KR100609006B1 - Method for measuring quantity of ???­?? protein and diagnosis kit using the same - Google Patents

Abstract

The present invention relates to a method for quantifying βig-h3 protein and a diagnostic kit using the same. Specifically, a recombinant protein and an antibody thereof for the pars-1 domain of βig-h3 protein or βig-h3 are used in an antigen-antibody reaction method. Methods for measuring the amount of βig-h3 protein contained in the test sample body fluid; kidney disease, liver disease, and rheumatic disease, including recombinant proteins and antibodies thereof against the pars-1 domain of βig-h3 protein or βig-h3 It is about a diagnostic kit. Βig-h3 quantification method and diagnostic kit of the present invention can be usefully used as an effective test method that reflects the sensitivity and progression of various diseases early and sensitively, including kidney disease, liver disease and rheumatic diseases.

Description

Method for measuring quantity of βｉｇ­ｈ３ protein and diagnosis kit using the same             

1 is a schematic diagram showing the structure of βig-h3 recombinant protein,

I, II, III and IV: each domain,

▨ and ▩: region where the nucleotide sequence is conserved

A; βig-h 3, B; Human βig-h3, C; Mouse βig-h3

2 is a schematic diagram showing the geometry of βig-h3 D-IV proteins recombined by repetition of the IV domain of βig-h3,

A; βig-h 3, B; βig-h 3 D-IV (1 ×), C; βig-h3 D-IV (2x),

D; βig-h3 D-IV (3x), E; βig-h3 D-IV (4x)

3 is an electrophoresis photograph of the isolated βig-h3 recombinant protein,

One ; Human βig-h3, 2; Mouse βig-h3

Figure 4 is a photograph confirming the βig-h3 D-IV (1x, 2x, 3x, 4x) proteins by electrophoresis method,

One ; βig-h 3 D-IV (1 ×), 2; βig-h3 D-IV (2x),

3; βig-h3 D-IV (3x), 4; βig-h3 D-IV (4x)

5 is an electrophoretic photograph showing the results of identifying human βig-h3 and mouse βig-h3 by western blot using a primary antibody.

One ; Human βig-h3, 2; Mouse βig-h3

6 is a schematic diagram showing the principle of an enzyme-linked immunosorbent assay (ELISA),

7 is a graph showing the quantitative ratio of primary antibodies,

◆; 1: 200, ■; 1: 400, ▲; 1: 800,

×; 1: 1600, ※; 1: 2000, ●; 1: 3200

8 is a graph showing the quantitative ratio of secondary antibodies,

A; Primary antibody fixed at 1: 1600,

B; Primary antibody fixed at 1: 2000,

◆; Dilute secondary antibody to 1: 1000,

■; Dilute secondary antibody to 1: 2000,

●; Dilute secondary antibody to 1: 3000

9 is a graph showing the coating concentration of human βig-h3 protein,

◆; 0.5 µg / ml; 1.0 μg / ml

10 is a graph showing that human βig-h3 protein and mouse βig-h3 protein can be used as standard proteins through cross-over experiments.

◆; Human βig-h3 protein coating concentration 0.5 μg / ml, primary anti human βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

■; Human βig-h3 protein coating concentration 0.5 μg / ml, primary anti mouse βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

▲; Mouse βig-h3 protein coating concentration 0.5 μg / ml, primary anti human βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

×; Mouse βig-h3 protein coating concentration 0.5 μg / ml, primary anti mouse βig-h3 antibody 1: 2000, secondary antibody 1: 2000

11 is a graph showing that recombinant βig-h3 D-IV (1x) protein and recombinant βig-h3 D-IV (4x) protein can be used as standard proteins through cross-over experiments.

◆ of A; βig-h3 D-IV (1x) coating concentration 0.5 μg / ml, primary anti human βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

■ of A; βig-h3 D-IV (4x) coating concentration 0.5 μg / ml, primary anti human βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

◆ of B; βig-h3 D-IV (1x) coating concentration 0.5 μg / ml, primary anti mouse βig-h3 antibody 1: 2000, secondary antibody 1: 2000,

■ of B; βig-h3 D-IV (4x) coating concentration 0.5 μg / ml, primary anti mouse βig-h3 antibody 1: 2000, secondary antibody 1: 2000

12 is an immunohistochemical staining picture showing the expression of βig-h3 in the kidney tissue,

▶ of A; Expression patterns in the basal membrane of S3 proximal tubular cells,

▶ of B; Expression of Glomerular Bowman's Capsule in the Basal Membrane

B of; Expression of Basal Membrane in Cortical Thick Ascending Limb Cells

Figure 13 is a graph showing the results of measuring βig-h3 in the urine of diabetic rats,

■; Control,

□; Diabetic rats treated with streptozotocin

14 is a graph showing the results of measuring βig-h3 in the urine of the diabetic rat of FIG. 13 for each individual,

FIG. 15 shows a case in which normal kidney, transplanted kidney size is small (Nephron underdose), chronic rejection, recurrent GN, and cyclosporine toxicity are shown. This is a graph measuring the concentration of βig-h3 protein in urine

FIG. 16 is a graph measuring the concentration of βig-h3 protein according to the date of a patient undergoing plasma exchange therapy after focal segmental glomerulosclerosis (FSGS) relapse after renal transplantation.

Figure 17 shows before and after renal transplantation in the urine of living (Living donor) or deceased (Cadaver donor), the kidney of the transplanted kidney is small (Underdose) or rejected (Rejection) patients It is a graph measuring the concentration of βig-h3 protein.

The present invention relates to a method for quantifying βig-h3 protein and a diagnostic kit using the same, and specifically, a recombinant protein and a antibody thereof for the pars-1 domain of βig-h3 protein or βig-h3 to be tested for antigen-antibody reaction. Methods for measuring the amount of βig-h3 protein contained in the sample body fluid and diagnosis of kidney disease, liver disease, and rheumatic diseases, including recombinant proteins and antibodies thereof against the pars-1 domain of βig-h3 protein or βig-h3 It's about kits.

βig-h3 is found in many types of cells, including human melanoma cells, mammalian epithelial cells, keratinocytes and lung fibroblasts. It is an extracellular matrix protein induced by TGF-β. The transforming growth factor-β (TGF-β) is a substance involved in the growth and differentiation of various kinds of cells. In mammals, three kinds of TGF-β1, TGF-β2 and TGF-β3 exist, and growth regulation and immunity It is known to have a variety of complex functions, such as regulating responses, stimulating bone formation, inducing cartilage specific macromolecules, and promoting wound healing (Bennett, NT et al., Am. J. Surg . 1993) . , 165, 728). This TGF-β is expressed in epithelial cells of wound healing, which has been shown to stimulate the expression of integrin molecules in keratinocytes during re-epithelialization. In recent studies on TGF-β expression, TGF-β mRNA is expressed in the epithelium of normal skin or epithelial layer regenerated in acute and chronic wounds, while TGF-β1 mRNA is not expressed in normal skin and chronic wounds but regenerated in acute wounds. It was found that in the epithelial layer, TGF-β2 mRNA is not expressed (Schmid, P. et al., J. Pathol ., 1993 , 171, 191). Although no orthodoxy has yet been established for this mechanism, TGF-β is expected to play a large role in re-epithelialization.

Βig-h3, which is known as a related gene of TGF-β, was first identified by Stonier et al., And cDNA library screening in A549 cell line, a human lung adenocarcinoma cell line treated with TGF-β1. The data were identified during screening and reported to show a 20-fold increase in two days after treatment with TGF-β1 (Stonier, J, et al., DNA cell Biol ., 1992 , 11, 511). By DNA sequencing, the βig-h3 protein is a carboxy-terminal Arg-Gly-Asp (RGD) capable of ligand recognition of amino-terminal secretory sequences and several integrins. It was found that it consists of 683 amino acids set forth in SEQ ID NO: 1 having the sequence .

βig-h3 contains four internal repeated domains homologous with RGD motifs, which are secreted proteins of various species including mammals, insects, sea urchins, plants, yeast and bacteria Or as highly conserved sequences in membrane proteins. Proteins including the conservative sequence include periostin, fasciclin I, sea urchin HLC-2, algal-CAM and mycobacterium MPB70. Included. (Kawamoto, T. et al., Biochem. Biophys. Acta ., 1998 , 1395, 288). The homologous domains found conservatively in these proteins (hereinafter abbreviated as "fas-1 domains") are two highly conserved branches consisting of 110 to 140 amino acids and about 10 amino acids. (H1 and H2). Of these proteins, βig-h3, periostin and pascycline I have four pars-1 domains, two HLC-2s and only one pars-1 domain MPB70. Although the biological function of these proteins is not known precisely, some of them are known to mediate cell attachment and detachment as cell adhesion molecules. For example, βig-h3, periostin, and pascycline I have been reported to mediate the attachment of fibroblasts, osteoblasts and neurons, respectively, and algal-CAM has been found in the embryos of avian Volvolx. It has been found to be a cell adhesion molecule present (LeBaron, RG, et al., J. Invest. Dermatol ., 104, 844, 1995; Horiuchi, K. et al., J. Bone Miner. Res ., 1999 , 14, 1239; Huber, O. et al., EMBO J. , 1994 , 13, 4212).

Purified βig-h3 protein promotes adhesion and proliferation of dermal fibroblasts while inhibiting the adhesion of A549, HeLa and WI-38 cells in serum-free medium. In particular, βig-h3 is known to inhibit the growth, colony formation and appearance of tumor cells, for example, in nude mice transfected with βig-h3 expression vector in Chinese hamster ovary cells Significantly reduced tumorigenic capacity of these cells is disclosed in US Pat. Nos. 5,599,788 and 5,714,588. Also disclosed in these patents are methods for promoting the spread and adhesion of cells, particularly fibroblasts, to wounds by contacting the wound with an effective amount of βig-h3 for quick and effective wound healing. Thus, βig-h3 is a cell adhesion molecule that is highly induced by TGF-β in many cells, resulting in cell growth, cell differention, wound healing, morphogenesis and cell adhesion. plays a very important role in cell adhesion.

