KR102475926B1 - Biomarker Composition for Diagnosing Systemic lupus erythematous and Method of providing information for diagnosis of Systemic lupus erythematous using the same - Google Patents

Abstract

It relates to a biomarker composition for diagnosing systemic lupus erythematosus and a method for providing information necessary for diagnosing systemic lupus erythematosus using the same, wherein the expression of ApoH protein or a gene encoding the same is measured in a urine sample of an individual suspected of systemic lupus erythematosus. Since it is possible to accurately diagnose systemic lupus erythematosus through a simple and convenient method of measuring the level and comparing it with a healthy control group, it will be widely used in the diagnosis and treatment of systemic lupus erythematosus.

Description

Biomarker Composition for Diagnosing Systemic lupus erythematous and Method of providing information for diagnosis of Systemic lupus erythematous using the same}

It relates to a biomarker composition for diagnosing systemic lupus erythematosus and a method for providing necessary information for diagnosing systemic lupus erythematosus using the biomarker composition.

Systemic lupus erythematous (SLE) is a chronic inflammatory autoimmune disease characterized by the production of autoantibodies. Skin symptoms are the most common, but other diseases of the body such as joints, lungs, heart, kidneys, bone marrow, and blood vessels are common. It is a systemic disease that affects multiple organs.

These systemic lupus erythematosus treatment drugs are prescribed according to the extent of tissue or organ damage, and commonly used drugs include nonsteroidal anti-inflammatory drugs (NSAIDs), low-dose glucocorticoids (adrenocortical hormones), antimalarial drugs, etc., and severe (life-threatening level) In some cases, high-dose steroids and immunosuppressants are used. However, side effects such as myopathy, osteoporosis, hypertension, diabetes, atherosclerotic vascular disease, and increased risk of infection occur when the glucocorticoid and steroid preparations are administered for a long period of time. Therefore, early diagnosis from the onset of the first symptoms of systemic lupus erythematosus is an important situation.

However, systemic lupus erythematosus is difficult to diagnose due to the presence of various clinical features, severity, and autoantibodies.

Currently, serological biomarkers for the diagnosis of systemic lupus erythematosus are antinuclear antibodies (ANA) and anti-double-stranded DNA antibodies (anti-dsDNA). Although anti-dsDNA is considered a highly specific marker for systemic lupus erythematosus, it has been reported to have low sensitivity and low positive predictive value for the diagnosis of systemic lupus erythematosus. Therefore, there is an urgent need to develop novel systemic lupus erythematosus diagnostic biomarkers with strong sensitivity and specificity.

Therefore, in order to develop a biomarker capable of accurately diagnosing systemic lupus erythematosus in a simple and convenient way, the present inventors analyzed various proteins in urine samples of systemic lupus erythematosus patients and healthy controls, and compared them with healthy controls. The present invention was completed by confirming and selecting ApoH (Apolipoprotein H), which significantly increased expression.

An object of the present invention is to provide a biomarker composition for diagnosing systemic lupus erythematous (SLE) that can accurately diagnose systemic lupus erythematous (SLE) by a simple and convenient method.

Another object is to provide a kit for diagnosing systemic lupus erythematosus comprising the composition.

Another object is to provide a method for providing information necessary for diagnosing systemic lupus erythematosus using the biomarker composition.

In order to achieve the above object, one aspect provides a biomarker composition for diagnosing systemic lupus erythematous, including an agent capable of measuring the expression level of ApoH (Apolipoprotein H) protein or a gene encoding the same. .

The term “Apolipoprotein H (ApoH)”, also called beta-2 glycoprotein 1 or β2GPI, refers to an abundant plasma (serum) glycoprotein found both free and linked to lipoproteins. DNA and amino acid sequences of ApoH from various mammalian species are known, including mouse, rat, dog, cow, chimpanzee and human. One of the functions of ApoH is to bind cardiolipin, a lipid, and there is a positively charged amino acid in the structure of ApoH, which is involved in phospholipid binding.

