KR101188439B1 - Kits for Glaucoma Diagnosis - Google Patents

Abstract

The present invention relates to a kit for diagnosing glaucoma comprising an antigen specifically binding to an autoantibody against a valosin-containing protein (VCP), collagen type VI or α-enolase, and a method for diagnosing glaucoma using the same. The present invention enables early diagnosis and molecular diagnosis of glaucoma, and can easily determine whether to develop glaucoma easily and quickly.

Glaucoma, Diagnostics, Autoantibodies, VCP, Collagen, α-Enolase

Description

Kit for Glaucoma Diagnosis

 The present invention relates to a glaucoma diagnostic kit and a method for diagnosing glaucoma.

Glaucoma is a disease in which intraocular pressure rises significantly, and symptoms such as eye hardening, retinal atrophy, papillary depression, and blindness may occur. The intraocular pressure is maintained only when the liquid is generated in the eye and discharged out of the eye at a constant speed. The intraocular pressure rises when there is an abnormality in the generation and discharge path of the waterproofing, and when the intraocular pressure increases, the optic nerve papilla is pushed back, Injury results. Dark spots occur depending on the degree of damage to the optic nerve, and as the disease progresses further, blindness occurs. This type of glaucoma is the most common form of open angle glaucoma (OAG), mainly after the age of 40, and the frequency of occurrence increases with age. Glaucoma is not detected in the early stages of the disease and can only be recovered when the disease progresses. Although glaucoma is not always accompanied by ocular hypertension, many patients with glaucoma often have intraocular pressures in excess of 21 mmHg, and long-term steroid eye drops after myopia correction with excimer may be accompanied by elevated intraocular pressure in some patients. It can also cause steroid-induced glaucoma.

Glaucoma is not a result of everyday life, it is not transmitted or life-threatening, but it is a terrible ocular disease that most often causes blindness, along with diabetic retinopathy. In particular, early diagnosis and early treatment of glaucoma are important because glaucoma cannot be recovered once the optic nerve is damaged. Although many ophthalmologists are currently studying for the cause and effective treatment, the exact pathogenesis of glaucoma is not known yet. Recently, genetic factors play an important role in the pathogenesis of glaucoma. In addition, research has been conducted on the linkage of autoimmune with glaucoma, but it only shows the diagnosis of the pattern of autoantibody response, not the diagnosis using a specific protein.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop novel biomarkers related to the development of glaucoma, and finally, by identifying three autoantibodies that are produced by glaucoma-specifically, the present invention has been completed.

It is therefore an object of the present invention to provide a diagnostic kit for glaucoma.

Another object of the present invention is to provide a method for diagnosing glaucoma.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a kit for diagnosing glaucoma comprising a vasosin-containing protein (VCP), collagen type VI or α-enolase.

According to another aspect of the present invention, the present invention provides antigen-antibody reactions from human samples to provide the information necessary for diagnosing glaucoma, and provides a VCP (valosin-containing protein), collagen type VI or α-enola as a glaucoma marker. Provided are methods for detecting autoantibodies to an agent.

The present inventors have tried to develop a novel biomarker related to the development of glaucoma, and eventually identified three autoantibodies that are generated glaucoma-specifically.

The present invention enables the molecular diagnosis of glaucoma. The term “glaucoma” as used herein refers to a chronic disease in which the optic nerve is damaged. More specifically, the intraocular pressure increases when there is an abnormality in the production and discharge paths of the eye, and when the intraocular pressure increases, the optic nerve papilla is pushed backward, resulting in damage to the optic nerve. Dark spots develop in the disease, and as the disease progresses further, blindness occurs. Optic nerve papillary changes and visual field changes show typical open-angle glaucoma, but normal or low intraocular pressure is called normal-tension glaucoma. Even in this case, the optic nerve is damaged by an unknown etiology. In particular, normal-tension glaucoma is found to occur frequently in Asians, including Koreans and Japanese. Symptoms of glaucoma include eye hardening, retinal atrophy, papillary depression, and blindness.

In the present invention, glaucoma is judged by a molecular diagnostic method, not by a doctor's opinion.

Glaucoma-specific biomarkers presented by the present invention are autoantibodies against valosin-containing protein (VCP), collagen type VI or α-enolase. According to the present invention, autologous antibodies against VCP, collagen type VI or α-enolase are produced in the human body when glaucoma develops, and the present invention detects such autoantibodies to determine glaucoma. The detection of autoantibodies can be carried out using antigens that specifically bind to autoantibodies, namely VCP, collagen type VI or α-enolase, or their antigenic determining sites.

As used herein, the term “antigen determinant” is used interchangeably with epitope and refers to a portion of an antigen that is specifically recognized by a particular antibody. An epitope typically consists of 3-30 amino acid sequences. The epitope of VCP, collagen type VI or α-enolase that can be used in the present invention can be readily determined by studying the position at which the autoantibodies that bind these proteins bind to these three antigenic proteins.

