KR20100087275A - Kits for glaucoma diagnosis - Google Patents

Abstract

PURPOSE: A kit for diagnosing glaucoma and a method for diagnosing glaucoma using the same are provided to easily and quickly determine occurrence of glaucoma. CONSTITUTION: A kit for diagnosing glaucoma contains collagen type VI. The kit for diagnosing glaucoma contains an antigen which specifically binds to collagen type VI. The glaucoma is open-angle glaucoma. A method for diagnosing glaucoma comprises a step of reacting antigen-antibody reaction from human sample and detecting autoantibody to collagen type VI. The sample is blood, serum, or plasma.

Description

Kits for Glaucoma Diagnosis{Kits 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 sclerosis, retinal atrophy, nipple depression, and blindness may also appear. The intraocular pressure is maintained only when a fluid called waterproofing is created in the eye and discharged out of the eye at a constant rate. When there is an abnormality in the generation and discharge path of such waterproofing, the intraocular pressure rises. It results in impaired consequences. Depending on the degree of damage to the optic nerve, dark spots occur in the field of view, and as the disease progresses, blindness occurs. Such glaucoma is the most common form of open angle glaucoma (OAG), and it occurs mainly after the 40s, and the incidence of glaucoma increases with age. Glaucoma has no subjective symptoms in the early stage, and is often detected only after the disease has worsened, making it impossible to recover. Although glaucoma is not always accompanied by ocular hypertension, many glaucoma patients often exhibit intraocular pressure in excess of 21 mmHg, and long-term steroid injection after myopic surgery with excimer is accompanied by an increase in intraocular pressure in some patients. It can also cause steroid-induced glaucoma.

Glaucoma is not caused by everyday lifestyles, is not contagious and is not life-threatening, but, along with diabetic retinopathy, is a terrifying eye disease that most often causes blindness. In particular, once an onset of glaucoma occurs, the damaged optic nerve cannot be restored again, so early diagnosis and early treatment are important. Even now, many ophthalmologists are studying the cause for identification and effective treatment, but the exact etiology of glaucoma is not yet known. However, it has recently been reported that genetic factors play an important role in the pathogens of glaucoma. In addition, studies on the linkage of glaucoma to autoimmunity have been conducted, but it is only showing the diagnosis of the pattern of autoantibody reactions, not the diagnosis using specific proteins.

Throughout this specification, a number of papers and patent documents are referenced and citations are indicated. The disclosure contents of cited papers and patent documents are incorporated by reference in this specification as a whole, and the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly described.

The present inventors made efforts to develop a novel biomarker related to the onset of glaucoma, and finally, by identifying three types of autoantibodies generated specifically for glaucoma, the present invention was completed.

Accordingly, an object of the present invention is to provide a kit for diagnosing 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 by the following detailed description, claims and drawings.

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

According to another aspect of the present invention, the present invention provides information necessary for glaucoma diagnosis through an antigen-antibody reaction from a human sample, as a glaucoma marker VCP (valosin-containing protein), collagen type VI or α-enola. It provides a method of detecting an autoantibody against a drug.

The present inventors made efforts to develop novel biomarkers related to the onset of glaucoma, and finally identified three autoantibodies that are produced specifically for glaucoma.

The present invention allows molecular diagnosis of glaucoma. The term “glaucoma” as used herein refers to a chronic disease in which the optic nerve is damaged. More specifically, when there is an abnormality in the production and discharge path of waterproofing in the eye, the intraocular pressure rises, and when the intraocular pressure increases, the optic nerve nipple is pushed back, resulting in damage to the optic nerve. Dark spots occur in the disease, and as the disease progresses gradually, blindness occurs. In addition, changes in the optic nerve papilla and visual field 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, it has been found that normal tension glaucoma occurs at a high frequency in Asians, including Koreans and Japanese. Symptoms of glaucoma include sclerosis, retinal atrophy, nipple depression, and blindness.

In the present invention, glaucoma is determined by a molecular diagnosis method, not by a doctor's findings.