The term "systemic lupus erythematous (SLE)" refers to a disease in which an inflammatory response due to autoimmunity occurs in various tissues of the body. Depending on the tissue involved, various symptoms such as skin rash, photosensitivity, arthritis, mouth ulcer, nephritis, cytopenia, vasculitis, and serositis appear. Although the exact cause of systemic lupus erythematosus is not known, it is known that genetic factors are involved in the pathogenesis. In addition to genetic factors, certain viral infections, exposure to ultraviolet rays, excessive stress, some drugs including antibiotics, or environmental factors caused by several hormones have also been reported to play an important role in the pathogenesis.

The term “diagnosis” means confirming the presence or character of a pathological condition.

The term "biomarker" may also be used as a diagnostic marker, and refers to a substance capable of diagnosing the presence or absence of systemic lupus erythematosus in a biological sample. Polypeptides or nucleic acids (eg mRNA, etc.), lipids, glycolipids, glycoproteins or sugars (eg For example, organic biomolecules such as monosaccharides, disaccharides, oligosaccharides, etc.) may be included.

The expression level of the ApoH protein or the gene encoding it can be measured in a urine sample of an individual.

In one embodiment, as a result of comparing the expression levels of ApoH in urine samples of SLE patients without nephritis, SLE patients with nephritis and healthy controls, the expression of ApoH in urine samples of SLE patients regardless of whether or not nephritis was present. It can be seen that the level increased significantly.

In another embodiment, as a result of ROC analysis for SLE diagnosis, it can be confirmed that the expression level of ApoH in urine shows a sensitivity of 91.5% and a specificity of 92.0%.

The ApoH protein may be a mature form in which the N-terminal signal peptide is removed from the full-length ApoH protein.

The ApoH protein may be represented by the amino acid sequence of SEQ ID NO: 1.

The mature ApoH protein may be represented by the amino acid sequence of SEQ ID NO: 2.

The measurement of the expression level of the ApoH protein is a process of confirming the presence and expression level of the ApoH protein in a biological sample of an individual in order to diagnose systemic lupus erythematosus, using a molecule that specifically binds to the protein. to check the amount of

Agents capable of measuring the expression level of the ApoH protein include a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer ( aptamer) or nanoparticles, but is not limited thereto.

Analysis methods for this include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket ( rocket) immunoelectrophoresis, immunoprecipitation assay, immunohistochemical analysis, complement fixation assay, FACS (Fluorescence activated cell sorter) or protein chip, etc. It is not limited thereto.

Measuring the expression level of the gene encoding the ApoH protein is a process of confirming the presence and expression level of the ApoH gene in a biological sample of an individual in order to diagnose systemic lupus erythematosus. It is used to determine the amount of a gene.

An agent capable of measuring the expression level of the gene encoding the ApoH protein may be a primer pair, a probe, or an antisense nucleotide specifically binding to the gene, but is not limited thereto.

Analysis methods for this include reverse transcriptase-polymerase chain reaction (RT-PCR), real time-polymerase chain reaction, and RNase protection assay (RNase protection assay; RPA), Northern blotting, and DNA chips, but are not limited thereto.

Another aspect provides a kit for diagnosing systemic lupus erythematosus comprising the biomarker composition.

The kit can diagnose systemic lupus erythematosus using a biological sample isolated from an individual. Specifically, the kit can diagnose systemic lupus erythematosus using a urine sample collected from an individual. Early diagnosis of systemic lupus erythematosus may be possible in a simpler and more convenient way by using a urine sample that can be easily collected from an individual.

The kit may include tools and reagents commonly used in immunological analysis, as well as preparations for measuring the expression level of the protein or gene.

Examples of the tools or reagents may include, but are not limited to, suitable carriers, labeling substances capable of generating detectable signals, chromophores, solubilizers, detergents, buffers, and stabilizers. When the labeling substance is an enzyme, it may include a substrate capable of measuring enzyme activity and a reaction terminating agent. The carrier includes a soluble carrier and an insoluble carrier, and an example of the soluble carrier is a physiologically acceptable buffer known in the art, such as PBS, and an example of the insoluble carrier is polystyrene, polyethylene, polypropylene, polyester, poly polymers such as acrylonitrile, fluororesin, crosslinked dextran, polysaccharide, latex-coated magnetic microparticles, other paper, glass, metal, agarose, and combinations thereof.