VCP, collagen type VI and α-enolase used in the present invention are preferably derived from mammals, more preferably from humans, cattle, pigs, horses, mice, rats and rabbits, and most It is preferably derived from humans. Specific examples of VCP, collagen type VI and α-enolase used in the present invention are illustrated in SEQ ID NO: 1, 2 and 3, respectively.

Autoantibody-specific antigens included in kits of the invention can be prepared through a variety of methods known in the art. For example, the three antigens can be obtained through genetic recombination.

An exemplary method for obtaining VCP, collagen type VI or α-enolase antigen used in the present invention through genetic recombination is as follows: nucleotide sequence encoding VCP, collagen type VI or α-enolase An expression vector is prepared. Vectors can be constructed with prokaryotic or eukaryotic cells as hosts. In the case of prokaryotic cells as hosts, the vector is a powerful promoter capable of promoting transcription (e.g., tac promoter, lac Promoter, lac UV5 promoter, lpp promoter, p _L ^λ promoter, p _R ^λ promoter, rac 5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter and T7 promoter, etc.), ribosomal binding site for initiation of detoxification And transcription / detox termination sequences. If E. coli is used as the host cell, E. coli The promoter and operator site of the tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol . , 158: 1018-1024 (1984)) and the left promoter of phage λ (p _L ^λ promoter, Herskowitz, I. and Hagen, . Ann Rev Genet, 14:. . 399-445 (1980)) may be used as a control region. On the other hand, vectors that can be used in the present invention are plasmids (eg, pSC101, ColE1, pBR322, pUC8 / 9, pHC79, pUC19, pET, etc.), phage (eg, λgt4? ΛB, λ-Charon, etc., which are often used in the art. , λΔz1 and M13, etc.) or viruses (eg SV40, etc.).

In the case of eukaryotic cells as hosts, the vector may be a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K). promoter, SV40 promoter, cytomegalovirus promoter and the HSV tk Promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

The vector used in the present invention may be fused with other sequences to facilitate purification of the antigen expressed therefrom. Sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA), and most preferably Is 6 × His. Because of the additional sequence for such purification, proteins expressed in the host are rapidly and easily purified through affinity chromatography.

Host cells capable of continuously cloning and expressing the vector in a stable manner are known in the art and may use any host cell, for example E. coli JM109, E. coli Bacillus sp. Strains such as BL21 (DE3), E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and Salmonella typhimurium Enterobacteria and strains such as Um, Serratia Marcesons and various Pseudomonas species. In addition, in the case of transforming a vector into a eukaryotic cell, as a host cell, yeast ( Saccharomyce) cerevisiae ), insect cells and human cells (eg, CHO cell lines (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like.

The method of transporting a vector into a host cell is provided by CaCl when the host cell is a prokaryotic cell.₂ Method (Cohen, S.N. et al.,Proc . Natl . Acac . Sci . USA9: 2110-2114 (1973)), one method (Cohen, S.N. et al.,Proc. Natl . Acac . Sci . USA9: 2110-2114 (1973); And Hanahan, D.,J. Mol . Biol ., 166: 557-580 (1983)) and electroporation methods (Dower, W. J. et al.,Nucleic . Acids Res ., 16: 6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, the micro-injection method (Capecchi, M.R.,Cell, 22: 479 (1980)), calcium phosphate precipitation (Graham, F. L. et al.,Virology, 52: 456 (1973)), electroporation (Neumann, E. et al.,EMBO J., 1: 841 (1982)), liposome-mediated transfection (Wong, T.K. et al.,Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal,Mol . Cell Biol ., 5: 1188-1190 (1985)), and gene balm (Yang et al.,Proc . Natl . Acad . Sci .87: 9568-9572 (1990)) and the like can be injected into a host cell.

Vectors injected into a host cell can be expressed in the host cell, in which case a large amount of antigen is obtained. For example, when the expression vector contains a lac promoter, the host cell may be treated with IPTG to induce gene expression.

According to a preferred embodiment of the invention, the antigen included in the kit of the invention is a VCP antigen. As demonstrated in the examples below, among the three markers of the present invention, VCP autoantibodies are most specific for glaucoma.

The kit of the present invention can be applied to a variety of glaucoma, preferably congenital glaucoma, traumatic glaucoma, glaucoma symptom, ocular hypertension, primary open-angle glaucoma, normal-tension glaucoma, lens cystic glaucoma with pseudo-explosion of the lens, chronic simple Glaucoma, low-tension glaucoma, pigmented glaucoma, primary closed-angle glaucoma, acute closed-angle glaucoma, chronic closed-angle glaucoma, intermittent closed-angle glaucoma, glaucoma that develops on the trauma of the eye, glaucoma that develops on the inflammation of the eye or on drugs Applied to glaucoma, more preferably to primary open-angle glaucoma or normal-tension glaucoma.

