Patent Application
20110236913
The present invention relates to development of a protein used for diagnosing IgA nephropathy and thin-glomerular-basement-membrane (hereinafter, referred to as “TGBM”) nephropathy, and used as a biomarker for diagnosing the severity, and more particularly to a biomarker protein increased/decreased in urine of IgA nephropathy patients or TGBM nephropathy patients compared to those in normal people, and a diagnostic kit using the biomarker protein, which diagnose IgA nephropathy and TGBM nephropathy early, and previously predict and determine the progress stage.
IgA nephropathy is one of the various diagnoses of glomerulonephritis, and is known to be the most frequent glomerulonephritis in foreign countries as well as Korea. Its cause is not well known. When bacteria invade a body, an immunity substance referred to as an “antibody” is required to struggle against the bacteria. Herein, a phenomenon in which immunoglobulin A (IgA), one of such antibodies, destroys a glomerulus by going to a kidney's glomerulus without struggling against bacteria is referred to as IgA nephropathy. At present, the representative biomarkers for determining the progress stage of IgA nephropathy include proteinuria and creatine. However, proteinuria is highly related to a marker for other diseases in that it is also a marker indicating occurrence of hypertension and cardiovascular diseases as well as IgA nephropathy, and thus it is difficult to directly determine the progress stage of diabetic nephropathy. Also, TGBM nephropathy causes symptoms, such as haematuria and proteinuria. Although such symptoms are similar to those of IgA nephropathy, TGBM is a disease showing a relatively not so serious prognosis. However, it is impossible to accurately diagnose IgA nephropathy by only such a history (indicating a cause, symptoms, and progress with the passage of time, etc. of a patient's disease), and thus a kidney biopsy is required for accurate diagnosis.
Accordingly, when a novel diagnosis marker having clinically high specificity and sensitivity, and a monoclonal antibody for detecting the marker are developed, it is possible to develop a kit which can diagnose diabetic nephropathy early and predict the progress stage.
Accordingly, the present inventors attempted to conduct research for overcoming problems of the above described conventional technologies. As a result, they have completed the present invention by identifying biomarkers of diseases such as IgA nephropathy and TGBM nephropathy through a target proteomics technique.
IgA nephropathy is the most frequent glomerulonephritis in Korea. It may occur in all age groups, but mainly occurs in people in their teens and twenties. Its clinical features are various. Intermittent visible haematuria may occur accompanied by upper respiratory tract infection, or microscopic haematuria and proteinuria persistently occur. In some cases, IgA nephropathy is expressed as nephritic syndrome, acute nephritis, or hypertension. Also, in many cases, it is progressed while being unknown to patients, and then is found as a chronic renal failure. It has been reported that IgA nephropathy is gradually progressed, and then is progressed to a terminal kidney disease in about 30% or more of patients within 20 years. However, IgA nephropathy shows various clinical progresses. There are known risk factors that have an effect on prognosis such as persistent proteinuria, depression in renal function, persistent hypertension, old age, being male, and histological findings, etc. IgA nephropathy is a kind of immune complex-mediated glomerular disease. Its basic cause is a control disorder of an immune reaction against an antigen coming into a mucous membrane, etc., by which polymeric IgA1 is excessively produced in bone marrow, and at the same time the structure defect of IgA1 occurs. As a result, these are deposited in mesangium, and damage is caused by mediators. Optical microscopic findings on IgA nephropathy characteristically include focal or diffuse cell proliferation, and expansion of extracellular matrix in mesangium. The cell proliferation-variously occurs and extremely causes crescent formation, and also vascular lesion and tubulointerstitial lesion are observed. Immunofluorescence is necessary for a diagnosis of IgA nephropathy because it shows that IgA is significantly deposited in mesangium. In general, IgA is deposited together with C3, IgG, IgM, etc. Electron microscopic findings include that high electron dense depositions are observed in mesangium or its periphery, and intra subcutaneous depositions in a local mesangium periphery are observed.
