-amylase, β-amylase, α1-microglobulin, or lysozyme, an inhibitor of any thereof, or an inhibitor of such enzyme; drug or toxin, which is aminoglycoside, polymyxin B, aprotinin, or trichosantin; carrier protein, which is albumin, lactoferrin, hemoglobin, odorant-binding protein, transthyretin, or liver type fatty acid binding proteins (L-FABP); and receptor-associated protein (RAP), which is cytochrome-c, calcium (Ca2+), advanced glycation end products (AGE), cubilin, or Na+—H+ exchanger isoform 3 (NHE3) or binding fragment of such substance.

9. The method according to claim 1, wherein said renal disease is nephritis.

10. The method according to claim 1, wherein said renal disease is nephropathy.

11. The method according to claim 1, wherein said renal disease is renal tubular disorder.