The glaucoma diagnostic method of the present invention can be carried out through various immunoassay methods known in the art using VCP, collagen type VI or α-enolase, or epitopes thereof. The method of the present invention basically comprises immunoassay methods (eg, radioimmunoassay methods, radioimmunoprecipitation methods, enzyme-linked immunosorbent methods (ELISA), dot blot analysis, Western blot, using antigen-antibody responses). , Quantitatively or qualitatively according to inhibition or competition assays and Sandswitch assays ( Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-). linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984).

For example, when following a radioimmunoassay, a marker in a sample (e.g., serum), i.e., VCP, collagen type, with an antibody labeled with a radioisotope (e.g. I ¹²⁵ , P ³² , S ³⁵ ) The production of immune complexes formed by binding to autoantibodies against VI or α-enolase can be measured to qualitatively or quantitatively detect autoantibodies in the sample.

In addition, in accordance with the ELISA method, (i) adsorbing an antigen (ie, VCP, collagen type VI or α-enolase, or an epitope thereof) to a well plate with autoantibodies in the sample; (Ii) adding a sample to the well plate to react and wash; (Iii) adding and reacting the secondary antibody to which the enzyme or fluorescent substance is bound; And (iii) determining whether or not the secondary antibody is bound.

The enzyme bound to the secondary antibody may include an enzyme catalyzing a chromogenic reaction, a fluorescence reaction, a luminescent reaction, or an infrared reaction, but is not limited thereto. For example, an alkaline phosphatase,? -Galactosidase, Radish peroxidase, luciferase, and cytochrome P ₄₅₀ . In the case where alkaline phosphatase is used as an enzyme binding to the secondary antibody, it is preferable that the substrate is selected from the group consisting of bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS -Bl-phosphate and ECF (enhanced chemifluorescence) are used. When horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'- Azine-di [3-ethylbenzthiazoline sulfonate]), o - phenylene diamine (OPD) and naphthol / pie Ronin, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS a substrate such as phenzaine methosulfate may be used All.

On the other hand, the kit of the present invention can be developed in a rapid kit (rapid kit) method using a strip-type solid substrate. Rapid kits generally include solid substrates (eg, glass, fiberglass, metals (eg, gold, alloys of gold and copper, aluminum), metal oxides, ceramics, quartz, silicon, semiconductors, Si / SiO ₂ wafers, germanium, In the gallium arsenide, carbon, carbon nanotubes, polymers (e.g. polystyrene, polyethylene, polypropylene, polyacrylamide), sepharose, agarose), the liquid phase sample is operated by flow or movement. Rapid kits are commonly referred to as chromatographic analysis methods. For example, a capture antibody fixed on the inner surface of the micropores during autoantibodies in the serum of the sample reacts with the antigen bound to the gold particles and then moves through the micropores of the nitrocellulose membrane by capillary action. By combining with an antibody) to form a color band is to visually discriminate between positive and negative.

Samples used in the method of the present invention include various biological samples. For example, biological samples that can be used in the present invention preferably include blood, lymph, bone marrow fluid, saliva, milk, urine, feces, ocular fluid, semen, thymic fluid, ascites and amniotic fluid, and cellular tissue fluid. . Preferably the biological sample applied to the present invention is blood, plasma, or serum, most preferably serum.

According to the present invention, it is possible to easily and quickly determine whether to develop glaucoma. In addition, glaucoma-related drugs can be treated in patients based on the data from the methods of the present invention.

The features and advantages of the present invention are summarized as follows:

(Iii) The present invention is based on the discovery that autoantibodies against VCP, collagen type VI or α-enolase serve as biomarkers for diagnosis of glaucoma.

(Ii) The present invention enables early diagnosis and molecular diagnosis of glaucoma using antigens that specifically bind to the autoantibodies, namely VCP, collagen type VI or α-enolase, or epitopes thereof.

(Iii) The present invention can easily determine whether glaucoma develops easily and quickly.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Analysis of Autoantibodies against Bovine Optic Nerve Protein Using Western Blotting

Isolation of protein in bovine optic nerve

The optic nerve section connected to the back of the cow's eye was cut. The tissue was dissolved in a solution (RIPA buffer, GenDEPOT, USA) and ultracentrifuged for 20 minutes at 13,000 rpm using an ultracentrifuge. After centrifugation, only the supernatant was collected and the protein was quantified using the BCA protein analysis kit (GenDEPOT, USA).