As described above, βig-h3 is a substance that is expected to be useful in various ways, but only a very small amount can be collected in vivo. In order to solve this problem, a method of producing and expressing in a eukaryotic cell system by genetic recombination has been developed.However, when expressed in a eukaryotic cell system, cells that produce βig-h3 are much slower than cells that do not produce cells. It is difficult to secure a positive cell number. Therefore, the present inventors have established a purification method for mass expression of a recombinant protein including all and part of the domain of βig-h3 protein using Escherichia coli as a host, and confirmed that the recombinant protein supports cell attachment and diffusion. (Korean Patent Application No. 2000-25664).

On the other hand, cell adhesion activity of βig-h3 as a cell adhesion molecule was first reported in human dermal fibroblasts, and then also in chondrocytes, peritoneal fibroblasts, and human MRC5 fibroblasts. It became. This cell adhesion activity of βig-h3 was initially thought to be mediated by the RGD motif present at the carboxyl-terminus of βig-h3, but the RGD motif is not necessary to promote the dispersion of chondrocytes and is a carboxyl-terminal pro RGD motifs are not indispensable for mediating the cell adhesion activity of βig-h3 as it has been reported that carboxyl-terminus processing can inhibit cell adhesion by the mature form of βig-h3, which lacks the RGD motif. This was confirmed. In addition, recent studies have shown that βig-h3 increases the dispersion of fibroblasts through integrin α1β1, whereas the RGD motif of βig-h3 is not required for βig-h3-mediated cell dispersion, and βig-h3 independently integrins. It has been found that it can interact with α1β1 to promote cell adhesion and expansion (Ohno, S., et al., Biochim. Biophys. Acta, 1999 , 1451, 196). In addition, conservative H1 and H2 peptides in βig-h3 were also found to have no effective effect on βig-h3-mediated cell adhesion. These results indicate that essential amino acids for the cell adhesion activity of βig-h3 are present in regions other than the H1 and H2 regions, as well as the repetitive parse-1 domain of βig-h3 as well as the parse-1 domains of other proteins. Computational analysis of homology comparisons also revealed that in addition to H1 and H2, there are several highly conserved amino acids that may be involved in cell adhesion activity.

Therefore, the present inventors have studied to identify conservative motifs involved in cell adhesion and desorption activity and to prepare peptides containing the same, and as a result, functional cell receptors using the second and fourth domains of βig-h3, known as cell adhesion molecules, have been identified. As peptides, peptides NKDIL and EPDIM and derivatives thereof that bind to α3β1 integrin to mediate cell adhesion and desorption activity, and are aspartic, two highly conserved amino acids located near the H2 region in the second and fourth domains of βig-h3 An acid (aspartic acid, Asp) and isoleucine (isoleucine, Ile) has been applied for a patent application has been found to act as an essential amino acid for cell adhesion and desorption activity (Korean Patent Application No. 2000-25665).

On the other hand, until now, βig-h3 has not been reported to be directly related to other diseases except βig-h3 in relation to some tumors in humans. In particular, βig-h3 protein expression, kidney disease, liver disease and There is no known direct relationship with the onset of rheumatic disease, and there has been no report on the possibility of linking the disease by measuring the amount of βig-h3 protein in body fluids.

Therefore, the present inventors quantitate a method of βig-h3 using a recombinant protein by connecting several fourth pars-1 domains of βig-h3 or βig-h3 as a standard protein for measuring βig-h3, and βig using the same. The present invention has been developed by developing a diagnostic kit for quantifying -h3 and using the above method to detect acutely various diseases including kidney disease, liver disease and rheumatism and to determine the prognosis of response to treatment. Completed.

An object of the present invention is to provide a method for quantifying βig-h3 protein using a recombinant protein for the pars-1 domain of βig-h3 protein or βig-h3 and a diagnostic kit using the above quantitative method.

In order to achieve the above object, the present invention provides a method for quantifying βig-h3 protein.

The present invention also provides a diagnostic kit for renal disease, liver disease and rheumatic disease using the quantitative method of βig-h3 protein.

Hereinafter, the present invention will be described in detail.

The quantification method of βig-h3 of the present invention

1) preparing a recombinant protein for the pars-1 domain of βig-h3 protein or βig-h3 (step 1);

2) preparing an antibody against the protein of step 1 (step 2); And

3) measuring the amount of βig-h3 protein contained in the test sample by using the protein of step 1 and the antibody of step 2 in an quantitative method using an antigen-antibody reaction (step 3).

The βig-h3 protein in step 1 is a human βig-h3 protein having an amino acid sequence of SEQ ID NO: 3 or a mouse βig-h3 protein having an amino acid sequence of SEQ ID NO: 5 . The βig-h3 protein components of human and mouse are shown in FIG . 1, in which the hatched and cross hatched cells represent the highly conserved sequences of repeating Pars-1 domains I, II, III, and IV. Blanks indicate RGD motifs.

The βig-h3 protein has four parse-1 domains, wherein the parse-1 domain of βig-h3 in step 1 is one or more domains selected from the first to fourth parse-1 domains of the βig-h3 protein. Can be used, and it is preferable that the fourth parse-1 domain is used. In addition, the fourth parse-1 domain may be used alone or as a recombination protein of several repeats, preferably 1 to 10 linkages, and more preferably using 1 to 4 linkages. In the present invention, as a preferred embodiment, the case of using a protein recombined by linking one, two, three or four fourth domains of βig-h3 is illustrated.

In the present invention, amino acids described in SEQ ID NO: 7 , SEQ ID NO: 8 , SEQ ID NO: 9, and SEQ ID NO: 10 each including one to four fourth pars-1 domains 502 to 632 in the amino acid sequence of βig-h3. Proteins having sequences were prepared, which were "βig-h3 D-IV (1x)", "βig-h3 D-IV (2x)", "βig-h3 D-IV (3x)" and "βig-h3", respectively. D-IV (4x) "(see Figure 4 ).

In preparing the standard protein of the present invention, the expression vector and the transformation may be performed by conventional methods.

The antibody to the βig-h3 protein or the pars-1 domain of βig-h3 in step 2 was prepared as a primary antibody using the protein or recombinant protein of step 1 as an antigen. The preparation of the primary antibody can be carried out by conventional methods, and monoclonal antibodies or polyclonal antibodies can be used.

In order to measure the amount of βig-h3 protein contained in the test sample of step 3, all quantitative methods based on antigen-antibody binding can be used, and immunoblotting ( Current Protocols in Molecular Biology , vol 2, chapter) 10.8; David et al., Cells (a Laboratory manual) , vol 1, chapter 73), Current Protocols in Molecular Biology , vol 2, chapter 10.16; Cells (a Laboratory manual) , vol 1, chapter 72) , Current Protocols in Molecular Biology , vol 2, chapter 11.2; ELISA Theory and Practice , John R. Crowther; The ELISA Guidebook, John R. Crowther), Radioimmuno assay (RIA) (Nuklearmedizin 1986 Aug; 25 (4) 125-127, Tumor markers as target substances in the radioimmunologic detection of malignancies.von Kleist S; Mariani G. Ann Oncol 1999; 10 Suppl 4: 37-40), protein chips (Daniel Figeys et.al , Electrophoresis 2001, 22 Albala JS. Expert Rev Mol Diagn 2001 Jul; 1 (2): 145-152), rapid assay (Kasahar) a Y and Ashihara Y, Clinica Chimica Acta 267 (1997), 87-102; Korean Patent Application 2000-46639) or microarray (Vivian G. cheung et al, Nature genetics 1999, 21, 15-19; Robert J. Lipshutz et al , Nature genetics 1999, 21, 20-24; Christine Debouck and Peter N. Goodfellow, Nature genetics 1999, 21, 48-50; It is preferable to use a method selected from the group consisting of DNA Microarrays , M. Schena, and more preferably to use an ELISA method. In addition to the ELISA method, known biological microchips and automated microarray systems can be used to analyze samples in large quantities, and in urine can be developed as a convenient self-diagnosis method. have.

According to an embodiment of the present invention, measuring the amount of βig-h3 protein by a competition assay using an ELISA among the various methods listed above

1) coating the substrate with a βig-h3 protein or a recombinant protein for the pars-1 domain of βig-h3 (step 1);

2) reacting the antibody against the protein of step 1 with a test sample (step 2);

3) adding the reactant of step 2 to the encoded protein of step 1, reacting and washing (step 3); And

4) adding a secondary antibody to the reactant of step 3 to react and measuring absorbance (step 4).

The substrate of step 1 may be any substrate that is commonly used, nitrocellulose membrane, a plate synthesized with a polyvinyl resin (e.g. 96 well plate), a well plate synthesized with a polystyrene resin and Glass slide glass and the like can be used.

In addition, the secondary antibody of step 4 is used by labeling the enzyme, fluorescent material, luminescent material, radioactive material or metal chelate. The labeling substance can be used for all commonly used, and the chromophore is peroxidase, alkaline phosphatase, β-D-galactosidase, malate dehydrogenase, staphylococcus nuclease, It is preferable to use horseradish peroxidase, catalase, acetylcholine esterase, etc., and fluorescent materials include isothiocyanate (fluorescein isothiochanate), phycobilin protein, rhodamine, and phycoerythrin ( It is preferable to use phycoerythrin, phycocyanin, orthophthalic aldehyde and the like.

