WO2008007600A1 - Method for detection of sle - Google Patents

Abstract

Disclosed is a method for detecting systemic lupus erythematosus (SLE) having higher sensitivity and specificity compared to a conventional detection method which determines an anti-Sm antibody and/or an anti-dsDNA antibody. The method comprises detecting an anti-aldolase A antibody which is a disease marker for SLE. An epitope against an anti-aldolase A antibody is located on a region lying between an amino acid residue at position-94 to an amino acid residue at position 183 from the N-terminus of an amio acid sequence constituting aldolase A (SEQ ID NO:1). The detection is conduced by using a polypeptide selected from the region and having an antigenicity against an anti-aldolase A antibody or a polypeptide containing the region as an antigenic polypeptide.

Description

 Specification

 Test method for systemic lupus erythematosus

 Technical field

 [0001] The present invention is a novel marker for systemic lupus erythematosus (SLE), and for the first time, an anti-aldolase A antibody and an epitope portion against the anti-aldolase A antibody were confirmed. It relates to the inspection method.

 [0002] This application claims priority from Japanese Patent Application No. 2006-192201, which is incorporated herein by reference.

 Background art

 [0003] Systemic lupus erythematosus (hereinafter, "SLE") is a type of collagen disease.

 Collagen disease is an autoimmune 'refractory disease of unknown origin, mainly due to inflammation of connective tissues and blood vessels, accompanied by the appearance of autoantibodies. Collagen diseases include rheumatoid arthritis (hereinafter referred to as “RA”), SLE, systemic sclerosis (scleroderma) (SSc), multiple myositis (PM) Z dermatomyositis (DM), rheumatic fever ( In addition to (RF) and polyarteritis nodosa (PN), there are many related diseases such as mixed connective tissue disease (MCTD), Shida Daren syndrome (SjS), and Behcet's disease. When collagen disease is suspected, general tests are first performed, and serological tests such as antinuclear antibodies are performed for a definitive diagnosis.

 [0004] SLE is a systemic autoimmune disease thought to be manifested by a wide range of abnormalities in immune regulation (Wakeland et al, Immunity 15, 97-408 (2001); Marrack et al, Nat. Med. 7, 899-905 (2001)). Autoantibodies against nuclear proteins, DNA, phospholipids, etc. appear in patients with SLE. Currently, methods for measuring anti-Sm antibodies and anti-dsDNA antibodies are commonly used for SLE testing. Although anti-Sm antibodies are highly specific for SLE, the sensitivity is 5-30%, and anti-Sm antibodies alone are not useful for diagnosis of SLE. Also, the test results for anti-dsDNA antibodies have a low specificity for SLE, a force that correlates with disease activity.

[0005] On the other hand, an anti-aldolase antibody can be used as a marker for RA, which is a type of collagen disease. (Patent Document 1). In addition, it is disclosed that it is useful as an antigenic polypeptide against an anti-aldolase antibody in a polypeptide strength RA that also has amino acid strength from the N-terminal to the 38th amino acid of human aldolase A (Patent Document 2). However, even though RA is a type of collagen disease in common with SLE, the clinical symptoms of these diseases are different, and even though each cause is regarded as a completely different disease, .

 [0006] Furthermore, there is a report focusing on anti-aldolase autoantibodies as a diagnostic marker for diabetic retinopathy (Non-patent Document 1). In addition, there has been a report on the isolation of an aldolase as a target antigen of an anti-aldolase antibody from Arno and Imama patients (Non-patent Document 2). However, these diseases are quite different from SLE.

 [0007] With regard to SLE, a method for elucidating and testing a disease marker with higher sensitivity and specificity compared to a conventional testing method using a disease marker is desired for diagnosis and prognostic estimation.

 Patent Document 1: Japanese Patent Laid-Open No. 11-94836

 Patent Document 2: JP 2000-178299 A

 Non-Patent Document 1: Bo- Young Ahn et al., Proteomics 2006, 6, 0000-0000 (www.proteomic s-journal.com)

 Non-Patent Document 2: Felix Mor et al., J. Immunology 2005, 3439-3445

 Disclosure of the invention

 Problems to be solved by the invention

[0008] The present invention relates to a test method for SLE, and provides a test method with higher sensitivity and specificity as compared with conventional test methods for measuring anti-Sm antibody and Z or anti-dsDNA antibody. To do.

 Means for solving the problem

 [0009] As a result of intensive studies to solve the above problems, the present inventors have confirmed for the first time that an anti-aldolase A antibody can be a disease marker for SLE. As a result of further investigations, the present inventors completed the present invention by discovering that the epithelial partial force against anti-aldolase A antibody, which is a marker for SLE, exists in a specific region of aldolase A.

That is, the present invention comprises the following. 1. The amino acid sequence shown in the region from position 94 to position 183 from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing (SEQ ID NO: 2), or one of the amino acid sequences shown in SEQ ID NO: 2 or Multiple amino acid strengths Region strength of amino acid sequence that is substituted, deleted, added or inserted, and has an amino acid sequence strength comprising at least 4 amino acids, and has antigenicity against anti-aldolase A antibody Polypeptide.

 2. A method for measuring an anti-aldolase A antibody, which comprises using at least one polypeptide according to item 1 above.