Western blotting of proteins against serum of normal and patient

Western blotting was performed according to standard methods (see Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine , 108-121, CRC press). First, 15 µg of the optic nerve protein extracted above was electrophoresed on a 9% acrylamide gel, then transferred to a nitro-cellulose membrane, and blocked using skim milk powder. Serum obtained from normal blood and peripheral blood of each patient group was attached to the nitro-cellulose membrane as the primary antibody, and the secondary antibody was then attached to the membrane using immunoglobin G of human being bound to horseradish peroxidase. The chemiluminescence kit (Chemiluminescence substrate kit, GenDEPOT, USA) was used to identify autoantibodies. The results are summarized in FIG. 1 and Table 1. FIG. In Table 1, + indicates that the protein is expressed and-indicates that the protein is not expressed. In addition, the number of + represents the degree of expression of the protein.

IgG number Molecular weight (kDa) Control group Normal POAG NTG Glaucoma One 85 - + ++ ++ + 2 90 - - + + ++ 3 100 - - ++ + + 4 150 - - ++ + + 5 185 - - ++ + ++ 6 200 - +++ +++ +++ +++

POAG: Patients with Primary open angle glaucoma

NTG: Patients with Normal Tension Glaucoma

Example  2: two-dimensional electrophoresis using bovine optic nerve protein and Western Blotting

Optic nerve proteins were isolated by two-dimensional electrophoresis (Park et al., Proteomics , 4 (11): 3446-3455 (2004)). First electrophoresis was performed using an IPG strip of pH range 4-7 (Amersham Pharmacia, USA) and secondly using 9% acrylamide gel with SDS. After two-dimensional electrophoresis, one gel was stained with Colloidal Coomassie Blue G-250 solution, ProteomeTech, Korea, and image was analyzed using an image analyzer (GS-710 imaging calibrated densitometer, Bio-Rad, USA). The remaining gels were subjected to western blotting to confirm the location of the autoantibodies via protein comparison on the gel via ImageMaster ^™ 2D Platinum software, Amersham Biosciences, USA.

Example  3: protein identification

Unlike western normal blotting, the bands of specific antibodies in glaucoma patients and western blotting after secondary electrophoresis were used to cut out spots from glaucoma patients. First, the sliced gel was washed and then the CBB stained on the gel was removed through 50% acetonitrile / 25 mM ammonium bicarbonate solution. Then, the gel was dehydrated and trypsin (Promega, USA) treated to rehydrate the gel. Tryptic-peptide was then extracted by adding ammonium bicarbonate buffer. After desalting the extracted peptides, proteins were identified through an HP 1100 HPLC nano-flow system (Agilent, CA) to which an LCQ DECA ion trap (Thermo Fisher Scientific, San Jose, Calif.) Was connected. The resin used was packed with a Zorbax 300SB-C18 resin (particle size 5 μm, Agilent, Calif.) In a silica column (100 mm length × 75 μm I.D., tip diameter 10 μm) and used for MS / MS scan. The peak list of MS / MS spectra was converted to data format using Bioworks 3.3 software (Thermo Fisher Scientific, USA) and protein identified using a database from the National Center for Biotechnology Information (NCBI). The antigenic proteins for autoantibodies specific to glaucoma patients identified in this experiment are summarized in Table 2.

Spot number Protein name s-1 Collagen type VI s-2 Valosin-containing protein (VCP) s-3, 4 α-enolase

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the results of Western blotting on the serum of normal, open angle glaucoma patients, normal-tension glaucoma patients, and those with glaucoma to bovine optic nerve protein. In FIG. 1, the arrow indicates a protein expressed in the glaucoma patient group when compared to the normal person, and S-2 indicates a protein that appears only in the glaucoma patient group when compared to other eye diseases.

Figure 2 shows the results of CBB staining by two-dimensional electrophoresis of bovine optic nerve protein. In FIG. 2, numbers indicate spots that differ between normal and patient groups by Western blotting after two-dimensional electrophoresis on bovine optic nerve protein.

Figure 3 shows the results of Western blotting using the serum of normal and glaucoma patients after two-dimensional electrophoresis using bovine optic nerve protein. In the figure, A is a control group, and B shows a spot showing a difference in the glaucoma group (open angle glaucoma patients and normal-tension glaucoma patients) compared to the normal.