The glaucoma-specific biomarker presented by the present invention is an autoantibody against valosin-containing protein (VCP), collagen type VI or α-enolase. According to the present invention, when glaucoma occurs, autoantibodies against VCP, collagen type VI or α-enolase are generated in the human body, and the present invention detects such autoantibodies to determine glaucoma. The detection of autoantibodies can be performed using antigens that specifically bind to autoantibodies, ie, VCP, collagen type VI or α-enolase, or their antigenic determining sites.

In the present specification, the term "antigen determinant" is used with the same meaning for an epitope, and refers to a portion of an antigen specifically recognized by a specific antibody. The epitope typically consists of a sequence of 3-30 amino acids. The epitope of VCP, collagen type VI or α-enolase that can be used in the present invention can be easily determined by studying the position where autoantibodies that bind to 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 derived from humans, cows, pigs, horses, mice, rats and rabbits, and most It is preferably derived from humans. Specific examples of the VCP, collagen type VI and α-enolase used in the present invention are exemplified in the first sequence, the second sequence, and the third sequence, respectively.

The autoantibody-specific antigen included in the kit of the present invention can be prepared through various methods known in the art. For example, the three antigens can be obtained through a gene recombination method.

An example of a method of obtaining the VCP, collagen type VI or α-enolase antigen used in the present invention through a gene recombination method is as follows: VCP, collagen type VI or α-enolase-encoding nucleotide sequence is included. Prepare the expression vector. Vectors can be constructed using prokaryotic or eukaryotic cells as a host. In the case of using prokaryotic cells as a host, the vector is a strong promoter capable of performing transcription (eg, 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.), ribosome binding sites for initiation of translation, and transcription/translation termination sequences. When E. coli is used as a host cell, the promoter and operator site of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol. , 158:1018-1024 (1984)) and the left-handed promoter of phage λ (p. _{The L} ^λ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet. , 14:399-445 (1980)) can be used as a regulatory site. Meanwhile, vectors that can be used in the present invention include plasmids that are often used in the art (eg pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), phage (eg, λgt4?λB, λ-Charon). , λΔz1 and M13, etc.) or a virus (eg, SV40, etc.).

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

The vector used in the present invention may be fused with other sequences in order 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 the like, most preferably Is 6x His. Because of the additional sequence for this purification, the protein expressed in the host is rapidly and easily purified through affinity chromatography.

Host cells capable of stably and continuously cloning and expressing the vector are known in the art, and any host cell can be used, for example, E. coli JM109, E. coli BL21 (DE3), E. coli RR1, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and other strains of the genus Bacillus, as well as Salmonella typhimurium, Serratia marsessons and various Pseudomonas species There are the same enterobacteriaceae and strains. In addition, when the vector is transformed into eukaryotic cells, as host cells, yeast ( Saccharomyce cerevisiae ), insect cells and human cells (e.g., CHO cell line (Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines) and the like can be used.

The method of transporting the vector into the host cell is CaCl ₂ method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9:2110-2114 (1973)), if the host cell is a prokaryotic cell. One method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA , 9:2110-2114 (1973); And Hanahan, D., J. Mol. Biol. , 166:557-580 (1983) ) And an electroporation method (Dower, WJ et al., Nucleic. Acids Res. , 16:6127-6145 (1988)). In addition, when the host cell is a eukaryotic cell, microinjection (Capecchi, MR, Cell , 22:479 (1980)), calcium phosphate precipitation (Graham, FL et al., Virology , 52:456 (1973)), Electroporation (Neumann, E. et al., EMBO J. , 1:841 (1982)), liposome-mediated transfection (Wong, TK et al., Gene , 10:87 (1980)), DEAE- Dextran treatment (Gopal, Mol. Cell Biol. , 5:1188-1190 (1985)), and Gene Bombadment (Yang et al., Proc. Natl. Acad. Sci. , 87:9568-9572 (1990)) ), etc., the vector can be injected into the host cell.

The vector injected into the 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 the lac promoter, the host cell may be treated with IPTG to induce gene expression.