In addition to the chromophore or fluorescent substance, the secondary antibody may be labeled with isolumino, lucigenin, luminol, aromatic aridiniumester, imidazole, and acridine salt. Light emitting materials such as luciferin, luciferase, luciferase and aquorin, or radiological materials such as ¹²⁵ I, ¹²⁷ I, ¹³¹ I, ¹⁴ C, ³ H, ³² P, ³⁵ S, as well as immunologically It will not specifically limit, if it can be used for a measurement method. It is also possible to use low molecular heptenes such as biotin, dinitrophenyl, pyridoxyl or fluoresamine in the antibody.

In addition, in the case of using the chromophore in step 4, in order to measure its activity, a chromophore substrate is used, and the chromophore substrate may use any substance capable of developing chromophore bound to the secondary antibody, and 4CN (4 -chloro-1-naphtol), DAB (Diaminobenzidine), AEC (Aminoethyl carbazole), ABTS [2, 2'-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], OPD (o-Phenylenediamine) and TMB ( Tetramethyl Benzidine) can be used.

The test sample of step 2 is a patient suffering from a disease related to βig-h3, and all kinds of body fluids may be the test material, and preferably, urine, blood or synovial fluid of a kidney disease, liver disease or rheumatism patient.

In the present invention, in order to confirm whether the quantitative method of βig-h3 protein accurately quantifies βig-h3 protein, a recombinant protein including at least one fourth pars-1 domain of mouse βig-h3 or βig-h3 is used as a standard protein. Was measured and compared with the case where human βig-h3 was used as the standard protein.

As a result of determining the optimal coating concentration of the human βig-h3 protein and the quantitative ratio of the antibody in the quantitative method of βig-h3 of the present invention, the quantitative ratio of the primary anti-human βig-h3 antibody is 1: 1600 and 1: 2000. Since the graph forms a straight line, the ratio is found to be the most suitable (see FIG. 7 ). The quantitative ratio of the secondary antibody is 1: 2000, so the graph forms a straight line. 8 ), the human βig-h3 protein was suitable for the quantitative range of the graph showing a straight line in both cases of 1.0 μg / ml and 0.5 μg / ml, but 0.5 μg / ml of Pearson's product moment correlation coefficient was greater than 1.0 μg / ml. The (R ² ) value appeared in a form close to 1, and it was confirmed that the coating concentration was most suitable when 0.5 μg / ml (see FIG. 9 ).

       From the above results, the present inventors confirmed that the optimal conditions were that the coating concentration of the human βig-h3 standard protein was 0.5 µg / ml and the dilution ratio of the primary anti-human βig-h3 antibody and the secondary antibody was 1: 2000, respectively. .

In the same manner as in the case of human βig-h3, the inventors used mouse βig-h3, recombinant βig-h3 D-IV (1x), ig-h3 D-IV (2x), ig-h3 D-IV (3x), and βig. -h3 D-IV (4x) was used to determine protein concentration, quantitative ratios of primary and secondary antibodies. Specifically, the coating concentration of each protein was 0.5 μg / ml, and the primary anti human βig-h3 antibody and the secondary antibody were 1: 2000, and the primary anti mouse βig-h3 antibody and the secondary antibody were 1 quantitatively. A quantitative experiment was performed at: 2000.

As a result, in all cases, the graphs formed a straight line, and the measurement range was also similar to each other from 11 ng / ml to 900 ng / ml (see FIGS . 11 and 12 ).

From the above results, the standard proteins are human βig-h3, mouse βig-h3, recombinant βig-h3 D-IV (1x), ig-h3 D-IV (2x), ig-h3 D-IV (3x) and βig Any of -h3 D-IV (4x) may be used, and since the primary antibody has an interaction, it was confirmed that either an anti-human βig-h3 antibody or an anti-mouse βig-h3 antibody may be used.

In the present invention, the coating concentration of the standard protein is preferably in the range of 0.1 to 2.0 µg / ml, more preferably 0.5 to 1.0 µg / ml. In addition, the ratio of the primary antibody and the secondary antibody is preferably 1: 400 to 1: 3200, more preferably 1: 2000.

The present invention provides a diagnostic kit for various diseases by measuring the amount of βig-h3 protein in the body fluid of a specimen suffering from kidney disease, liver disease or rheumatic disease.

The diagnostic kit of the present invention includes a recombinant protein for the par-1 domain of βig-h3 protein or βig-h3 and an antibody for the par-1 domain of βig-h3 protein or βig-h3. In addition, it may further include a buffer solution, a secondary antibody, a wash solution, a reaction stop solution or a color substrate.

The diagnostic kit of the present invention can diagnose various diseases through the measurement of βig-h3 protein in all body fluids including kidney disease, liver disease and rheumatic disease.

Since βGF-h3 expression is strongly induced by TGF-β, which plays an important role in the pathogenesis of renal disease, βig-h3 is quantified based on the correlation between renal disease and βig-h3 expression. Diagnose kidney disease In order to confirm this, by measuring βig-h3 in urine of diabetic patients, a representative disease causing kidney disease, the urine βig-h3 concentration of diabetic kidney disease, including microalbuminuria, was found in normal persons. The levels of βig-h3 were increased even in patients without clinically diabetic kidney disease. Urine βig-h3 in patients with diabetic kidney disease was higher than normal in most patients, and some of the patients without clinical kidney disease were high. These results suggest that urine βig-h3 reflects the degree of damage to the kidneys, and high levels of diabetes mellitus, especially in some diabetic patients who do not have clinical kidney disease, indicate that they are clinically manifested. There is no dysfunction, but it means that the kidneys are somewhat damaged. Therefore, it was confirmed that βig-h3 measurement of urine has a high diagnostic significance that reflects early kidney damage, and may reflect kidney damage earlier than any test that reflects existing kidney function.

In addition, in order to determine whether βig-h3 concentration in urine reflects kidney damage in diabetic patients early, when measuring βig-h3 concentration in a diabetic animal model, βig-h3 after 5 days after the onset of diabetes The concentration increased about 4 times (see FIG. 13 ). Looking at the change in βig-h3 concentration in each individual after diabetes was induced, it was found that all individuals significantly increased urine βig-h3 concentration after diabetes induction (see FIG. 14 ). On the 5th day of diabetes, urea and creatine levels are normal and the histological findings of the kidney do not show any abnormalities. Thus, a significant increase in βig-h3 levels in urine on day 5 suggests that early renal microscopic damage could not be detected.

In addition, in the present invention, to determine the correlation between kidney damage and βig-h3 concentration by measuring the concentration of βig-h3 from the urine of the patient before and after renal transplantation, high levels of βig-h3 before renal transplantation Showed a slow drop in the case of successful surgery and continued to maintain high values in case 5 patients who did not recover renal function after surgery (see Table 2 ). From the above results it can be seen that the concentration of βig-h3 of the present invention sensitively reflects the damage of the kidney.

In addition, in the present invention, as a result of measuring the concentration of βig-h3 by collecting urine of renal failure patients, it was confirmed that the βig-h3 concentration value in the urine is significantly higher than normal, all of the βig-h3 concentration in the urine is kidney It was confirmed that it has a very important diagnostic significance in many cases of early damage and sensitive kidney damage (see Table 3 ).

It is clinically very important to determine whether chronic hepatitis patients progress to cirrhosis, but there is no such method. Since TGF-β is the most important factor in the progression of cirrhosis, the concentration of βig-h3 induced by TGF-β may increase in the blood as cirrhosis progresses, which may reflect the progression of cirrhosis. have. In fact, it was confirmed that βig-h3 was expressed more in hepatic cirrhosis by immunohistochemistry of liver tissue of hepatitis patients. Therefore, in the present invention, the chronic hepatitis patients are divided into grades and stages based on the results of histology, and the correlation between the concentrations of βig-h3 in the blood and chronic hepatitis patients is higher than normal in the blood βig-h3. It was confirmed that the concentration was high, the βig-h3 concentration in the low grade and stage was higher than the βig-h3 concentration in the high grade and stage (see Table 5 ). Grade 3 and stage 3 are advanced liver cirrhosis, and the activity of cirrhosis has already peaked, whereas stages 1 and 2 and grades 1 and 2 are active states in which the inflammatory response is currently active. Can be. Therefore, it can be seen that the concentration of βig-h3 in the blood reflects the activity state of cirrhosis of the liver, and the progress of the patient's progression to cirrhosis can be observed by measuring the blood βig-h3 concentration regularly in the same patient.

In addition, the βig-h3 concentrations in synovial fluid of osteoarthritis and rheumatoid arthritis showed approximately 2-fold higher βig-h3 concentration in the synovial fluid of patients with rheumatoid arthritis. From the above results, it was confirmed that βig-h3 concentration in the synovial fluid can be useful for the diagnosis of degenerative arthritis and rheumatoid arthritis (see Table 6 ).

Therefore, the diagnostic kit for quantifying βig-h3 protein of the present invention is capable of early and sensitive diagnosis of renal disease, liver disease and rheumatic disease, and reflects the diagnosis, affective affect, and progression of the disease. It can be usefully used.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Standard Proteins and Primary Antibodies

<1-1> Isolation of Human βig-h3 and Mouse βig-h3

We have produced βig-h3 protein in humans and mice. A schematic of each protein component is shown in FIG . 1, in which the hatched and cross hatched cells represent the highly conserved sequences of repeating domains I, II, III, and IV, with the RGD motif represented by a blank. It was.

The inventors have pBluescript SK (-) the βig-h3 cDNA (pBS βig-h3) having the base sequence represented by the SEQ ID NO: 2 cloned into a vector (obtained by cloning from a papilloma cell cDNA of human skin) Nde I and Bgl II DNA fragments with blunt ends were prepared by digestion with restriction enzymes, and the fragments were subcloned into EcoR V and EcoR I sites of the pET-29β vector (available from Novagen). A protein having an amino acid sequence set forth in SEQ ID NO: 3 corresponding to 653 amino acids from 69 amino acids of βig-h3 was isolated and named human βig-h3.