The biomarker composition is as described above.

Another aspect provides a method for providing information necessary for diagnosing systemic lupus erythematosus, comprising measuring the expression level of an ApoH protein or a gene encoding the same in a biological sample obtained from a subject.

The term "biological sample" refers to any sample obtained from an individual capable of measuring the expression level of an ApoH protein or a gene encoding the same according to one aspect. Specifically, the biological sample may be urine. The biological sample may be prepared by treatment by a method commonly used in the art of the present invention.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.

Using the biomarker composition for diagnosing systemic lupus erythematosus according to one aspect, the expression level of the ApoH protein or the gene encoding it is measured in a urine sample of an individual suspected of having systemic lupus erythematosus, and compared with the expression level of a normal individual. By diagnosing lupus erythematosus, early diagnosis of systemic lupus erythematosus is possible through a convenient and simple method. As early diagnosis is possible and treatment is possible through the use of drugs with few side effects before tissue or organ damage becomes severe, it will be usefully utilized in the diagnosis and treatment of systemic lupus erythematosus.

Figure 1a is a result of confirming the differentially expressed protein (DEP) in urine samples of systemic lupus erythematosus (SLE) patients and healthy controls (HC) through SWATH analysis according to one aspect.
1b is a result of comparing the expression intensities of ORM1, AHSG, CP, and PTGDS, which are known as SLE markers, in urine samples of SLE patients and HC through SWATH data analysis according to one aspect.
1c is a result of comparing the expression intensity of ApoH in urine samples of SLE patients and HC through SWATH data analysis according to one aspect.
Figure 2a shows the expression of ApoH in urine samples of a healthy control group (HC), a systemic lupus erythematosus patient without nephritis (SLE), and a systemic lupus erythematosus patient with nephritis (LN) through an ELISA method according to one aspect. This is the result of strength comparison.
2B is a result of measuring the accuracy of ApoH for SLE diagnosis through ROC analysis according to one aspect.

Hereinafter, the present invention will be described in more detail by examples. However, these examples are intended to illustrate the present invention by way of example, and the scope of the present invention is not limited by these examples.

Example 1. Selection of test subjects

Among the patients who visited the rheumatology clinic of Asan Medical Center, a tertiary university hospital in Seoul, Korea in 2019, 76 SLE patients who met the SLE classification criteria defined by the American College of Rheumatology in 1998 were selected as experimental subjects. There were 63 female patients and 13 male patients, and the average age of the patients was 43 years (23-53 years). In addition, as a healthy control group, 25 subjects matching the sex and age of the patient group were selected. Written informed consent was obtained from all patients and control subjects before conducting the experiment, and urine samples were collected on the day of visit to Asan Medical Center after obtaining informed consent. This experiment was approved by the Institutional Review Board of Asan Medical Center (No. 2014-0568).

Example 2. Sample preparation for proteomic analysis

First, a 500 μl urine sample was lyophilized to reduce the volume in a centrifugal vacuum concentrator (LABCONCO, CentriVap, Kansas City, MO, USA). The dried sample was reconstituted with 100 μL lysis buffer by mixing 5% sodium dodecyl sulfate and 50 mM triethylammonium bicarbonate (pH 7.55, Thermo Fisher Scientific). Except for the trypsin/LysC mixture (Promega, Madison, WI, USA), the samples were subjected to S trap-based trypsin digestion according to the previously described method. The dried peptides were reconstituted with 0.1% formic acid, and absorbance at 205 nm wavelength was measured on a NanoDrop One spectrophotometer (ThermoFisher Scientific) to measure the peptide concentration. 40 μg peptides from each sample were assigned to individual assays and dried. All remaining peptides were combined for spectroscopic library generation.