There are patent disclosures concerning research on such a diagnosis method of IgA nephropathy. Korean laid-open patent No. 1999-82292 discloses a method of obtaining a novel gene from an IgA renopathy patient's leukocytes by using a differential display method, an IgA nephropathy diagnostic agent including oligonucleotide derived from a leukocyte, and a therapeutic agent. Korean registered patent No. 528664 discloses an IgA protein for diagnosing diabetic retinopathy, a diagnostic kit including an IgA protein antibody, and a diagnosis method. Korean laid-open patent No. 2004-54609 discloses a method for relating a gene expression profile to a protein expression profile in identifying a target protein related to a disease such as an autoimmune disease to identify new drug development and a diagnosis marker. US patent publication No. 2007/87448 discloses a general method for diagnosing the progress stage of a disease by collecting a physiological specimen from blood and urine of a subject, obtaining a spectroscopic peak through the mass spectrometry of the specimen, and using protein profile analysis information obtained from the peak. Korean registered patent No. 792630 discloses a method for early diagnosis and progress stage determination of diabetic nephropathy in diabetes mellitus, in which the occurrence, early diagnosis, progress stage, and symptom severity of diabetic nephropathy in diabetes mellitus are determined from blood of a person, and a monoclonal antibody prepared based on an identified protein is used for an immunoassay kit.
Meanwhile, although it is important to diagnose early and treat IgA nephropathy or TGBM nephropathy before the occurring of serious depression in renal function in IgA nephropathy patients or TGBM nephropathy patients, it is difficult to determine to carry out invasive test such as renal biopsy by only using hematuria (or proteinuria) with no symptom shown in young adults in order to diagnose the nephropathy. Furthermore, in a case of the renal biopsy, it is difficult to actually carry out the biopsy in all patients for various reasons. Thus, it has been required to develop a novel method or kit for diagnosing IgA nephropathy or TGBM nephropathy, which is simple and accurate.
Therefore, the present invention has been made in view of the above-mentioned problems and requirements, and a principle object of the present invention is to provide a novel biomarker protein or an immunogenic fragment thereof, which can diagnose IgA nephropathy and TGBM nephropathy early, and effectively diagnose their progress stages.
Another object of the present invention is to provide an antibody against the biomarker protein, and a composition and a diagnostic kit which use the same.
In accordance with an aspect of the present invention, there is provided a biomarker for diagnosing IgA nephropathy and TGBM nephropathy, which includes, as an active component, proteins selected from the group including proteins increased/decreased in urine of nephropathy subjects compared to those in normal people. In order to obtain a diagnosis biomarker protein, label-free quantification was used. For the convenience of analysis and for the reproducibility, in Examples, both a group of IgA nephropathy patients (hereinafter, referred to as a “IgAN group”), and a group of TGBM patients (hereinafter, referred to as a “TGBM group”) were limited to only teenagers. Also, instead of proteins of entire urine, only exosome within urine was separated and used for Examples.
In the present invention, a subject indicates an IgA nephropathy patient or a TGBM nephropathy patient, and an immunogenic fragment indicates a biomarker protein's fragment having at least one epitope recognized by the antibody against the biomarker protein of the present invention.
In order to accomplish another object of the present invention, the present invention provides a diagnostic agent for IgA nephropathy and/or TGBM nephropathy, which includes, as an active component, an antibody specifically bound to the biomarker protein of the present invention or the immunogenic fragment thereof. The antibody of the present invention may be a polyclonal antibody, but preferably a monoclonal antibody.
The polyclonal antibody may be produced by injecting a biomarker protein or a fragment thereof, as an immunogen, to a foreign host, according to a conventional method known to people skilled in the art. The foreign host includes mammals such as a mouse, a rat, a sheep, a rabbit, and the like. The immunogen is injected through intramuscular, intraperitoneal or subcutaneous injection, and is generally administered together with an adjuvant for improving antigenicity. From the foreign host, blood is periodically collected to collect blood serum showing improved titer and antigenic specificity, from which an antibody is separated and purified.
The monoclonal antibody may be produced by a technology for producing immortalized cell lines by fusion, known in the art [Koeher and Milstein 1975, Nature, 256:495)]. Hereinafter, the production method will be simply described. First, 20 μg of pure protein is obtained, and is immunized into Balb/C rat. Otherwise, peptide is synthesized, bound to bovine serum albumin, and immunized into a rat. Then, antigen-producing lymphocyte separated from the rat is fused with myeloma of a human or a mouse to produce immortalized hybridoma. Then, ELISA method is used to select and proliferate only a hybridoma cell producing a required monoclonal antibody, and from the culture, the monoclonal antibody is separated and purified.