Next, the inventors made a BamH I and Xho I site in βig-h3 cDNA and performed a polymerase chain reaction (PCR) in the same manner as described above to describe SEQ ID No. 4 having a nucleotide sequence corresponding to 641 amino acids from 23 amino acids. A DNA fragment was prepared, and a protein having an amino acid sequence as set forth in SEQ ID NO: 5 expressed by inserting the fragment into the BamH I and Xho I sites of the pET-29β vector was named mouse βig-h3.

In order to express the human βig-h3 protein and mouse βig-h3 protein, the present inventors transformed E. coli BL21 (DE3) using a host, and transformed strains had an optical concentration (OD) at 595 nm. Incubated in LB medium containing kanamicine at 37 ° C. and 50 μg / ml until 0.5 to 0.6. During the incubation process, 1 mM IPTG (isopropyl-β-D-(-) thiogalactopyranoside) was expressed by inducing βig-h3 protein at 37 ° C. for 3 hours.

       After expressing βig-h3, E. coli pellets were purified by 50 mM Tris-HCl (pH 8.0), 100 mM sodium chloride, 1 mM EDTA, 1% Triton X-100, 1 mM phenylmethane-sulphate. The cells were resuspended in cell lysis buffer consisting of phenylmethane sulfonyl fluoride (hereinafter referred to as “PMSF”) and 0.5 mM DTT and sonicated to disrupt the cells. This procedure was repeated five times. It was.

       The solution was centrifuged to obtain insoluble bodies containing βig-h3 containing 20 mM tris hydrochloric acid (Tris-HCl) containing 0.5 M sodium chloride, 5 mM imidazol and 8 M urea. After dissolving in buffer, the protein was purified using Ni-NTA resin (Qiagen). In 20 mM Tris hydrochloric acid buffer solution containing 50 mM sodium chloride, the protein was purified by dialysis from high to low concentrations of urea in turn and confirmed by SDS-PAGE.

As a result, it was confirmed that the human βig-h3 and mouse βig-h3 proteins of the present invention are isolated ( FIG. 2 ).

<1-2> Preparation and Separation of βig-h3 D-IV (1x) and βig-h3 D-IV (4x)

The present inventors performed PCR amplification of a DNA fragment described in SEQ ID NO: 6 encoding a fourth domain from 498 to 637 of human βig-h3 described in SEQ ID NO: 1, and then cloned into a pET-29β vector. An expression vector of the domain was prepared and named "βig-h3 D-IV".

Next, the nucleotide sequence corresponding to the fourth domain was synthesized by PCR to smooth the 3 'terminal portion with the Klenow fragment, and then the expression vector pβig-h3 D-IV including the fourth domain prepared above. Was inserted into the EcoR V region of pβig-h3 D-IV (2x). After inserting the pβig-h3 D-IV (2x) fragment into EcoR V and Xho I, smoothing the 3 'terminal portion with the Klenow fragment in the same manner as above, and then pβig-h3 D-IV and pβig-h3 D Each was inserted into the EcoR V region of -IV (2x), which was named pβig-h3 D-IV (3x) and pβig-h3 D-IV (4x) ( FIG. 3 ). His-tag was made by linking six histidine residues to the carboxy terminus of the DNA fragment to purify the protein expressed using Ni-NTA resin (Qiagen).

The expression vector was transformed into E. coli BS21 (DE3), and the E. coli transformants were cultured in a medium to which kanamycin was added at a concentration of 50 µg / ml. The E. coli transformants were suspended in a cell lysis buffer solution containing 1 mM Tris-HCL (pH 8.0), 100 mM EDTA, 1% Triton X-100, 1 mM PMSF and 0.5 mM DTT, followed by sonication. Was crushed. After repeating the above procedure five times, the supernatant was separated by centrifugation, and the supernatant was purified using a column filled with Ni-NTA resin and confirmed using SDS-PAGE.

As a result, βig-h3 D-IV (1x), βig-h3 D-IV (2x), and βig-h3 each having an amino acid sequence represented by SEQ ID NO: 7 to SEQ ID NO: 10 including the fourth domain of the present invention. It was confirmed that D-IV (3x) and βig-h3 D-IV (4x) proteins were expressed ( FIG. 4 ).

<1-3> Preparation and Isolation of Primary Antibody

Antibodies were prepared by subcutaneous injection into rabbits using human βig-h3 and mouse βig-h3 proteins isolated in Example <1-1> as antigens. The first injection mixed 200 μg of protein with complete Freund's adjuvant and the next four injections mixed 100 μg of protein with incomplete Freund's adjuvant. Injections were made every three weeks. Serum was collected for 2 hours at room temperature after blood collection, and centrifuged at 10,000 x g for 10 minutes to obtain a supernatant containing primary antibody, which was stored and used in a -20 ° C freezer ( FIG. 5 ).

Example 2 Determination of Coating Concentration of Human βig-h3 Protein and Quantitative Ratio of Antibody

<2-1> Quantitative Ratio Determination of Primary Antibodies

       To obtain a quantitative ratio of primary antibody to human βig-h3 protein, human βig-h3 was diluted in 0.5 μg / ml of the coating buffer solution (20 mM carbonate-bicarbonate solution, pH 9.6, 0.02% sodium azide) and then 96 200 μl was added to the well plate and coated overnight at 4 ° C. Diluted buffer solution (saline-phosphate buffer / Twin 80) at a concentration of 1: 200, 1: 400, 1: 800, 1: 1600, 1: 2000, and 1: 3200 in primary anti-human βig-h3 antibody in coated plates. After dilution), the secondary antibody fixed at 1: 5000 was added and reacted at room temperature for 1 hour and a half. After the reaction, a substrate solution (o-phenylenediamine was dissolved in methanol at 10 mg / ml, diluted 1: 100 in distilled water, and mixed with 10 μl of 30% hydrogen peroxide was added), followed by reaction at room temperature for 1 hour. The reaction was stopped with 50 μl of 8 N sulfuric acid solution and immediately subjected to ELISA analysis (O.D 492 nm).

As a result, the quantitative ratio of the primary anti-human βig-h3 antibody was found to be the most suitable ratio since the graph forms a straight line at 1: 1600 and 1: 2000 ( FIG. 7 ).

<2-2> Quantitative Ratio Determination of Secondary Antibodies

       In order to obtain a quantitative ratio of the secondary antibody, human βig-h3 protein was coated at 0.5 μg / ml, the primary anti human βig-h3 antibody was fixed by diluting 1: 1600 and 1: 2000, and the concentration of the secondary antibody was determined. ELISA analysis was performed by diluting to 1: 1000, 1: 2000, and 1: 3000.

As a result, the graph is a straight line when the ratio is 1: 2000, and it was confirmed that the ratio was the most appropriate ratio ( FIG. 8 ).

<2-3> Determination of Coating Concentration of Human βig-h3 Protein

       In order to determine the coating concentration of the human βig-h3 protein, the inventors diluted the primary anti human βig-h3 antibody to 1: 2000 and the secondary antibody to 1: 2000, and the coating concentration of the human βig-h3 protein was 0.5 μg / ml. And 1.0 µg / ml was used for ELISA analysis.

As a result, in both cases of 1.0 μg / ml and 0.5 μg / ml, the graph shows a straight line, which is suitable for the quantitative range, but 0.5 μg / ml of Pearson's product moment correlation coefficient (R ² ) is closer to 1 than 1.0 μg / ml. Appearing in form, it was confirmed that the coating concentration is most suitable when 0.5 μg / ㎖ ( Fig. 9 ).

       From the above results, the inventors performed quantitative experiments with the coating concentration of the human βig-h3 standard protein as 0.5 μg / ml and the dilution ratio of the primary anti-human βig-h3 antibody and the secondary antibody as 1: 2000, respectively.

Based on the results, log transformation was performed by the formula of Robard (1971) represented by Equation 1 below, and a range was measured by forming a straight line from 11 ng / ml to 900 ng / ml. It was confirmed that the possible range, it was confirmed that the reaction conditions have a sensitivity that can be measured up to 10 ng / ㎖ ( Figure 10 ).

ｌｏｇ ｂ = ｌｏｇ ｅ ^{ｂ / (100-ｂ)}

Where b represents the percentage value at each concentration relative to the absorbance of the wells without antigen.

Example 3 Mouse βig-h3 through Cross Experiment Quantitative scoping of recombinant βig-h3 D-IV (1x) and βig-h3 D-IV (4x) proteins

       Using the mouse βig-h3, recombinant βig-h3 D-IV (1x) and βig-h3 D-IV (4x) in the same manner as in Example 2 to determine the protein concentration, the quantitative ratio of the primary antibody and secondary antibody It was. Specifically, the coating concentration of each protein was 0.5 μg / ml, and the primary anti human βig-h3 antibody and the secondary antibody were 1: 2000, and the primary anti mouse βig-h3 antibody and the secondary antibody were 1 quantitatively. A quantitative experiment was performed at: 2000.

As a result, in all cases, the graphs formed a straight line, and the measuring ranges were also similar to each other from 11 ng / ml to 900 ng / ml ( FIGS. 11 and 12 ).

From the above results, the standard protein may be any one of human βig-h3, mouse βig-h3, recombinant βig-h3 D-IV (1x) and βig-h3 D-IV (4x). It was also confirmed that either anti-human βig-h3 antibody or anti-mouse βig-h3 antibody may be used because of the cross interaction.