Example 3. in-house middle-pH reverse phase fractionation for urine proteome spectroscopy library

및 입자 크기 5 μm; Waters Corporation, USA)에서 0.5 mL/min의 유속으로 수행하였다. 이동상 A로서 10mM triethylammonium bicarbonate(TEAB, pH 8.5) 및 이동상 B로서 90% 아세토니트릴(acetonitrile; pH 10) 중 10mM TEAB를 사용 하였다. 샘플을 200 μL 이동상 A에 용해시킨 다음, 2,100 μL의 샘플 루프에 주입하였다. 5-5% B 15분, 5-45% B 62.5분, 45-60% B 5분, 60-60% B 12.5분, 60-5% B 7.5분, 5-5% B 20분의 구배를 적용한 다음 컬럼을 100% 및 50% 이동상 B로 각각 30분 동안 세척하였다. 분획 과정에서 Shimadzu Prominence(Shimadzu, Tokyo, Japan)의 fraction collector를 사용하여, 30초 마다 용리액을 수집하였다. 생성된 168개의 분획을 24개의 분획이 되도록 결합(concatenate) 방식으로 혼합하였다. 각 분획을 건조하고 사용하기 전까지 -20℃에서 보관하였다. 액체 크로마토 그래피-탠덤 질량 분석법(Liquid chromatography-tandem mass spectrometry; LC-MS / MS) 분석 전에, iRT Kit(Biognosys AG, Schlieren, Switzerland)의 펩타이드(라이브러리 샘플 및 개별 샘플)를 제조업체의 지침에 따라 모든 샘플에 추가하였다.Fractions of the combined samples were run on an XBridge C18 column (4.6 mm id Х 250 mm length; pore size 130) using a Shimadzu Prominence HPLC instrument (Shimadzu, Japan).

and particle size 5 μm; Waters Corporation, USA) at a flow rate of 0.5 mL/min. 10 mM triethylammonium bicarbonate (TEAB, pH 8.5) as mobile phase A and 10 mM TEAB in 90% acetonitrile (pH 10) as mobile phase B were used. The sample was dissolved in 200 μL mobile phase A and then injected into a 2,100 μL sample loop. Gradients of 5-5% B 15 min, 5-45% B 62.5 min, 45-60% B 5 min, 60-60% B 12.5 min, 60-5% B 7.5 min, 5-5% B 20 min. After application, the column was washed with 100% and 50% mobile phase B for 30 minutes each. In the fractionation process, the eluent was collected every 30 seconds using a fraction collector from Shimadzu Prominence (Shimadzu, Tokyo, Japan). The resulting 168 fractions were mixed in a concatenate manner to make 24 fractions. Each fraction was dried and stored at -20 °C until use. Prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, peptides (library samples and individual samples) from the iRT Kit (Biognosys AG, Schlieren, Switzerland) were all purified according to the manufacturer's instructions. added to the sample.

Example 3. Mass spectrometry analysis for quantitative proteomics

)를, 분석 칼럼으로 Eksigent 컬럼(C18-CL, 0.3 x 150 mm, 입자 크기 3 μm, 구멍 크기 120

)으로 하고 컬럼 온도는 40 ℃로 유지한 상태에서 ekspert^TM nanoLC 425(Eksigent, Dublin, CA)를 사용하였다. 샘플을 100% 용리액 A : 0.1% 포름산을 사용하여 10 ㎕/min의 유속으로 트랩 컬럼에 로딩하였다. 10분 후, 펩타이드는 용리액 A 및 용리액 B : 100% acetonitrile 중 0.1% 포름산을 사용하여 5 ㎕/min에서 87분 단위(용리액 B는 68분에 걸쳐 3 내지 25%, 5분에 걸쳐 25 내지 35%, 2분에 걸쳐 35 내지 80%, 3분 동안 80% 유지, 1분 동안 80 내지 3% 유지, 8분 동안 3% 유지)로 분리하였다. 질량 분석기는 MS1(400 ~ 1250 m/z) 및 MS2(100 ~ 1500 m/z) 스캔에 대해 각각 250 ms 및 50 ms 수집 시간, 및 15초 동적 제외로 데이터 의존 수집(data dependent acquisition; DDA) top30 모드에서 작동하였다. 롤링 충돌 에너지는 +2에서 +5까지의 전하 상태로 분획에 사용되었다. 다른 매개변수는 다음과 같이 설정하였다: 이온 소스 가스 1(GS1) 15; 이온 소스 가스 2(GS2) 20; 커튼 가스(CUR) 30; 온도 (TEM) 250 ℃; 이온 스프레이 전압 플로팅(ISVF) 5500.For in-house urine proteome spectroscopy library generation, samples were analyzed using a SCIEX TripleTOF 5600+ system mass spectrometer with the following LC-MS/MS setup: For LC separations, an Eksigent Micro Trap cartridge (ChromXP C18CL, 5 μm, 120