<110> Lumieye Genetics, Inc. <120> Kits for Glaucoma Diagnosis <160> 3 <170> Kopatentin 1.71 <210> 1 <211> 806 <212> PRT <213> Homo sapiens <400> 1 Met Ala Ser Gly Ala Asp Ser Lys Gly Asp Asp Leu Ser Thr Ala Ile   1 5 10 15 Leu Lys Gln Lys Asn Arg Pro Asn Arg Leu Ile Val Asp Glu Ala Ile              20 25 30 Asn Glu Asp Asn Ser Val Val Ser Leu Ser Gln Pro Lys Met Asp Glu          35 40 45 Leu Gln Leu Phe Arg Gly Asp Thr Val Leu Leu Lys Gly Lys Lys Arg      50 55 60 Arg Glu Ala Val Cys Ile Val Leu Ser Asp Asp Thr Cys Ser Asp Glu  65 70 75 80 Lys Ile Arg Met Asn Arg Val Val Arg Asn Asn Leu Arg Val Arg Leu                  85 90 95 Gly Asp Val Ile Ser Ile Gln Pro Cys Pro Asp Val Lys Tyr Gly Lys             100 105 110 Arg Ile His Val Leu Pro Ile Asp Asp Thr Val Glu Gly Ile Thr Gly         115 120 125 Asn Leu Phe Glu Val Tyr Leu Lys Pro Tyr Phe Leu Glu Ala Tyr Arg     130 135 140 Pro Ile Arg Lys Gly Asp Ile Phe Leu Val Arg Gly Gly Met Arg Ala 145 150 155 160 Val Glu Phe Lys Val Val Glu Thr Asp Pro Ser Pro Tyr Cys Ile Val                 165 170 175 Ala Pro Asp Thr Val Ile His Cys Glu Gly Glu Pro Ile Lys Arg Glu             180 185 190 Asp Glu Glu Glu Ser Leu Asn Glu Val Gly Tyr Asp Asp Ile Gly Gly         195 200 205 Cys Arg Lys Gln Leu Ala Gln Ile Lys Glu Met Val Glu Leu Pro Leu     210 215 220 Arg His Pro Ala Leu Phe Lys Ala Ile Gly Val Lys Pro Pro Arg Gly 225 230 235 240 Ile Leu Leu Tyr Gly Pro Pro Gly Thr Gly Lys Thr Leu Ile Ala Arg                 245 250 255 Ala Val Ala Asn Glu Thr Gly Ala Phe Phe Phe Leu Ile Asn Gly Pro             260 265 270 Glu Ile Met Ser Lys Leu Ala Gly Glu Ser Glu Ser Asn Leu Arg Lys         275 280 285 Ala Phe Glu Glu Ala Glu Lys Asn Ala Pro Ala Ile Ile Phe Ile Asp     290 295 300 Glu Leu Asp Ala Ile Ala Pro Lys Arg Glu Lys Thr His Gly Glu Val 305 310 315 320 Glu Arg Arg Ile Val Ser Gln Leu Leu Thr Leu Met Asp Gly Leu Lys                 325 330 335 Gln Arg Ala His Val Ile Val Met Ala Ala Thr Asn Arg Pro Asn Ser             340 345 350 Ile Asp Pro Ala Leu Arg Arg Phe Gly Arg Phe Asp Arg Glu Val Asp         355 360 365 Ile Gly Ile Pro Asp Ala Thr Gly Arg Leu Glu Ile Leu Gln Ile His     370 375 380 Thr Lys Asn Met Lys Leu Ala Asp Asp Val Asp Leu Glu Gln Val Ala 385 390 395 400 Asn Glu Thr His Gly His Val Gly Ala Asp Leu Ala Ala Leu Cys Ser                 405 410 415 Glu Ala Ala Leu Gln Ala Ile Arg Lys Lys Met Asp Leu Ile Asp Leu             420 425 430 Glu Asp Glu Thr Ile Asp Ala Glu Val Met Asn Ser Leu Ala Val Thr         435 440 445 Met Asp Asp Phe Arg Trp Ala Leu Ser Gln Ser Asn Pro Ser Ala Leu     450 455 460 Arg Glu Thr Val Val Glu Val Pro Gln Val Thr Trp Glu Asp Ile Gly 465 470 475 480 Gly Leu Glu Asp Val Lys Arg Glu Leu Gln Glu Leu Val Gln Tyr Pro                 485 490 495 Val Glu His Pro Asp Lys Phe Leu Lys Phe Gly Met Thr Pro Ser Lys             500 505 510 Gly Val Leu Phe Tyr Gly Pro Pro Gly Cys Gly Lys Thr Leu Leu Ala         515 520 525 Lys Ala Ile Ala Asn Glu Cys Gln Ala Asn Phe Ile Ser Ile Lys Gly     530 535 540 Pro Glu Leu Leu Thr Met Trp Phe Gly Glu Ser Glu Ala Asn Val Arg 545 550 555 560 Glu Ile Phe Asp Lys Ala Arg Gln Ala Ala Pro Cys Val Leu Phe Phe                 565 570 575 Asp Glu Leu Asp Ser