Example 4 Correlation between Kidney Disease and βig-h3 Expression

<4-1> βig-h3 Measurement in Diabetic Patients

       The present inventors focused on the fact that βig-h3 expression is strongly induced by TGF-β, which plays an important role in the pathogenesis of renal disease, and attempted to confirm the correlation between renal disease and βig-h3 expression. To this end, the present inventors first measured βig-h3 in urine with respect to diabetic patients, a representative disease causing kidney disease. Specifically, 110 μl of urine and 110 μl of primary antibody (1: 1000) were added to a round plate and incubated for 1 and a half hours at 37 ° C., 200 μl of which was again coated on a plate previously coated with βig-h3. The reaction is added for 30 minutes at room temperature. After the reaction, the ELISA assay (O.D 492 nm) was performed by stopping the reaction with the secondary antibody-substrate stopper solution.

Urine βig-h3 Concentration in Patients with Kidney Disease Target group βig-h3 (ng / ml) normal  31.0 (n = 93, ± 8.6) Type II DM  101.9 (n = 51, ± 17.1) Type II DM + microalbuminuria  127.4 (n = 30, ± 27.7) Type II DM + overt proteinuria  105.4 (n = 19, ± 14.9) Type II DM + CRF  153.6 (n = 93, ± 28.1)

As a result, the urine βig-h3 concentration in diabetic kidney disease, including microalbuminuria, was 5 times higher than that of normal people, and βig in patients without clinically diabetic kidney disease. The concentration of -h3 was increased. Urine βig-h3 in patients with diabetic kidney disease was higher than normal in most patients, and some of the patients without clinical kidney disease were high. These results suggest that urine βig-h3 reflects the degree of damage to the kidneys, and high levels of diabetes mellitus, especially in some diabetic patients who do not have clinical kidney disease, indicate that they are clinically manifested. There is no dysfunction, but it means that the kidneys are somewhat damaged. Therefore, it was confirmed that βig-h3 measurement of urine has a high diagnostic significance that reflects early kidney damage, and may reflect kidney damage earlier than any test that reflects existing kidney function.

<4-2> βig-h3 Measurement in a Diabetic-Induced Animal Model

We measured βig-h3 concentration in a diabetic-induced animal model to determine if βig-h3 concentration in urine reflects kidney damage in diabetic patients early.

Diabetes was induced by intraperitoneal injection of streptozotosin, a diabetes-causing drug, at 60 mg / kg in SD rats (Sprague-Dawley rats). Urine was collected for 24 hours and βig-h3 concentration was measured in the same manner as in Example <4-1>.

As a result, the βig-h3 concentration before diabetes induction was 56.9 ± 6.4 ng / creatine mg, but the mean value at 5 days after diabetes induction was 230.4 ± 131.8 ng / creatinine mg, which increased the βig-h3 concentration by about 4 times ( FIG. 13 ). . Looking at the change in βig-h3 concentration in each individual after diabetes was induced, it can be seen that the urine βig-h3 concentration significantly increased in all individuals after diabetes induction ( FIG. 14 ). On the 5th day of diabetes, urea and creatine levels are normal and the histological findings of the kidney do not show any abnormalities. Thus, a significant increase in βig-h3 levels in urine on day 5 suggests that early renal microscopic damage could not be detected.

<4-3> βig-h3 Measurement in Kidney Transplant Surgery Patients

The present inventors attempted to determine the correlation between kidney damage and βig-h3 concentration by measuring the concentration of βig-h3 in the urine of patients before and after renal transplantation. To this end, βig-h3 concentration was measured in patients before and after kidney transplantation in the same manner as in Example <4-1>, and the results are shown in Table 2 .

Changes in βig-h3 Concentration Before and After Renal Transplantation   Working patient -6 -5 -4 -3 -2 -One 0 One 2 3 4 5 6 Surgical Success One 376.9 199.2 105.6 59.1 67.6 84.5 63.1 61.2 39.7 9.9 O 2 149.2 147.3 133.5 159.5 148.3 147.3 96.0 74.0 40.7 20.3 27.9 26.4 O 3 107.8 95.8 101.4 102.3 102.2 106.1 106.6 125.5 83.5 49.4 36.5 33.3 23.2 O 4 298.8 208.1 140.5 169.9 188.4 76.3 24.4 O 5 188.6 160.7 469.3 290.9 494.7 324.4 - Ｘ

As a result, the high βig-h3 concentration before renal transplantation showed a slow drop in the case of successful surgery, and the high level was maintained in the fifth patient who did not recover the renal function after the surgery. . From the above results it can be seen that the concentration of βig-h3 of the present invention sensitively reflects the damage of the kidney.

<4-4> βig-h3 Measurement in Patients with Renal Failure

The present inventors have measured the concentration of βig-h3 in the same manner as in Example <4-1> by collecting urine of renal failure patients, and found that all patients showed significantly higher levels of βig-h3 than normal ( Table 3 ).

Urine βig-h3 Concentration in Patients with Renal Failure Target group βig-h3 (ng / mg) normal  31.0 (n = 93, ± 8.6) Chronic kidney failure 335.4 (n = 9, ± 56.0)

<4-5> βig-h3 Measurement in Patients with Kidney Disease

In order to determine whether βig-h3 is expressed differently from normal in patients with kidney-related diseases, in the case of normal post-renal transplantation, when the size of the transplanted kidney is small, chronic rejection, recurrent pyelitis, and cyclosporine toxicity were observed. Classified as the case, the concentration of βig-h3 from the urine of the patient was measured in the same manner as in Example <4-1>.

As a result, the mean value of patients with normal renal function after renal transplantation was 39.4 ng / creatine mg, while in patients with chronic rejection, recurrent pyelonephritis, and cyclosporine toxicity, the concentrations of βig-h3 increased significantly, respectively, 140.8, 175.4 and 90.9, respectively. ng / creatinine mg is shown ( FIG. 15 , Table 4 ).

βig-h3 Normal after kidney transplant (n = 47) Small kidney size (n = 16) Chronic Rejection (n = 15) Recurrent pyelitis (n = 6) Cyclosporin toxicity (n = 6) Average 39.4 ± 18.2 54.7 ± 23.0 140.8 ± 81.1 175.4 ± 65.8 90.9 ± 22.4 lowest 9.4 17.9 48.8 83.2 64.6 maximum 84.7 100.0 374.4 249.8 119.4

In addition, the present inventors examined whether the increase in βig-h3 concentration by relapse after renal transplantation decreased again in response to treatment. After kidney transplantation, pyelonephritis (local segmental glomerulosclerosis) recurred, the urine βig-h3 concentration in patients undergoing plasma exchange was gradually decreased, indicating that the urine βig-h3 concentration decreased in response to treatment. As can be seen, it represents the possibility as an index of the treatment response ( FIG. 16 ).

<4-6> Analysis of Effect on βig-h3 Concentration after Renal Transplant

The present inventors measured the concentration of βig-h3 in urine every day immediately after renal surgery to determine how the concentration of urine βig-h3 changes after renal transplantation.

As a result, the urine βig-h3 concentration gradually decreased after surgery in the case of successful transplantation of the kidney of a living person or brain of a brain dead person. In the case of kidney transplantation of the brain dead person returning to the value was a normal value between 6 to 7 days ( Fig. 17 ).

In addition, when the transplanted kidney is normal due to the small size of the transplanted kidney, but the renal function is insufficient, the blood creatine level is still high while the urine βig-h3 concentration returns to normal. This means that the transplanted kidney is so small that it does not filter out all the waste in the patient, but the kidney itself is normal and therefore the concentration of βig-h3 reflecting kidney damage has returned to normal ( FIG. 17 ). On the other hand, when kidney transplantation failed, urine βig-h3 concentration showed a tendency to be severe.

Based on the above results, the measurement of urine βig-h3 concentration can be used as a good indicator for early diagnosis of renal disease abnormality, progression of kidney disease and determination of treatment effect.

       From the above results, it was confirmed that βig-h3 concentration in urine reflects kidney damage sensitively and has a very important diagnostic significance in various cases of kidney damage.

Example 5 Correlation between Liver Disease and βig-h3 Expression

       It is clinically very important to determine whether chronic hepatitis patients progress to cirrhosis, but there is no such method. Since TGF-β is the most important factor for the progression of cirrhosis, the concentration of βig-h3 induced by TGF-β may increase in the blood as cirrhosis progresses, which may reflect the progression of cirrhosis. . In fact, it is known that βig-h3 is expressed more in hepatic cirrhosis by immunohistochemistry of liver tissue of hepatitis patients. Accordingly, the present inventors divided chronic hepatitis patients into grades and stages based on the results of histological examination and confirmed the correlation with blood βig-h3 concentration.

Blood of chronic hepatitis patients was collected and the concentration of βig-h3 was measured in the same manner as in Example <4-1>, and the results are shown in Table 5 below.

Blood βig-h3 Levels in Patients with Chronic Hepatitis Rating βig-h3 (ng / mg) step βig-h3 (ng / mg) 0 (normal)  146.2 (n = 172, ± 28.5) 0 (normal)  146.2 (n = 172, ± 28.5) One  196.6 (n = 16, ± 30.6) One 193.4 (n = 20, ± 30.2) 2  190.0 (n = 43, ± 72.8) 2 192.2 (n = 36, ± 79.1) 3  167.5 (n = 7, ± 21.9) 3 172.5 (n = 10, ± 21.9)

As a result, patients with chronic hepatitis were found to have higher βig-h3 concentrations in blood than normal, and those with lower grades and βig-h3 concentrations in stages (1 and 2) were higher than those in stages (3). It confirmed that it was high. Grade 3 and stage 3 are advanced liver cirrhosis, and the activity of cirrhosis has already peaked, whereas stages 1 and 2 and grades 1 and 2 are active states in which the inflammatory response is currently active. Can be. Therefore, it can be seen that the concentration of βig-h3 in the blood reflects the activity state of cirrhosis of the liver, and the progress of the patient's progression to cirrhosis can be observed by measuring the blood βig-h3 concentration regularly in the same patient.