) as an analytical column, an Eksigent column (C18-CL, 0.3 x 150 mm, particle size 3 μm, pore size 120

) and ekspert ^TM nanoLC 425 (Eksigent, Dublin, CA) was used while maintaining the column temperature at 40 °C. Samples were loaded onto the trap column using 100% eluent A: 0.1% formic acid at a flow rate of 10 μl/min. After 10 min, the peptides were separated from eluent A and eluent B: 0.1% formic acid in 100% acetonitrile at 5 μl/min in 87 min increments (eluent B was 3 to 25% over 68 min, 25 to 35% over 5 min). %, 35-80% over 2 minutes, 80% hold for 3 minutes, 80-3% hold for 1 minute, 3% hold for 8 minutes). The mass spectrometer undergoes data dependent acquisition (DDA) with 250 ms and 50 ms acquisition times, and 15 s dynamic exclusion for MS1 (400 to 1250 m/z) and MS2 (100 to 1500 m/z) scans, respectively. It worked in top30 mode. Rolling collision energies were used for fractionation with charge states from +2 to +5. Other parameters were set as follows: ion source gas 1 (GS1) 15; ion source gas 2 (GS2) 20; curtain gas (CUR) 30; temperature (TEM) 250 °C; Ion Spray Voltage Plot (ISVF) 5500.

For individual samples, all mass spectrometry runs were run in Sequential window acquisition of all theoretical mass spectra (SWATH) mode, using 100 variable windows, according to the SCIEX Technical Notes. LC setup was performed under the same conditions except for the gradient times described above. The gradient of eluent B was 3-25% over 38 min, 25-32% over 5 min, 32-80% over 2 min, 80% hold over 3 min, 80-3% over 1 min, 3 min. An 80% retention was applied. The SWATH parameters were set as follows: lower limit m/z 400; upper limit m/z 1250; window overlap (Da) 1.0; CES was set at 5 for the small windows, 8 for the large windows, and 10 for the largest windows. MS2 spectra were collected in the 100-1500 m/z range for 2.5 ms in high-sensitivity mode, with a total cycle time of 2.8 seconds.

Example 4. in-house Generation of urine proteome spectroscopy library

All DDA raw files were retrieved from MaxQuant (PMID: 27809316) v1.6.7. 0, and the following search parameters were used: specific trypsin was selected as enzyme, carbamidomethylation of cysteine was set as fixed modification, and N-terminal protein acetylation and oxidation (M) was set as variable modification. . In addition, the false discovery rate (FDR) was set to 0.01 at both the protein and peptide spectrum match (PSM) levels, proteins identified as one or more unique peptides were used, and other settings were kept at default values. The spectroscopy library was built from MaxQuant DDA search results using Spectronaut Enterprise + x64 (13.12.200217) with default settings.

Example 5. SWATH Data Analysis

SWATH-MS data for individual samples were processed using DIA-NN 1.7.10 with an in-house urine proteome spectroscopy library. Spectra were searched with default settings, but the m/z range was 400-1250 for precursors and 100-1500 for fragment ions. Protein quantification values were obtained using the MaxLFQ algorithm (PMID: 24942700) implemented in the diann R package (https://github.com/vdemichev/diann-rpackage). Protein intensity values were filtered for precursor and protein groups passing a q-value greater than or equal to 0.01. Normalization of protein intensity values was performed in the following way:

The protein intensity value obtained by the MaxLFQ algorithm was multiplied by the protein ratio value, and the creatine concentration was calculated by dividing this value. The protein ratio is the total peptide amount quantified using nanodrops divided by the amount of peptide prepared for MS analysis.