Ile Ala Lys Ala Arg Gly Gly Asn Ile Gly Asp             580 585 590 Gly Gly Gly Ala Ala Asp Arg Val Ile Asn Gln Ile Leu Thr Glu Met         595 600 605 Asp Gly Met Ser Thr Lys Lys Asn Val Phe Ile Ile Gly Ala Thr Asn     610 615 620 Arg Pro Asp Ile Ile Asp Pro Ala Ile Leu Arg Pro Gly Arg Leu Asp 625 630 635 640 Gln Leu Ile Tyr Ile Pro Leu Pro Asp Glu Lys Ser Arg Val Ala Ile                 645 650 655 Leu Lys Ala Asn Leu Arg Lys Ser Pro Val Ala Lys Asp Val Asp Leu             660 665 670 Glu Phe Leu Ala Lys Met Thr Asn Gly Phe Ser Gly Ala Asp Leu Thr         675 680 685 Glu Ile Cys Gln Arg Ala Cys Lys Leu Ala Ile Arg Glu Ser Ile Glu     690 695 700 Ser Glu Ile Arg Arg Glu Arg Glu Arg Gln Thr Asn Pro Ser Ala Met 705 710 715 720 Glu Val Glu Glu Asp Asp Pro Val Pro Glu Ile Arg Arg Asp His Phe                 725 730 735 Glu Glu Ala Met Arg Phe Ala Arg Arg Ser Val Ser Asp Asn Asp Ile             740 745 750 Arg Lys Tyr Glu Met Phe Ala Gln Thr Leu Gln Gln Ser Arg Gly Phe         755 760 765 Gly Ser Phe Arg Phe Pro Ser Gly Asn Gln Gly Gly Ala Gly Pro Ser     770 775 780 Gln Gly Ser Gly Gly Gly Thr Gly Gly Ser Val Tyr Thr Glu Asp Asn 785 790 795 800 Asp Asp Asp Leu Tyr Gly                 805 <210> 2 <211> 1028 <212> PRT <213> Homo sapiens <400> 2 Met Arg Ala Ala Arg Ala Leu Leu Pro Leu Leu Leu Gln Ala Cys Trp   1 5 10 15 Thr Ala Ala Gln Asp Glu Pro Glu Thr Pro Arg Ala Val Ala Phe Gln              20 25 30 Asp Cys Pro Val Asp Leu Phe Phe Val Leu Asp Thr Ser Glu Ser Val          35 40 45 Ala Leu Arg Leu Lys Pro Tyr Gly Ala Leu Val Asp Lys Val Lys Ser      50 55 60 Phe Thr Lys Arg Phe Ile Asp Asn Leu Arg Asp Arg Tyr Tyr Arg Cys  65 70 75 80 Asp Arg Asn Leu Val Trp Asn Ala Gly Ala Leu His Tyr Ser Asp Glu                  85 90 95 Val Glu Ile Ile Gln Gly Leu Thr Arg Met Pro Gly Gly Arg Asp Ala             100 105 110 Leu Lys Ser Ser Val Asp Ala Val Lys Tyr Phe Gly Lys Gly Thr Tyr         115 120 125 Thr Asp Cys Ala Ile Lys Lys Gly Leu Glu Gln Leu Leu Val Gly Gly     130 135 140 Ser His Leu Lys Glu Asn Lys Tyr Leu Ile Val Val Thr Asp Gly His 145 150 155 160 Pro Leu Glu Gly Tyr Lys Glu Pro Cys Gly Gly Leu Glu Asp Ala Val                 165 170 175 Asn Glu Ala Lys His Leu Gly Val Lys Val Phe Ser Val Ala Ile Thr             180 185 190 Pro Asp His Leu Glu Pro Arg Leu Ser Ile Ile Ala Thr Asp His Thr         195 200 205 Tyr Arg Arg Asn Phe Thr Ala Ala Asp Trp Gly Gln Ser Arg Asp Ala     210 215 220 Glu Glu Ala Ile Ser Gln Thr Ile Asp Thr Ile Val Asp Met Ile Lys 225 230 235 240 Asn Asn Val Glu Gln Val Cys Cys Ser Phe Glu Cys Gln Pro Ala Arg                 245 250 255 Gly Pro Pro Gly Leu Arg Gly Asp Pro Gly Phe Glu Gly Glu Arg Gly             260 265 270 Lys Pro Gly Leu Pro Gly Glu Lys Gly Glu Ala Gly Asp Pro Gly Arg         275 280 285 Pro Gly Asp Leu Gly Pro Val Gly Tyr Gln Gly Met Lys Gly Glu Lys     290 295 300 Gly Ser Arg Gly Glu Lys Gly Ser Arg Gly Pro Lys Gly Tyr Lys Gly 305 310 315 320 Glu Lys Gly Lys Arg Gly Ile Asp Gly Val Asp Gly Val Lys Gly Glu                 325 330 335 Met Gly Tyr Pro Gly Leu Pro Gly Cys Lys Gly Ser Pro Gly Phe Asp             340 345 350 Gly Ile Gln Gly Pro Pro Gly Pro Lys Gly Asp Pro Gly Ala Phe Gly         355 360 365 Leu Lys