Example 6 Correlation between Rheumatoid Arthritis Patients and βig-h3 Expression

The present inventors measured the concentration of βig-h3 in synovial fluid of patients with osteoarthritis and rheumatoid arthritis in the same manner as in Example <4-1> ( Table 6 ).

Βig-h3 concentration in synovial fluid βig-h3 (ng / mg) Degenerative arthritis  11.0 (n = 29, ± 0.3) Rheumatoid arthritis  21.0 (n = 20, ± 2.5)

As a result, the βig-h3 concentration in the synovial fluid of the patients with rheumatoid arthritis was about 2 times higher. From these results, the βig-h3 concentration in the synovial fluid could be useful for the diagnosis of degenerative arthritis and rheumatoid arthritis. It was confirmed that there is.

As described above, the βig-h3 assay of the present invention uses human βig-h3, mouse βig-h3, βig-h3 D-IV (1x) or βig-h3 D-IV (4x) as a standard protein. It is possible to accurately diagnose the concentration of βig-h3 in these inexpensive and diverse body fluids, and the βig-h3 concentration in the sample is sensitive to early renal, hepatic and rheumatic diseases such as TGF-β-related medical symptoms. Therefore, it can be usefully used as an effective test method that reflects the diagnosis, the degree of injury, and the degree of progression of the disease.