Example 6. Measurement of urine ApoH levels

Concentrations of urine ApoH adjusted for urine creatinine concentration (ENZO, ADI-907-030A) were measured in 59 SLE patients and 25 healthy subjects using a commercially available ELISA kit (abcam, ab108814).

Example 7. Statistical Analysis

For differential analysis of protein intensity between samples, the free software Perseus (version 1.6.14.0) was used. Values of normalized protein intensity were converted to a log2 scale. Each type of sample was grouped, and a minimum of 90% was required in at least one group. Missing values were replaced with random numbers drawn from a normal distribution using default parameters (width: 0.3, downshift: 1.8). To find statistically significant differences between samples, a T-test was performed using Benjamini-Hochberg FDR (0.05 cutoff). Visualization of volcano plots and box plots was performed using InstantClue software.

Continuous values were expressed as mean (standard deviation) for parametric data and median (interquartile range) for non-parametric data. Receiver operating characteristics (ROC) analysis was used to determine the predictive value of factors associated with SLE diagnosis. The cutoff value was determined at the level at which Youden's index was maximum. A P-value of less than 0.05 was considered statistically significant in all analyses. All analyzes were performed using IBM SPSS Version 20.0 (IBM Corp., Armonk, NY, USA).

Experimental Example 1. SWATH analysis for SLE biomarker discovery

In order to select biomarker candidates, urine samples of 25 healthy controls (HC) and 76 systemic lupus erythematosus (SLE) patients were selected, and proteins were extracted by the method described in the above example, and protein qualitative and Quantitative analysis was performed.

To construct the spectroscopic library, urine samples were collected and 2,323 proteins and 8,371 peptides were found through DDA analysis. A total of 1,157 protein groups (581±31 and 479±16 in HC) were quantified in individual samples using an in-house spectroscopic library.

To identify differentially expressed proteins (DEPs) among the 1,157 quantified proteins, fold-changes and p-values were calculated by Benjamini-Hochberg FDR of two groups. As a result, 153 proteins (FDR ≤ 0.05, log2 fold change ± 1) whose expression was increased in SLE compared to HC were identified (Fig. 1a). Among the 153 DEPs identified, Alpha-1-acid glycoprotein 1 (ORM1), Alpha-2-HS-glycoprotein (AHSG), Ceruloplasmin (CP), and Prostaglandin-H2 D-isomerase (PTGDS), previously known as SLE markers, were also identified. was found, and it was confirmed that the expression was increased similar to the previously known results (Fig. 1b). Interestingly, compared to HC, it was confirmed that the expression of ApoH (beta-2 glycoprotein), previously unknown as a SLE marker, increased in the urine of SLE patients (FIG. 1c).

Experimental Example 2. Confirmation and Accuracy Measurement of Urine ApoH as SLE Diagnostic Marker

As a result of comparison with healthy controls using the ELISA method, it was confirmed that the urinary ApoH expression level increased in SLE patients with and without lupus nephritis (LN) (FIG. 2a). SLE patients had higher urinary ApoH expression levels than healthy controls [352.674 (287.631-534.075) ng/mg vs 128.768 (107.969-172.821) ng/mg, p < 0.001]. On the other hand, among SLE patients, there was no difference in urinary ApoH expression level between patients with or without LN [378.351 (327.963-623.663) ng/mg vs 326.755 (263.835-406.932) ng/mg, p = 0.066 ].

Also, the ROC analysis of the SLE results is as shown in FIG. 2B. The expression level of ApoH in urine had an AUC of 0.946 (95% CI, 0.882-1.000) at a cutoff value of 228.53 ng/mg (sensitivity 91.5%, specificity 92.0%).