Gly Glu Lys Gly Glu Pro Gly Ala Asp Gly Glu Ala Gly Arg     370 375 380 Pro Gly Ser Ser Gly Pro Ser Gly Asp Glu Gly Gln Pro Gly Glu Pro 385 390 395 400 Gly Pro Pro Gly Glu Lys Gly Glu Ala Gly Asp Glu Gly Asn Pro Gly                 405 410 415 Pro Asp Gly Ala Pro Gly Glu Arg Gly Gly Pro Gly Glu Arg Gly Pro             420 425 430 Arg Gly Thr Pro Gly Thr Arg Gly Pro Arg Gly Asp Pro Gly Glu Ala         435 440 445 Gly Pro Gln Gly Asp Gln Gly Arg Glu Gly Pro Val Gly Val Pro Gly     450 455 460 Asp Pro Gly Glu Ala Gly Pro Ile Gly Pro Lys Gly Tyr Arg Gly Asp 465 470 475 480 Glu Gly Pro Pro Gly Ser Glu Gly Ala Arg Gly Ala Pro Gly Pro Ala                 485 490 495 Gly Pro Pro Gly Asp Pro Gly Leu Met Gly Glu Arg Gly Glu Asp Gly             500 505 510 Pro Ala Gly Asn Gly Thr Glu Gly Phe Pro Gly Phe Pro Gly Tyr Pro         515 520 525 Gly Asn Arg Gly Ala Pro Gly Ile Asn Gly Thr Lys Gly Tyr Pro Gly     530 535 540 Leu Lys Gly Asp Glu Gly Glu Ala Gly Asp Pro Gly Asp Asp Asn Asn 545 550 555 560 Asp Ile Ala Pro Arg Gly Val Lys Gly Ala Lys Gly Tyr Arg Gly Pro                 565 570 575 Glu Gly Pro Gln Gly Pro Pro Gly His Gln Gly Pro Pro Gly Pro Asp             580 585 590 Glu Cys Glu Ile Leu Asp Ile Ile Met Lys Met Cys Ser Cys Cys Glu         595 600 605 Cys Lys Cys Gly Pro Ile Asp Leu Leu Phe Val Leu Asp Ser Ser Glu     610 615 620 Ser Ile Gly Leu Gln Asn Phe Glu Ile Ala Lys Asp Phe Val Val Lys 625 630 635 640 Val Ile Asp Arg Leu Ser Arg Asp Glu Leu Val Lys Phe Glu Pro Gly                 645 650 655 Gln Ser Tyr Ala Gly Val Val Gln Tyr Ser His Ser Gln Met Gln Glu             660 665 670 His Val Ser Leu Arg Ser Pro Ser Ile Arg Asn Val Gln Glu Leu Lys         675 680 685 Glu Ala Ile Lys Ser Leu Gln Trp Met Ala Gly Gly Thr Phe Thr Gly     690 695 700 Glu Ala Leu Gln Tyr Thr Arg Asp Gln Leu Leu Pro Pro Ser Pro Asn 705 710 715 720 Asn Arg Ile Ala Leu Val Ile Thr Asp Gly Arg Ser Asp Thr Gln Arg                 725 730 735 Asp Thr Thr Pro Leu Asn Val Leu Cys Ser Pro Gly Ile Gln Val Val             740 745 750 Ser Val Gly Ile Lys Asp Val Phe Asp Phe Ile Pro Gly Ser Asp Gln         755 760 765 Leu Asn Val Ile Ser Cys Gln Gly Leu Ala Pro Ser Gln Gly Arg Pro     770 775 780 Gly Leu Ser Leu Val Lys Glu Asn Tyr Ala Glu Leu Leu Glu Asp Ala 785 790 795 800 Phe Leu Lys Asn Val Thr Ala Gln Ile Cys Ile Asp Lys Lys Cys Pro                 805 810 815 Asp Tyr Thr Cys Pro Ile Thr Phe Ser Ser Pro Ala Asp Ile Thr Ile             820 825 830 Leu Leu Asp Gly Ser Ala Ser Val Gly Ser His Asn Phe Asp Thr Thr         835 840 845 Lys Arg Phe Ala Lys Arg Leu Ala Glu Arg Phe Leu Thr Ala Gly Arg     850 855 860 Thr Asp Pro Ala His Asp Val Arg Val Ala Val Val Gln Tyr Ser Gly 865 870 875 880 Thr Gly Gln Gln Arg Pro Glu Arg Ala Ser Leu Gln Phe Leu Gln Asn                 885 890 895 Tyr Thr Ala Leu Ala Ser Ala Val Asp Ala Met Asp Phe Ile Asn Asp             900 905 910 Ala Thr Asp Val Asn Asp Ala Leu Gly Tyr Val Thr Arg Phe Tyr Arg         915 920 925 Glu Ala Ser Ser Gly Ala Ala Lys Lys Arg Leu Leu Leu Phe Ser Asp     930 935 940 Gly Asn Ser Gln Gly Ala Thr Pro Ala Ala Ile Glu Lys Ala Val Gln 945 950 955 960 Glu Ala Gln Arg Ala Gly Ile Glu Ile Phe Val Val Val Val Gly Arg                 