<110> KIM, In-San          REGEN Biotech. Inc. <120> Method for measuring quantity of βｉｇ­ｈ3 and diagnosis ki <130> 2p-01-29 <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 683 <212> PRT <213> Homo sapiens <400> 1 Met Ala Leu Phe Val Arg Leu Leu Ala Leu Ala Leu Ala Leu Ala Leu   1 5 10 15 Gly Pro Ala Ala Thr Leu Ala Gly Pro Ala Lys Ser Pro Tyr Gln Leu              20 25 30 Val Leu Gln His Ser Arg Leu Arg Gly Arg Gln His Gly Pro Asn Val          35 40 45 Cys Ala Val Gln Lys Val Ile Gly Thr Asn Arg Lys Tyr Phe Thr Asn      50 55 60 Cys Lys Gln Trp Tyr Gln Arg Lys Ile Cys Gly Lys Ser Thr Val Ile  65 70 75 80 Ser Tyr Glu Cys Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly                  85 90 95 Cys Pro Ala Ala Leu Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val             100 105 110 Val Gly Ser Thr Thr Thr Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu         115 120 125 Arg Pro Glu Met Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser     130 135 140 Asn Glu Ala Trp Ala Ser Leu Pro Ala Glu Val Leu Asp Ser Leu Val 145 150 155 160 Ser Asn Val Asn Ile Glu Leu Leu Asn Ala Leu Arg Tyr His Met Val                 165 170 175 Gly Arg Arg Val Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr             180 185 190 Ser Met Tyr Gln Asn Ser Asn Ile Gln Ile His His Tyr Pro Asn Gly         195 200 205 Ile Val Thr Val Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala     210 215 220 Thr Asn Gly Val Val His Leu Ile Asp Lys Val Ile Ser Thr Ile Thr 225 230 235 240 Asn Asn Ile Gln Gln Ile Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu                 245 250 255 Arg Ala Ala Val Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn             260 265 270 Gly Gln Tyr Thr Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu Lys Ile         275 280 285 Pro Ser Glu Thr Leu Asn Arg Ile Leu Gly Asp Pro Glu Ala Leu Arg     290 295 300 Asp Leu Leu Asn Asn His Ile Leu Lys Ser Ala Met Cys Ala Glu Ala 305 310 315 320 Ile Val Ala Gly Leu Ser Val Glu Thr Leu Glu Gly Thr Thr Leu Glu                 325 330 335 Val Gly Cys Ser Gly Asp Met Leu Thr Ile Asn Gly Lys Ala Ile Ile             340 345 350 Ser Asn Lys Asp Ile Leu Ala Thr Asn Gly Val Ile His Tyr Ile Asp         355 360 365 Glu Leu Leu Ile Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala     370 375 380 Glu Ser Asp Val Ser Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly Leu 385 390 395 400 Gly Asn His Leu Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu                 405 410 415 Asn Ser Val Phe Lys Asp Gly Thr Pro Pro Ile Asp Ala His Thr Arg             420 425 430 Asn Leu Leu Arg Asn His Ile Ile Lys Asp Gln Leu Ala Ser Lys Tyr         435 440 445 Leu Tyr His Gly Gln Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg     450 455 460 Val Phe Val Tyr Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala 465 470 475 480 Ala His Asp Lys Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg                 485 490 495 Val Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp             500 505 510 Asn Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr         515 520 525 Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn     530 535 540 Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly 545 550 555 560 Asp Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu                 565 570 575 Ile Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu             580 585 590 Gln Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val         595 600 605 Asn Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val     610 615 620 Val His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Arg Pro Gln 625 630 635 640 Glu Arg Gly Asp Glu Leu Ala Asp Ser Ala Leu Glu Ile Phe Lys Gln                 645 650 655 Ala Ser Ala Phe Ser Arg Ala Ser Gln Arg Ser Val Arg Leu Ala Pro             660 665 670 Val Tyr Gln Lys Leu Leu Glu Arg Met Lys His         675 680 <210> 2 <211> 2691 <212> DNA <213> Homo sapiens <400> 2 gcttgcccgt cggtcgctag ctcgctcggt gcgcgtcgtc ccgctccatg gcgctcttcg 60 tgcggctgct ggctctcgcc ctggctctgg ccctgggccc cgccgcgacc ctggcgggtc 120 ccgccaagtc gccctaccag ctggtgctgc agcacagcag gctccggggc cgccagcacg 180 gccccaacgt gtgtgctgtg cagaaggtta ttggcactaa taggaagtac ttcaccaact 240 gcaagcagtg gtaccaaagg aaaatctgtg gcaaatcaac agtcatcagc tacgagtgct 300 gtcctggata tgaaaaggtc cctggggaga agggctgtcc agcagcccta ccactctcaa 360 acctttacga gaccctggga gtcgttggat ccaccaccac tcagctgtac acggaccgca 420 cggagaagct gaggcctgag atggaggggc ccggcagctt caccatcttc gcccctagca 480 acgaggcctg ggcctccttg ccagctgaag tgctggactc cctggtcagc aatgtcaaca 540 ttgagctgct caatgccctc cgctaccata tggtgggcag gcgagtcctg actgatgagc 600 tgaaacacgg catgaccctc acctctatgt accagaattc caacatccag atccaccact 660 atcctaatgg gattgtaact gtgaactgtg cccggctcct gaaagccgac caccatgcaa 720 ccaacggggt ggtgcacctc atcgataagg tcatctccac catcaccaac aacatccagc 780 agatcattga gatcgaggac acctttgaga cccttcgggc tgctgtggct gcatcagggc 840 tcaacacgat gcttgaaggt aacggccagt acacgctttt ggccccgacc aatgaggcct 900 tcgagaagat ccctagtgag actttgaacc gtatcctggg cgacccagaa gccctgagag 960 acctgctgaa caaccacatc ttgaagtcag ctatgtgtgc tgaagccatc gttgcggggc 1020 tgtctgtaga gaccctggag ggcacgacac tggaggtggg ctgcagcggg gacatgctca 1080 ctatcaacgg gaaggcgatc atctccaata aagacatcct agccaccaac ggggtgatcc 1140 actacattga tgagctactc atcccagact cagccaagac actatttgaa ttggctgcag 1200 agtctgatgt gtccacagcc attgaccttt tcagacaagc cggcctcggc aatcatctct 1260 ctggaagtga gcggttgacc ctcctggctc ccctgaattc tgtattcaaa gatggaaccc 1320 ctccaattga tgcccataca aggaatttgc ttcggaacca cataattaaa gaccagctgg 1380 cctctaagta tctgtaccat ggacagaccc tggaaactct gggcggcaaa aaactgagag 1440 tttttgttta tcgtaatagc ctctgcattg agaacagctg catcgcggcc cacgacaaga 1500 gggggaggta cgggaccctg ttcacgatgg accgggtgct gaccccccca atggggactg 1560 tcatggatgt cctgaaggga gacaatcgct ttagcatgct ggtagctgcc atccagtctg 1620 caggactgac ggagaccctc aaccgggaag gagtctacac agtctttgct cccacaaatg 1680 aagccttccg agccctgcca ccaagagaac ggagcagact cttgggagat gccaaggaac 1740 ttgccaacat cctgaaatac cacattggtg atgaaatcct ggttagcgga ggcatcgggg 1800 ccctggtgcg gctaaagtct ctccaaggtg acaagctgga agtcagcttg aaaaacaatg 1860 tggtgagtgt caacaaggag cctgttgccg agcctgacat catggccaca aatggcgtgg 1920 tccatgtcat caccaatgtt ctgcagcctc cagccaacag acctcaggaa agaggggatg 1980 aacttgcaga ctctgcgctt gagatcttca aacaagcatc agcgttttcc agggcttccc 2040 agaggtctgt gcgactagcc cctgtctatc aaaagttatt agagaggatg aagcattagc 2100 ttgaagcact acaggaggaa tgcaccacgg cagctctccg ccaatttctc tcagatttcc 2160 acagagactg tttgaatgtt ttcaaaacca agtatcacac tttaatgtac atgggccgca 2220 ccataatgag atgtgagcct tgtgcatgtg ggggaggagg gagagagatg tactttttaa 2280 atcatgttcc ccctaaacat ggctgttaac ccactgcatg cagaaacttg gatgtcactg 2340 cctgacattc acttccagag aggacctatc ccaaatgtgg aattgactgc ctatgccaag 2400 tccctggaaa aggagcttca gtattgtggg gctcataaaa catgaatcaa gcaatccagc 2460 ctcatgggaa gtcctggcac agtttttgta aagcccttgc acagctggag aaatggcatc 2520 attataagct atgagttgaa atgttctgtc aaatgtgtct cacatctaca cgtggcttgg 2580 aggcttttat ggggccctgt ccaggtagaa aagaaatggt atgtagagct tagatttccc 2640 tattgtgaca gagccatggt gtgtttgtaa taataaaacc aaagaaacat a 2691 <210> 3 <211> 585 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE (222) (1) .. (585) <223> 69 to 653 amino acid sequence of human ID No. 1 <400> 3 Tyr Gln Arg Lys Ile Cys Gly Lys Ser Thr Val Ile Ser Tyr Glu Cys   1 5 10 15 Cys Pro Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly Cys Pro Ala Ala              20 25 30 Leu Pro Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val Val Gly Ser Thr          35 40 45 Thr Thr Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu Arg Pro Glu Met      50 55 60 Glu Gly Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser Asn Glu Ala Trp  65 70 75 80 Ala Ser Leu Pro Ala Glu Val Leu Asp Ser Leu Val Ser Asn Val Asn                  85 90 95 Ile Glu Leu Leu Asn Ala Leu Arg Tyr His Met Val Gly Arg Arg Val             100 105 110 Leu Thr Asp Glu Leu Lys His Gly Met Thr Leu Thr Ser Met Tyr Gln         115 120 125 Asn Ser Asn Ile Gln Ile His His Tyr Pro Asn Gly Ile Val Thr Val     130 135 140 Asn Cys Ala Arg Leu Leu Lys Ala Asp His His Ala Thr Asn Gly Val 145 150 155 160 Val His Leu Ile Asp Lys Val Ile Ser Thr Ile Thr Asn Asn Ile Gln                 165 170 175 Gln Ile Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu Arg Ala Ala Val             180 185 190 Ala Ala Ser Gly Leu Asn Thr Met Leu Glu Gly Asn Gly Gln Tyr Thr         195 200 205 Leu Leu Ala Pro Thr Asn Glu Ala Phe Glu Lys Ile Pro Ser Glu Thr     210 215 220 Leu Asn Arg Ile Leu Gly Asp Pro Glu Ala Leu Arg Asp Leu Leu Asn 225 230 235 240 Asn His Ile Leu Lys Ser Ala Met Cys Ala Glu Ala Ile Val Ala Gly                 245 250 255 Leu Ser Val Glu Thr Leu Glu Gly Thr Thr Leu Glu Val Gly Cys Ser             260 265 270 Gly Asp Met Leu Thr Ile Asn Gly Lys Ala Ile Ser Asn Lys Asp         275 280 285 Ile Leu Ala Thr Asn Gly Val Ile His Tyr Ile Asp Glu Leu Leu Ile     290 295 300 Pro Asp Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala Glu Ser Asp Val 305 310 315 320 Ser Thr Ala Ile Asp Leu Phe Arg Gln Ala Gly Leu Gly Asn His Leu                 325 330 335 Ser Gly Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu Asn Ser Val Phe             340 345 350 Lys Asp Gly Thr Pro Pro Ile Asp Ala His Thr Arg Asn Leu Leu Arg         355 360 365 Asn His Ile Ile Lys Asp Gln Leu Ala Ser Lys Tyr Leu Tyr His Gly     370 375 380 Gln Thr Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg Val Phe Val Tyr 385 390 395 400 Arg Asn Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala Ala His Asp Lys                 405 410 415 Arg Gly Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg Val Leu Thr Pro             420 425 430 Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser         435 440 445 Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn     450 455 460 Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg 465 470 475 480 Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu                 485 490 495 Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser             500 505 510 Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys         515 520 525 Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro     530 535 540 Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile 545 550 555 560 Thr Asn Val Leu Gln Pro Pro Ala Asn Arg Pro Gln Glu Arg Gly Asp                 565 570 575 Glu Leu Ala Asp Ser Ala Leu Glu Ile             580 585 <210> 4 <211> 1857 <212> DNA <213> Mouse Intracisternal A-particle <400> 4 gcaggtcccg ccaagtcacc ctaccagctg gtgctgcagc atagccggct ccggggtcgc 60 cagcacggcc ccaatgtatg tgctgtgcag aaggtcattg gcaccaacaa gaaatacttc 120 accaactgca agcagtggta ccagaggaag atctgcggca agtcgacagt catcagttat 180 gagtgctgtc ctggatatga aaaggtccca ggagagaaag gttgcccagc agctcttccg 240 ctctcaaatc tgtatgagac catgggagtt gtgggatcga ccaccacaca gctgtataca 300 gaccgcacag aaaagctgag gcctgagatg gagggacccg gaagcttcac catctttgct 360 cctagcaatg aggcctggtc ttccttgcct gcggaagtgc tggactccct ggtgagcaac 420 gtcaacatcg aactgctcaa tgctctccgc taccacatgg tggacaggcg ggtcctgacc 480 gatgagctca agcacggcat gaccctcacc tccatgtacc agaattccaa catccagatc 540 catcactatc ccaatgggat tgtaactgtt aactgtgccc ggctgctgaa ggctgaccac 600 catgcgacca acggcgtggt gcatctcatt gacaaggtca tttccaccat caccaacaac 660 atccagcaga tcattgaaat cgaggacacc tttgagacac ttcgggccgc cgtggctgca 720 tcaggactca ataccgtgct ggagggcgac ggccagttca cactcttggc cccaaccaac 780 gaggcctttg agaagatccc tgccgagacc ttgaaccgca tcctgggtga cccagaggca 840 ctgagagacc tgctaaacaa ccacatcctg aagtcagcca tgtgtgctga ggccattgta 900 gctggaatgt ccatggagac cctggggggc accacactgg aggtgggctg cagtggggac 960 aagctcacca tcaacgggaa ggctgtcatc tccaacaaag acatcctggc caccaacggt 1020 gtcattcatt tcattgatga gctgcttatc ccagattcag ccaagacact gcttgagctg 1080 gctggggaat ctgacgtctc cactgccatt gacatcctca aacaagctgg cctcgatact 1140 catctctctg ggaaagaaca gttgaccttc ctggcccccc tgaattctgt gttcaaagat 1200 ggtgtccctc gcatcgacgc ccagatgaag actttgcttc tgaaccacat ggtcaaagaa 1260 cagttggcct ccaagtatct gtactctgga cagacactgg acacgctggg tggcaaaaag 1320 ctgcgagtct ttgtttatcg aaatagcctc tgcattgaaa acagctgcat tgctgcccat 1380 gataagaggg gacggtttgg gaccctgttc accatggacc ggatgttgac acccccaatg 1440 gggacagtta tggatgtcct gaagggagac aatcgtttta gcatgctggt ggccgccatc 1500 cagtctgcag gactcatgga gatcctcaac cgggaagggg tctacactgt ttttgctccc 1560 accaatgaag cgttccaagc catgcctcca gaagaactga acaaactctt ggcaaatgcc 1620 aaggaactta ccaacatcct gaagtaccac attggtgatg aaatcctggt tagcggaggc 1680 atcggggccc tggtgcggct gaagtctctc caaggggaca aactggaagt cagctcgaaa 1740 aacaatgtag tgagtgtcaa