Through this, it was found that systemic lupus erythematosus patients could be accurately diagnosed through the expression level of urinary ApoH, regardless of whether lupus nephritis was accompanied or not.

<110> THE ASAN FOUNDATION University of Ulsan Foundation For Industry Cooperation <120> Biomarker Composition for Diagnosing Systemic lupus erythematous and Method of providing information for diagnosis of Systemic lupus erythematous using the same <130> PN210111KR <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 345 <212> PRT <213> Homo sapiens <400> 1 Met Ile Ser Pro Val Leu Ile Leu Phe Ser Ser Phe Leu Cys His Val 1 5 10 15 Ala Ile Ala Gly Arg Thr Cys Pro Lys Pro Asp Asp Leu Pro Phe Ser 20 25 30 Thr Val Val Pro Leu Lys Thr Phe Tyr Glu Pro Gly Glu Glu Ile Thr 35 40 45 Tyr Ser Cys Lys Pro Gly Tyr Val Ser Arg Gly Gly Met Arg Lys Phe 50 55 60 Ile Cys Pro Leu Thr Gly Leu Trp Pro Ile Asn Thr Leu Lys Cys Thr 65 70 75 80 Pro Arg Val Cys Pro Phe Ala Gly Ile Leu Glu Asn Gly Ala Val Arg 85 90 95 Tyr Thr Thr Phe Glu Tyr Pro Asn Thr Ile Ser Phe Ser Cys Asn Thr 100 105 110 Gly Phe Tyr Leu Asn Gly Ala Asp Ser Ala Lys Cys Thr Glu Glu Gly 115 120 125 Lys Trp Ser Pro Glu Leu Pro Val Cys Ala Pro Ile Ile Cys Pro Pro 130 135 140 Pro Ser Ile Pro Thr Phe Ala Thr Leu Arg Val Tyr Lys Pro Ser Ala 145 150 155 160 Gly Asn Asn Ser Leu Tyr Arg Asp Thr Ala Val Phe Glu Cys Leu Pro 165 170 175 Gln His Ala Met Phe Gly Asn Asp Thr Ile Thr Cys Thr Thr His Gly 180 185 190 Asn Trp Thr Lys Leu Pro Glu Cys Arg Glu Val Lys Cys Pro Phe Pro 195 200 205 Ser Arg Pro Asp Asn Gly Phe Val Asn Tyr Pro Ala Lys Pro Thr Leu 210 215 220 Tyr Tyr Lys Asp Lys Ala Thr Phe Gly Cys His Asp Gly Tyr Ser Leu 225 230 235 240 Asp Gly Pro Glu Glu Ile Glu Cys Thr Lys Leu Gly Asn Trp Ser Ala 245 250 255 Met Pro Ser Cys Lys Ala Ser Cys Lys Val Pro Val Lys Lys Ala Thr 260 265 270 Val Val Tyr Gln Gly Glu Arg Val Lys Ile Gln Glu Lys Phe Lys Asn 275 280 285 Gly Met Leu His Gly Asp Lys Val Ser Phe Phe Cys Lys Asn Lys Glu 290 295 300 Lys Lys Cys Ser Tyr Thr Glu Asp Ala Gln Cys Ile Asp Gly Thr Ile 305 310 315 320 Glu Val Pro Lys Cys Phe Lys Glu His Ser Ser Leu Ala Phe Trp Lys 325 330 335 Thr Asp Ala Ser Asp Val Lys Pro Cys 340 345 <210> 2 <211> 326 <212> PRT <213> artificial sequence <220> <223> Mature apolipoprotein H <400> 2 Gly Arg Thr Cys Pro Lys Pro Asp Asp Leu Pro Phe Ser Thr Val Val 1 5 10 15 Pro Leu Lys Thr Phe Tyr Glu Pro Gly Glu Glu Ile Thr Tyr Ser Cys 20 25 30 Lys Pro Gly Tyr Val Ser Arg Gly Gly Met Arg Lys Phe Ile Cys Pro 35 40 45 Leu Thr Gly Leu Trp Pro Ile Asn Thr Leu Lys Cys Thr Pro Arg Val 50 55 60 Cys Pro Phe Ala Gly Ile Leu Glu Asn Gly Ala Val Arg Tyr Thr Thr 65 70 75 80 Phe Glu Tyr Pro Asn Thr Ile Ser Phe Ser Cys Asn Thr Gly Phe Tyr 85 90 95 Leu Asn Gly Ala Asp Ser Ala Lys Cys Thr Glu Glu Gly Lys Trp Ser 100 105 110 Pro Glu Leu Pro Val Cys Ala Pro Ile Ile Cys Pro Pro Pro Ser Ile 115 120 125 Pro Thr Phe Ala Thr Leu Arg Val Tyr Lys Pro Ser Ala Gly Asn Asn 130 135 140 Ser Leu Tyr Arg Asp Thr Ala Val Phe Glu Cys Leu Pro Gln His Ala 145 150 155 160 Met Phe Gly Asn Asp Thr Ile Thr Cys Thr Thr His Gly Asn Trp Thr 165 170 175 Lys Leu Pro Glu Cys Arg Glu Val Lys Cys Pro Phe Pro Ser Arg Pro 180 185 190 Asp Asn Gly Phe Val Asn Tyr Pro Ala Lys Pro Thr Leu Tyr Tyr Lys 195 200 205 Asp Lys Ala Thr Phe Gly Cys His Asp Gly Tyr Ser Leu Asp Gly Pro 210 215 220 Glu Glu Ile Glu Cys Thr Lys Leu Gly Asn Trp Ser Ala Met Pro Ser 225 230 235 240 Cys Lys Ala Ser Cys Lys Val Pro Val Lys Lys Ala Thr Val Val Tyr 245 250 255 Gln Gly Glu Arg Val Lys Ile Gln Glu Lys Phe Lys Asn Gly Met Leu 260 265 270 His Gly Asp Lys Val Ser Phe Phe Cys Lys Asn Lys Glu Lys Lys Cys 275 280 285 Ser Tyr Thr Glu Asp Ala Gln Cys Ile Asp Gly Thr Ile Glu Val Pro 290 295 300 Lys Cys Phe Lys Glu His Ser Ser Leu Ala Phe Trp Lys Thr Asp Ala 305 310 315 320 Ser Asp Val Lys Pro Cys 325