965 970 975 Gln Val Asn Glu Pro His Ile Arg Val Leu Val Thr Gly Lys Thr Ala             980 985 990 Glu Tyr Asp Val Ala Tyr Gly Glu Ser His Leu Phe Arg Val Pro Ser         995 1000 1005 Tyr Gln Ala Leu Leu Arg Gly Val Phe His Gln Thr Val Ser Arg Lys    1010 1015 1020 Val Ala Leu Gly 1025 <210> 3 <211> 434 <212> PRT <213> Homo sapiens <400> 3 Met Ser Ile Leu Lys Ile His Ala Arg Glu Ile Phe Asp Ser Arg Gly   1 5 10 15 Asn Pro Thr Val Glu Val Asp Leu Phe Thr Ser Lys Gly Leu Phe Arg              20 25 30 Ala Ala Val Pro Ser Gly Ala Ser Thr Gly Ile Tyr Glu Ala Leu Glu          35 40 45 Leu Arg Asp Asn Asp Lys Thr Arg Tyr Met Gly Lys Gly Val Ser Lys      50 55 60 Ala Val Glu His Ile Asn Lys Thr Ile Ala Pro Ala Leu Val Ser Lys  65 70 75 80 Lys Leu Asn Val Thr Glu Gln Glu Lys Ile Asp Lys Leu Met Ile Glu                  85 90 95 Met Asp Gly Thr Glu Asn Lys Ser Lys Phe Gly Ala Asn Ala Ile Leu             100 105 110 Gly Val Ser Leu Ala Val Cys Lys Ala Gly Ala Val Glu Lys Gly Val         115 120 125 Pro Leu Tyr Arg His Ile Ala Asp Leu Ala Gly Asn Ser Glu Val Ile     130 135 140 Leu Pro Val Pro Ala Phe Asn Val Ile Asn Gly Gly Ser His Ala Gly 145 150 155 160 Asn Lys Leu Ala Met Gln Glu Phe Met Ile Leu Pro Val Gly Ala Ala                 165 170 175 Asn Phe Arg Glu Ala Met Arg Ile Gly Ala Glu Val Tyr His Asn Leu             180 185 190 Lys Asn Val Ile Lys Glu Lys Tyr Gly Lys Asp Ala Thr Asn Val Gly         195 200 205 Asp Glu Gly Gly Phe Ala Pro Asn Ile Leu Glu Asn Lys Glu Gly Leu     210 215 220 Glu Leu Leu Lys Thr Ala Ile Gly Lys Ala Gly Tyr Thr Asp Lys Val 225 230 235 240 Val Ile Gly Met Asp Val Ala Ala Ser Glu Phe Phe Arg Ser Gly Lys                 245 250 255 Tyr Asp Leu Asp Phe Lys Ser Pro Asp Asp Pro Ser Arg Tyr Ile Ser             260 265 270 Pro Asp Gln Leu Ala Asp Leu Tyr Lys Ser Phe Ile Lys Asp Tyr Pro         275 280 285 Val Val Ser Ile Glu Asp Pro Phe Asp Gln Asp Asp Trp Gly Ala Trp     290 295 300 Gln Lys Phe Thr Ala Ser Ala Gly Ile Gln Val Val Gly Asp Asp Leu 305 310 315 320 Thr Val Thr Asn Pro Lys Arg Ile Ala Lys Ala Val Asn Glu Lys Ser                 325 330 335 Cys Asn Cys Leu Leu Leu Lys Val Asn Gln Ile Gly Ser Val Thr Glu             340 345 350 Ser Leu Gln Ala Cys Lys Leu Ala Gln Ala Asn Gly Trp Gly Val Met         355 360 365 Val Ser His Arg Ser Gly Glu Thr Glu Asp Thr Phe Ile Ala Asp Leu     370 375 380 Val Val Gly Leu Cys Thr Gly Gln Ile Lys Thr Gly Ala Pro Cys Arg 385 390 395 400 Ser Glu Arg Leu Ala Lys Tyr Asn Gln Leu Leu Arg Ile Glu Glu Glu                 405 410 415 Leu Gly Ser Lys Ala Lys Phe Ala Gly Arg Asn Phe Arg Asn Pro Leu             420 425 430 Ala lys          

Claims (7)

Kit for diagnosing glaucoma containing a vasosin-containing protein (VCP). delete The kit of claim 1, wherein the glaucoma is open angle glaucoma. A method for detecting autoantibodies to valosin-containing proteins (VCPs) as glaucoma markers by antigen-antibody reactions from human samples to provide information necessary for glaucoma diagnosis. delete 5. The method of claim 4, wherein the glaucoma is open angle glaucoma. 5. The method of claim 4, wherein said sample is blood, serum, or plasma.

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