taaggagcct gttgccgaaa ccgacatcat ggccacaaac 1800 ggtgtggtct atgccatcaa cactgttctg cagccgccag ccaaccgacc acaagaa 1857 <210> 5 <211> 609 <212> PRT <213> Mouse Intracisternal A-particle <220> <221> PEPTIDE (222) (1) .. (609) <223> 23 to 641 amino acid sequence of mouse <400> 5 Ala Gly Pro Ala Lys Ser Pro Tyr Gln Leu Val Leu Gln His Ser Arg   1 5 10 15 Leu Arg Gly Arg Gln His Gly Pro Asn Val Cys Ala Val Gln Lys Val              20 25 30 Ile Gly Thr Asn Arg Lys Tyr Phe Thr Asn Cys Lys Gln Trp Tyr Gln          35 40 45 Arg Lys Ile Cys Gly Lys Ser Thr Val Ile Ser Tyr Glu Cys Cys Pro      50 55 60 Gly Tyr Glu Lys Val Pro Gly Glu Lys Gly Cys Pro Ala Ala Leu Pro  65 70 75 80 Leu Ser Asn Leu Tyr Glu Thr Leu Gly Val Val Gly Ser Thr Thr Thr                  85 90 95 Gln Leu Tyr Thr Asp Arg Thr Glu Lys Leu Arg Pro Glu Met Glu Gly             100 105 110 Pro Gly Ser Phe Thr Ile Phe Ala Pro Ser Asn Glu Ala Trp Ala Ser         115 120 125 Leu Pro Ala Glu Val Leu Asp Ser Leu Val Ser Asn Val Asn Ile Glu     130 135 140 Leu Leu Asn Ala Leu Arg Tyr His Met Val Gly Arg Arg Val Leu Thr 145 150 155 160 Asp Glu Leu Lys His Gly Met Thr Leu Thr Ser Met Tyr Gln Asn Ser                 165 170 175 Asn Ile Gln Ile His His Tyr Pro Asn Gly Ile Val Thr Val Asn Cys             180 185 190 Ala Arg Leu Leu Lys Ala Asp His His Ala Thr Asn Gly Val Val His         195 200 205 Leu Ile Asp Lys Val Ile Ser Thr Ile Thr Asn Asn Ile Gln Gln Ile     210 215 220 Ile Glu Ile Glu Asp Thr Phe Glu Thr Leu Arg Ala Ala Val Ala Ala 225 230 235 240 Ser Gly Leu Asn Thr Met Leu Glu Gly Asn Gly Gln Tyr Thr Leu Leu                 245 250 255 Ala Pro Thr Asn Glu Ala Phe Glu Lys Ile Pro Ser Glu Thr Leu Asn             260 265 270 Arg Ile Leu Gly Asp Pro Glu Ala Leu Arg Asp Leu Leu Asn Asn His         275 280 285 Ile Leu Lys Ser Ala Met Cys Ala Glu Ala Ile Val Ala Gly Leu Ser     290 295 300 Val Glu Thr Leu Glu Gly Thr Thr Leu Glu Val Gly Cys Ser Gly Asp 305 310 315 320 Met Leu Thr Ile Asn Gly Lys Ala Ile Ile Ser Asn Lys Asp Ile Leu                 325 330 335 Ala Thr Asn Gly Val Ile His Tyr Ile Asp Glu Leu Leu Ile Pro Asp             340 345 350 Ser Ala Lys Thr Leu Phe Glu Leu Ala Ala Glu Ser Asp Val Ser Thr         355 360 365 Ala Ile Asp Leu Phe Arg Gln Ala Gly Leu Gly Asn His Leu Ser Gly     370 375 380 Ser Glu Arg Leu Thr Leu Leu Ala Pro Leu Asn Ser Val Phe Lys Asp 385 390 395 400 Gly Thr Pro Pro Ile Asp Ala His Thr Arg Asn Leu Leu Arg Asn His                 405 410 415 Ile Ile Lys Asp Gln Leu Ala Ser Lys Tyr Leu Tyr His Gly Gln Thr             420 425 430 Leu Glu Thr Leu Gly Gly Lys Lys Leu Arg Val Phe Val Tyr Arg Asn         435 440 445 Ser Leu Cys Ile Glu Asn Ser Cys Ile Ala Ala His Asp Lys Arg Gly     450 455 460 Arg Tyr Gly Thr Leu Phe Thr Met Asp Arg Val Leu Thr Pro Pro Met 465 470 475 480 Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met Leu                 485 490 495 Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg Glu             500 505 510 Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala Leu         515 520 525 Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu Ala     530 535 540 Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly Gly 545 550 555 560 Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu Glu                 565 570 575 Val Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val Ala             580 585 590 Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr Asn         595 600 605 Val <210> 6 <211> 391 <212> DNA <213> Artificial Sequence <220> Β ig-h3 D-IV <400> 6 gtttgggacc ctgttcacca tggaccggat gttgacaccc ccaatgggga cagttatgga 60 tgtcctgaag ggagacaatc gttttagcat gctggtggcc gccatccagt ctgcaggact 120 catggagatc ctcaaccggg aaggggtcta cactgttttt gctcccacca atgaagcgtt 180 ccaagccatg cctccagaag aactgaacaa actcttggca aatgccaagg aacttaccaa 240 catcctgaag taccacattg gtgatgaaat cctggttagc ggaggcatcg gggccctggt 300 gcggctgaag tctctccaag gggacaaact ggaagtcagc tcgaaaaaca atgtagtgag 360 tgtcaataag gagcctgttg ccgaaaccga c 391 <210> 7 <211> 140 <212> PRT <213> Artificial Sequence <220> Β ig-h3 D-IV (1X) amino acid sequence <400> 7 Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn   1 5 10 15 Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu              20 25 30 Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu          35 40 45 Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp      50 55 60 Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile  65 70 75 80 Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln                  85 90 95 Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn             100 105 110 Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val         115 120 125 His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn     130 135 140 <210> 8 <211> 280 <212> PRT <213> Artificial Sequence <220> Β ig-h3 D-IV (2X) amino acid sequence <400> 8 Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn   1 5 10 15 Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu              20 25 30 Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu          35 40 45 Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp      50 55 60 Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile  65 70 75 80 Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln                  85 90 95 Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn             100 105 110 Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val         115 120 125 His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Leu Thr Pro Pro     130 135 140 Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met 145 150 155 160 Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg                 165 170 175 Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala             180 185 190 Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu         195 200 205 Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly     210 215 220 Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu 225 230 235 240 Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val                 245 250 255 Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr             260 265 270 Asn Val Leu Gln Pro Pro Ala Asn         275 280 <210> 9 <211> 420 <212> PRT <213> Artificial Sequence <220> Β ig-h3 D-IV (3X) amino acid sequence <400> 9 Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn   1 5 10 15 Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu              20 25 30 Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu          35 40 45 Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp      50 55 60 Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile  65 70 75 80 Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln                  85 90 95 Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn             100 105 110 Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val         115 120 125 His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Leu Thr Pro Pro     130 135 140 Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met 145 150 155 160 Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg                 165 170 175 Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala             180 185 190 Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu         195 200 205 Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly     210 215 220 Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu 225 230 235 240 Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val                 245 250 255 Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr             260 265 270 Asn Val Leu Gln Pro Pro Ala Asn Leu Thr Pro Pro Met Gly Thr Val         275 280 285 Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met Leu Val Ala Ala     290 295 300 Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg Glu Gly Val Tyr 305 310 315 320 Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala Leu Pro Pro Arg                 325 330 335 Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu Ala Asn Ile Leu             340 345 350 Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly Gly Ile Gly Ala         355 360 365 Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu Glu Val Ser Leu     370 375 380 Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val Ala Glu Pro Asp 385 390 395 400 Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr Asn Val Leu Gln                 405 410 415 Pro Pro Ala Asn             420 <210> 10 <211> 560 <212> PRT <213> Artificial Sequence <220> Β ig-h3 D-IV (4X) amino acid sequence <400> 10 Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn   1 5 10 15 Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu              20 25 30 Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu          35 40 45 Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp      50 55 60 Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile  65 70 75 80 Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln                  85 90 95 Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn             100 105 110 Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val         115 120 125 His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn Leu Thr Pro Pro     130 135 140 Met Gly Thr Val Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met 145 150 155 160 Leu Val Ala Ala Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg                 165 170 175 Glu Gly Val Tyr Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala             180 185 190 Leu Pro Pro Arg Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu         195 200 205 Ala Asn Ile Leu Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly     210 215 220 Gly Ile Gly Ala Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu 225 230 235 240 Glu Val Ser Leu Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val                 245 250 255 Ala Glu Pro Asp Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr             260 265 270 Asn Val Leu Gln Pro Pro Ala Asn Leu Thr Pro Pro Met Gly Thr Val         275 280 285 Met Asp Val Leu Lys Gly Asp Asn Arg Phe Ser Met Leu Val Ala Ala     290 295 300 Ile Gln Ser Ala Gly Leu Thr Glu Thr Leu Asn Arg Glu Gly Val Tyr 305 310 315 320 Thr Val Phe Ala Pro Thr Asn Glu Ala Phe Arg Ala Leu Pro Pro Arg                 325 330 335 Glu Arg Ser Arg Leu Leu Gly Asp Ala Lys Glu Leu Ala Asn Ile Leu             340 345 350 Lys Tyr His Ile Gly Asp Glu Ile Leu Val Ser Gly Gly Ile Gly Ala         355 360 365 Leu Val Arg Leu Lys Ser Leu Gln Gly Asp Lys Leu Glu Val Ser Leu     370 375 380 Lys Asn Asn Val Val Ser Val Asn Lys Glu Pro Val Ala Glu Pro Asp 385 390 395 400 Ile Met Ala Thr Asn Gly Val Val His Val Ile Thr Asn Val Leu Gln                 405 410 415 Pro Pro Ala Asn Leu Thr Pro Pro Met Gly Thr Val Met Asp Val Leu             420 425 430 Lys Gly Asp Asn Arg Phe Ser Met Leu Val Ala Ala Ile Gln Ser Ala         435 440 445 Gly Leu Thr Glu Thr Leu Asn Arg Glu Gly Val Tyr Thr Val Phe Ala     450 455 460 Pro Thr Asn Glu Ala Phe Arg Ala Leu Pro Pro Arg Glu Arg Ser Arg 465 470 475 480 Leu Leu Gly Asp Ala Lys Glu Leu Ala Asn Ile Leu Lys Tyr His Ile                 485 490 495 Gly Asp Glu Ile Leu Val Ser Gly Gly Ile Gly Ala Leu Val Arg Leu             500 505 510 Lys Ser Leu Gln Gly Asp Lys Leu Glu Val Ser Leu Lys Asn Asn Val         515 520 525 Val Ser Val Asn Lys Glu Pro Val Ala Glu Pro Asp Ile Met Ala Thr     530 535 540 Asn Gly Val Val His Val Ile Thr Asn Val Leu Gln Pro Pro Ala Asn 545 550 555 560

Claims (12)

delete delete delete delete delete delete delete A diagnostic kit for kidney disease, liver disease or rheumatoid disease comprising a recombinant protein for the pars-1 domain of βig-h3 protein or βig-h3 and an antibody to the pars-1 domain of βig-h3 protein or βig-h3. 9. The diagnostic kit according to claim 8, further comprising a buffer solution, a secondary antibody, a wash solution, a reaction stop solution or a color substrate. The diagnostic kit according to claim 8, wherein the βig-h3 protein is a human βig-h3 protein having an amino acid sequence as set out in SEQ ID NO: 3 or a mouse βig-h3 protein having an amino acid sequence as set out in SEQ ID NO: 5 . . The diagnostic kit according to claim 8, wherein the pars-1 domain of βig-h3 is one or two to ten repetitions of the fourth pars-1 domain of the βig-h3 protein. The diagnostic kit according to claim 11, wherein the pars-1 domain of βig-h3 is selected from the group consisting of SEQ ID NO: 7 , SEQ ID NO: 8 , SEQ ID NO: 9, and SEQ ID NO: 10 .

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