Claims (10)

Systemic lupus erythematosus comprising an agent capable of measuring the expression level of ApoH (Apolipoprotein H) protein or a gene encoding it, wherein the expression level of the ApoH protein or gene encoding it is measured in a urine sample of an individual (Systemic lupus erythematous) diagnostic composition. delete According to claim 1,
The ApoH protein is a composition for diagnosing systemic lupus erythematosus, wherein the N-terminal signal peptide is removed from the full-length ApoH protein. According to claim 3,
The composition for diagnosing systemic lupus erythematosus, wherein the ApoH protein consists of the amino acid sequence of SEQ ID NO: 2. According to claim 1,
Agents capable of measuring the expression level of the ApoH protein include a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a ligand, a peptide nucleic acid (PNA), an aptamer ( aptamer) or nanoparticles, a composition for diagnosing systemic lupus erythematosus. According to claim 1,
The agent capable of measuring the expression level of the gene encoding the ApoH protein is a primer pair, probe or antisense nucleotide that specifically binds to the gene, the composition for diagnosing systemic lupus erythematosus. A kit for diagnosing systemic lupus erythematosus, comprising the composition according to any one of claims 1 and 3 to 6. A method for providing information necessary for diagnosing systemic lupus erythematosus, comprising measuring the expression level of an ApoH protein or a gene encoding the same in a biological sample obtained from an individual, wherein the biological sample is urine. According to claim 8,
Information necessary for diagnosing systemic lupus erythematosus, further comprising diagnosing systemic lupus erythematosus when the expression level of the ApoH protein or the gene encoding it is higher than the expression level measured in a biological sample obtained from a normal individual. How to provide. delete

Images