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

The kit of the present invention can be applied to a variety of glaucoma, preferably congenital glaucoma, traumatic glaucoma, glaucoma prognosis, ocular hypertension, primary open right angle glaucoma, normal pressure glaucoma, capsular cystic glaucoma with false drop of the lens, chronic simple Glaucoma, low pressure glaucoma, pigmented glaucoma, primary obstructive right angle glaucoma, acute obstructive right angle glaucoma, chronic obstructive right angle glaucoma, intermittent obstructive right angle glaucoma, secondary glaucoma secondary to eye trauma, glaucoma secondary to eye inflammation, or secondary to drugs It is applied to glaucoma, more preferably to primary open right angle glaucoma or normal pressure glaucoma.

The glaucoma diagnosis method of the present invention can be performed 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 is basically an immunoassay method using an antigen-antibody reaction (e.g., a radioactive immunoassay method, a radioactive immuno-precipitation method, an enzyme-linked immunosorbent method (ELISA), dot blot analysis, western blot). , Inhibition or competition assays and Sanswitch assays) quantitatively or qualitatively (see 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, if the radioimmunoassay method is followed, a marker in the sample (e.g., serum), i.e., VCP, collagen type, using an antibody labeled with a ^{radioisotope (e.g., I 125} , P ³² , S ³⁵⁾ By measuring the production of an immune complex formed by binding with an autoantibody against VI or α-enolase, autoantibodies in a sample can be qualitatively or quantitatively detected.

In addition, in the case of the ELISA method, (i) adsorbing an antigen (ie, VCP, collagen type VI or α-enolase, or an epitope thereof) having the ability to bind to an autoantibody in the sample to a well plate; (Ii) adding a sample to the well plate for reaction and washing; (Iii) reacting by adding a secondary antibody to which an enzyme or a fluorescent substance is conjugated; And (iv) measuring the binding of the secondary antibody.

Enzymes bound to the secondary antibody include, but are not limited to, enzymes that catalyze a color development reaction, a fluorescence reaction, a luminescence reaction, or an infrared reaction, and examples include alkaline phosphatase, β-galactosidase, and hose. Radish peroxidase, luciferase and cytochrome P ₄₅₀ . When alkaline phosphatase is used as the enzyme that binds to the secondary antibody, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate (naphthol-AS) is used as a substrate. -B1-phosphate) and ECF (enhanced chemifluorescence) are used, and when horse radish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis -N-methylacridinium nitrate), resorupine benzyl ether, luminol, amplex red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o -phenylenediamine (OPD) and naphthol/pyronine, glucose oxidase and t-NBT (nitroblue tetrazolium) and m-PMS Substrates such as (phenzaine methosulfate) may be used.

Meanwhile, the kit of the present invention may be developed in a rapid kit method using a strip-type solid substrate. Rapid kits are generally solid substrates (e.g., glass, fiberglass, metals (e.g., gold, alloys of gold and copper, aluminum), metal oxides, ceramics, quartz, silicon, semiconductors, Si/SiO ₂ wafers, germanium, A liquid sample flows or moves in gallium arsenide, carbon, carbon nanotubes, polymers (eg, polystyrene, polyethylene, polypropylene, polyacrylamide), sepharose, and agarose. Typically, the rapid kit is referred to as a chromatographic analysis method. For example, the autoantibody contained in the sample's serum reacts with the antigen bound to the gold particles and then moves through the micropores of the nitrocellulose membrane by capillary action, while a capture antibody that is immobilized on the inner surface of the micropores (capture antibody) to form a color band, thereby visually discriminating 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, saliva, milk, urine, feces, ocular fluid, semen, thymus, ascites and amniotic fluid and 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, based on the data produced by the method of the present invention, glaucoma-related drugs can be treated to a patient.

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

(I) The present invention is based on the discovery that an autoantibody against VCP, collagen type VI or α-enolase serves as a biomarker for glaucoma diagnosis.

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

(Iii) The present invention can easily and quickly determine whether or not glaucoma occurs.