In order to accomplish a further object of the present invention, the present invention provides a method for diagnosing the progress stage of diabetic nephropathy of a subject, the method including the step of detecting the biomarker protein or the immunogenic fragment thereof, according to the present invention, from a fluid of the subject. In the diagnosis method of the present invention, human blood is used. Also, in the detecting step, from a blood solution of the subject, the biomarker protein or the immunogenic fragment thereof are directly detected through two dimensional (2-D) electrophoresis. Otherwise, the blood is contacted with the antibody of the present invention, thereby indirectly detecting the biomarker protein or the immunogenic fragment thereof through an antigen-antibody reaction.
An immunoassay method, which has been widely known as the antigen-antibody reaction, includes an enzyme immunoassay method (ELISA, Coated tube), a magnetic particle method (in which antibody-bound magnetic particles are bound to a tube, antigen-tracer and non-degradable pollutants are competitively reacted with each other to cause an enzyme reaction, and quantification is carried out), a latex particle method using antibody-bound latex particles, and the like. In order to accomplish a still further object of the present invention, the present invention provides a kit for diagnosing diabetic nephropathy, which includes, as an active component, an antibody specifically bound to the biomarker protein of the present invention or the immunogenic fragment thereof.
The diagnostic kit of the present invention is manufactured by a conventional manufacturing method known in the art, and typically includes freeze-dried antibody and buffer, stabilizer, inactive protein, and the like. The antibody may be labeled with radioisotope, fluorescence, enzyme, or the like. The monoclonal antibody of the present invention may be variously used for an immunoassay kit (ELISA, antibody coated tube test, lateral-flow test, potable biosensor), and also may be used to develop a protein chip having a detection spectrum for various diabetic complications through development of an antibody showing higher specificity and sensitivity.
In order to accomplish a still further object of the present invention, the present invention provides a composition for treating and preventing IgA nephropathy or TGBM nephropathy, which includes, as an active component, protein selected from the group including proteins increased/decreased in urine of IgA nephropathy patients or TGBM nephropathy patients.
The pharmaceutical composition of the present invention may be prepared as formulations such as parenteral injections by being mixed with pharmaceutically acceptable carrier, excipient, diluent, etc. according to a method known in the pharmacological field, and then may be administered through intravenous injection, etc. The dose of the pharmaceutical composition according to the present invention may be appropriately selected according to age, sex, severity, and disease symptoms of a patient, and preferably, 0.001 to 100 mg of protein per day per adult may be administered.
The technology of the present invention can easily diagnose IgA nephropathy or TGBM nephropathy from urine. There has been no commercially available efficient diagnostic agent for diagnosing IgA nephropathy patients or TGBM nephropathy patients so far. The biomarker protein composition of the present invention is very advantageous in that it can simply diagnose IgA/TGBM nephropathy from urine of IgA nephropathy patients or TGBM nephropathy patients, and can diagnose diabetic nephropathy early and previously predict the progress stage.
Also, the monoclonal antibody prepared based on the biomarker protein of the present invention can be used for an immunoassay kit (ELISA, antibody coated tube test, lateral-flow test, potable biosensor) capable of diagnosing IgA nephropathy and TGBM nephropathy early, and predicting the progress stage. Furthermore, the antibody can be used for developing a protein chip having a detection spectrum for early diagnosis and progress stage of IgA nephropathy and TGBM nephropathy from among various glomerular nephritis diseases, through development of an antibody showing higher specificity and sensitivity.
The foregoing and other objects, features and advantages of the present invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:
Hereinafter, the present invention will be described in detail with reference to Examples. These Examples are intended to only illustrate the present invention, and are not intended to limit the scope of the present invention.