1 shows the results of Western blotting for serum of a normal person, an open-angle glaucoma patient, a normal pressure glaucoma patient, and a glaucoma symptom of a bovine optic nerve protein. In FIG. 1, the arrows indicate proteins expressed in the glaucoma patient group when compared to the normal person, and S-2 indicates the protein that appears only in the glaucoma patient group when compared to other ophthalmic diseases.
2 shows the results of CBB staining by two-dimensional electrophoresis on bovine optic nerve proteins. In FIG. 2, the numbers indicate spots that differ between normal and patient groups by Western blotting after 2D electrophoresis on bovine optic nerve proteins.
3 shows the results of Western blotting using serum from normal and glaucoma patients after 2D electrophoresis using bovine optic nerve protein. In the figure, A is a control group, and B shows spots showing differences in glaucoma patients group (open angle glaucoma patients and normal pressure glaucoma patients) when compared to normal subjects.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Example 1: Analysis of autoantibodies against bovine optic nerve protein using western blotting

Protein isolation in bovine optic nerve

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

Western Blotting of Proteins for Serum of Normal Persons and Patients

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 subjected to electrophoresis on a 9% acrylamide gel, transferred to a nitro-cellulose membrane, and blocked using skim milk powder. Serum obtained from peripheral blood of normal individuals and each patient group as a primary antibody was attached to the nitro-cellulose membrane, and then the secondary antibody was attached to the membrane using human immunoglobin G bound to horseradish peroxidase. , Autoantibodies were confirmed using a chemiluminescence substrate kit (GenDEPOT, USA). The results are summarized in Fig. 1 and Table 1. In Table 1, + indicates that the protein was expressed, and-indicates that the protein was not expressed. In addition, the number of + indicates the degree of protein expression.

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

POAG: patients with primary open angle glaucoma

NTG: Normal tension glaucoma patients

Example 2: Two-dimensional electrophoresis and western blotting using bovine optic nerve protein

The optic nerve protein was isolated through two-dimensional electrophoresis (Park et al., Proteomics , 4(11):3446-3455(2004)). First, electrophoresis was performed using an IPG strip (Amersham Pharmacia, USA) having a pH range of 4-7, and secondly, separation was performed using a 9% acrylamide gel containing SDS. After two-dimensional electrophoresis, one gel was stained with CBB (Colloidal Coomassie Blue G-250 solution, ProteomeTech, Korea), and the image was analyzed through an image analyzer (GS-710 imaging calibrated densitometer, Bio-Rad, USA). Then, the rest of the gel was subjected to Western blotting, and ^{the position of the autoantibodies was confirmed through image software (ImageMaster™} 2D Platinum software, Amersham Biosciences, USA) for protein comparison on the gel.

Example 3: Protein identification

Unlike normal subjects after Western blotting, specific antibody bands appearing in glaucoma patients and spots appearing in glaucoma patients, unlike normal subjects, were cut out from the gel and identified through Western blotting performed after secondary electrophoresis. First, after washing the cut-out gel pieces, the CBB stained on the gel was removed through 50% acetonitrile/25 mM ammonium bicarbonate solution, and then the gel was dehydrated and treated with trypsin (Promega, USA) to rehydrate the gel Next, ammonium bicarbonate buffer solution was added to extract the trypsip-peptide. After desalting the extracted peptide, the protein was identified through an HP 1100 HPLC nano-flow system (Agilent, CA) to which an LCQ DECA ion trap (Thermo Fisher Scientific, San Jose, CA) was connected. The resin used was Zorbax 300SB-C18 resin (particle size 5 μm, Agilent, CA) packed 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 the MS/MS spectrum was converted into a data format using Bioworks 3.3 software (Thermo Fisher Scientific, USA), and protein identification was performed using a database of the National Center for Biotechnology Information (NCBI). Antigen proteins for autoantibodies that are specifically shown in glaucoma patients identified in this experiment are summarized in Table 2.

Spot number Protein name s-1 Collagen type 6 (collagen type VI) s-2 Valosin-containing protein (VCP) s-3, 4 α-enolase

As described above, specific parts of the present invention have been described in detail, and it is clear that these specific techniques are only preferred embodiments for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Accordingly, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

<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 (5)

Kit for diagnosing glaucoma comprising collagen type VI.
The kit of claim 1, wherein the glaucoma is open angle glaucoma.
A method for detecting autoantibodies to collagen type VI as glaucoma markers via antigen-antibody reactions from human samples to provide information necessary for diagnosing glaucoma.
4. The method of claim 3, wherein the glaucoma is open angle glaucoma.
4. The method of claim 3, wherein said sample is blood, serum, or plasma.

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