All urine samples of patients and normal people were provided from Kyung-pook national university hospital. From among urine samples collected from July 2006 to April 2008, through the renal biopsy of teenagers, urine samples of 5 IgA nephropathy patients, 7 TGBM nephropathy patients, and 12 normal people were selected. The first morning urine sample of each of the providers was collected, treated with protease inhibitor (1.67 Ml of 100 mM NaN3/2.5 Ml of 10 mM PMSF/50 μl of 1 mM Leupeptin), and stored at −80° C. The urine sample was standardized with a urine creatinine level. The standardized urine sample [IgA nephropathy patients (IgAN): 88.1 Ml, TGBM nephropathy patients (thinGBM): 65.2 Ml] was used through a strong vortex action immediately after being dissolved, and clinicopathologic information of used urine is noted in Table 1.
2-1. Purification of Exosome
The urine creatinine level of each urine sample was recognized by a body fluid analyzer. Then, urine samples of three groups were put together after their volumes were set in such a manner that the same creatinine level was included. The put-together urine sample was subjected to first centrifugation (17,000 g for 15 min at 4° C.) to remove fragments such as whole cells or large membrane proteins. The supernatant of the first centrifugation was subjected to second centrifugation (200,000×g for 1 h at 4° C.) to precipitate low-density membrane vesicle protein. The precipitated resultant product was resuspended by using 50 μg of isolation buffer (250 mM sucrose/10 mM triethanolamine/0.5 mM PMSF/1 μM leupeptin), transferred to 1.5 Ml polypropylene tube, added with 60 mg/Ml of DTT, and reacted at 60° C. for 10 minutes to separate THP. Then, the isolation buffer was added to carry out centrifugation (200,000 g for 1 h at 4° C.), and thereby low-density membrane vesicle protein was precipitated and separated.
2-2. Confirmation of Exosome Purity
In order to confirm the purity of separated exosome, three fractions below were obtained: {circle around (1)} urinary sediment (obtained by carrying out first centrifugation (17.000×g, 15 minutes) of 10 Ml of urine); {circle around (2)} urinary exosome (sediment which is obtained by carrying out first centrifugation (17,000×g, 15 minutes) of 10 Ml of urine to provide supernatant, and then carrying out second centrifugation (200,000×g, 1 hour) of the supernatant); and CO protein obtained by precipitating 10 Ml of first centrifuged supernatant through the addition of the same amount of acetone thereto. The obtained fractions were loaded on 12% acrylamide gel, and subjected to electrophoresis. Protein of the gel was transferred to nitrocellulose membrane, and was subjected to western blot. The resultant product was subjected to blocking at room temperature for 1 hour in 5% non-skim milk dissolved in TBS-T (20 mM Tris, 500 mM NaCl, 0.05% Tween-20), added with 5% non-skim milk/TBS-T solution containing anti-AQP2 (aquapolin 2) (Santa Cruz, Calif., USA) as a marker protein of exosome, and anti-NHE3 (Na/H exchanger 3) (Chemicon, Temecula, Calif., USA) primary antibody, and reacted at 4° C. for 16 hours. Then, the resultant product was conjugated with a secondary antibody bound to horseradish peroxidase (Santa Cruz, Calif., USA) chemiluminescence (ECL, Amersham Pharmacia Biotech), and imaged by a sensing method. In order to confirm the separation purity of exosome, antibodies of two proteins (NHE3 and AQP2) as exosome marker proteins were used to carry out western blot on three fractions: {circle around (1)} urinary sediment (obtained by carrying out first centrifugation (17.000×g, 15 minutes) of 10 Ml of urine); {circle around (2)} urinary exosome (sediment which is obtained by carrying out first centrifugation (17,000×g, 15 minutes) of 10 Ml of urine to provide supernatant, and then carrying out second centrifugation (200,000×g, 1 hour) of the supernatant); and {circle around (3)} protein obtained by precipitating 10 Ml of first centrifuged supernatant through the addition of the same amount of acetone thereto. The result is shown in
The obtained exosome sediment was suspended in 14 μl of 0.3% SDS solution, mixed with 5 μl of acrylamide solution (30%), 0.7 μl of 1% ammonium persulfate, and 0.3 μl of TEMED, and polymerized at room temperature for about 30 minutes. A small gel was cut into pieces with a size of 1 mm3 by a surgeon's knife, and washed with 25 mM of ABC buffer (pH 8.0) for 20 min and 25 mM of ABC (pH 8.0)/50% ACN buffer for 20 min. Then, the gel was dried by vacuum-drying, and subjected to a general in-gel digestion method. Each protein was added with 10 mM of DTT, reduced at 56° C. for 30 minutes, and then added with 55 mM of iodoacetamide (IAA), and alkylated by dark reaction at room temperature. Then, the gel pieces were washed with 25 mM of ABC (pH 8.0) buffer for 20 min and 25 mM of ABC (pH 8.0)/50% ACN buffer for 20 min, and dried by vacuum-drying. Trypsin was dissolved in 25 mM of ABC buffer to a concentration of 100 ng/μll, and reacted with gel pieces at 37° C. for 16 hours. Peptide cleaved by trypsin was extracted three times by using a solution of 25 mM ABC, 5% formic acid/25 mM ABC/50% ACN in water, and each extracted peptide solution was dried by vacuum-drying. Then, only peptide was collected by a solid-phase protein extracting method.
In nano-size liquid chromatography analysis, a nanoAcquity system (Waters Corporation, Milford, Mass.) was used, and herein, C18 trap column of 5 μm, 2 mm 180 μm (Symmetry), and C18 analyzing reversed-phase column of 1.7 μm, 25 cm×5 μm (BEH) (Waters Corporation) were used. The obtained peptide was analyzed by being dissolved in 0.1% formic acid. A mobile phase A (0.1% formic/water); and a mobile phase B (0.1% formic acid/CAN) were used. The phase was flowed into the trap column at a flow rate of 10 μL/min for 3 min. The mobile phase B was flowed at a concentration of 3 to 40% and a flow rate of 300 nl/min, for 360 min to separate peptide. The column was, washed with 90% mobile phase B for 15 min, and then restabilized with 3% mobile phase B for 20 min. The temperature of the column was maintained at 35° C., and an auxiliary pump was used to move 100 fmol (Glu1)-Fibrinopeptide B/μL at a flow rate of 300 nL/min to a nano-lock reference spray of a mass spectrometer. All samples were repetitively carried out 4 times. The analysis of tryptic peptide was carried out by using Q-Tof Premier mass spectrometer (Waters Corporation, Manchester, UK). The mass spectrometer has a resolution of 10,000 FWHM in v-mode. All analyses were performed in a cation mode, and TOF analysis was carried out in a range of m/z 50˜1990. [Glu1]—Fibrinopeptide B having a divalent charge was used to compensate an in-real time mass value, once per 30 sec. In low collision energy mass spectrometry, data is collected for 1 sec at 4 eV, and in high collision energy spectrometry, data is collected for 1 sec by increasing energy from 15 eV to 40 eV. Between the low collision energy spectrometry and the high collision energy spectrometry, there is a time difference of 0.1 sec.
The data obtained by liquid-phase chromatography mass spectrometer was analyzed by using ProteinLynx GlobalServer v2.3 (Waters Corporation) software. From the clustered and normalized data, protein was identified by using an algorithm of ProteinLynx GlobalServer v2.3, and then the accurate mass value of the identified result and the delay time of the liquid-phase chromatography were used for clustering. The peak intensity of identified tryptic peptide was used to calculate the amount of each protein, and then the calculated value was used to determine the expression of protein in each group.
From urine samples of 12 normal people, 5 IgA nephropathy patients, and 7 TGBM patients, exosome was separated, and was subjected to western blot by using antibodies such as anti-Aminopeptidase N, anti-Vasorin precursor, anti-Ceruloplasmin, anti-Alpha-1-antitrypsin, to determine the expression extent of the protein in each group. The entire process for identifying a biomarker for diagnosing a disease from urinary exosome is shown in
As a result of protein analysis in the above described Example, three repetitive tests through LC-MS/MS on a normal people group (a normal group) and two patient groups (IgAN group, and TGBM group) identified a total of 2059 proteins. Some of the proteins are noted in Tables 2 to 33.
In the present invention, from among proteins increased/decreased in IgA nephropathy patients and TGBM nephropathy patients, Aminopeptidase N, Vasorin precursor, Ceruloplasmin, and Alpha-1-antitrypsin were selected and subjected to western blot. The result is shown in
Although several exemplary embodiments of the present invention have been described for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2008-0096862 | Oct 2008 | KR | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/KR2009/005589 | 9/30/2009 | WO | 00 | 6/7/2011 |