 3. In the measurement method, at least one of the polypeptides described in the preceding paragraph 1 is immobilized on a solid phase as an antigen peptide, and a reaction product of the immobilized antigen peptide and an anti-aldolase A antibody present in a sample. The method for measuring an anti-aldolase A antibody according to item 2 above, wherein the anti-aldolase A antibody is measured by detecting.

 4. A systemic lupus erythematosus (SLE) test method using the method for measuring an anti-aldolase A antibody described in 2 or 3 above.

 5. A method for testing SLE, wherein an antibody against one or more of the polypeptides described in the preceding item 1 is detected as a disease marker for SLE.

 6. A polypeptide containing an amino acid sequence comprising one or more amino acid strength substitutions, deletions, additions or insertions of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 in the sequence listing as an antigen peptide As a method for SLE examination by detecting a reaction product between the immobilized antigen peptide and an anti-aldolase A antibody present in a sample.

7. A method for SLE detection by immobilizing full-length aldolase A as an antigen peptide on a solid phase and detecting a reaction product of the immobilized antigen peptide and an anti-aldolase A antibody present in a sample.

 8. A method for testing SLE that further comprises measuring anti-Sm antibody and Z or anti-dsDNA antibody in addition to the SLE testing method described in 1 above, as described in any one of 4 to 7 above.

9. Polypeptide comprising an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2 are substituted, deleted, added or inserted, or the preceding paragraph 1 A reagent for SLE test comprising at least one polypeptide described in 1. The invention's effect

 [0011] When a test is performed by measuring the anti-aldolase A antibody of the present invention, a test for SLE having high sensitivity and specificity can be performed. In addition, since aldolase A polypeptide can be applied to ELISA, many specimens can be processed and SLE can be tested earlier. Furthermore, by conducting a combination of the conventional tests for anti-Sm antibody and Z or anti-dsDNA antibody in parallel, a method for testing SLE with higher sensitivity and specificity can be provided.

 Brief Description of Drawings

 FIG. 1 is a diagram showing a protein development pattern by 2D-PAGE method for a protein extract of cultured cells (HUVEC). (Example 1)

 FIG. 2 is a diagram showing the result of Western blotting when SLE patient serum is reacted with the protein developed in FIG. 1. (Example 1)

 FIG. 3 is a diagram showing the result of Western blotting when the serum of a healthy person is reacted with the protein developed in FIG. 1. (Example 1)

 FIG. 4 is a diagram showing the results of Western blotting when a sample containing an anti-aldolase antibody is reacted with the protein developed in FIG. 1. (Example 1)

 FIG. 5 shows a partial deletion mutant of aldolase A. (Example 3)

 FIG. 6 shows the results of Western blotting for confirming the epitope of anti-aldolase A antibody. (Example 3)

 FIG. 7 shows ELISA results when aldolase A is used as an antigen peptide. (Example 4)

 FIG. 8 is a diagram showing ELIS A results when aldolase A is used as an antigen peptide. The SLE patients in Fig. 7 were grouped according to the presence or absence of renal impairment. (Example 4)

 BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is NCBI accession

This is an amino acid sequence constituting human aldolase A specified by No. NP_000025.

[0014] The polypeptide of the present invention refers to a polypeptide having antigenicity to the anti-aldolase A antibody of the present invention. The anti-aldolase A antibody of the present invention may be a disease marker for SLE Antibody. Specifically, the polypeptide of the present invention is derived from the amino acid sequence (SEQ ID NO: 2) of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and the N-terminal force is also shown in the region of positions 94 to 183. A selected polypeptide, which has an antigenicity against an anti-aldolase A antibody. A polypeptide having antigenicity to an antibody is required to contain at least 4 amino acids. Therefore, the polypeptide of the present invention is a polypeptide comprising at least 4 amino acids, having an amino acid sequence selected from the above-mentioned region, and exhibiting antigenicity against anti-aldolase A antibody. The number of amino acids contained in the polypeptide is not particularly limited.

 [0015] In addition, the polypeptide of the present invention is not limited to the above, and one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, added or inserted. It may be a polypeptide having an antigenicity against an anti-aldolase A antibody, which is selected from the region strength of the amino acid sequence.

 [0016] Furthermore, if the polypeptide of the present invention can detect the anti-aldolase A antibody of the present invention, among the amino acid sequences shown in SEQ ID NO: 1 in the sequence listing, the N-terminal force also has positions 94 to No. Amino acid sequence (SEQ ID NO: 2) shown in the region of position 183 or an amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted in the amino acid sequence shown in SEQ ID NO: 2 It may be aldolase A containing the sequence. Specifically, it may be full-length enzyme A.

 [0017] The polypeptide used in the method for measuring an anti-aldolase antibody of the present invention is at least one polypeptide selected as described above. In order to improve specificity and measurement sensitivity, a plurality of types of polypeptides may be used in combination. For example, it may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 or a polypeptide containing at least 4 amino acids compatible with the polypeptide of the present invention. Further, a mixture of a plurality of types of polypeptides selected from these types may be used.

[0018] Since autoantibodies that can be produced by patients with autoimmune diseases such as SLE are polyclonal antibodies, the antigenic peptides used in the above-described method for measuring anti-Aldolase antibodies of the present invention themselves have epitopes at a plurality of locations. It may have one or may contain multiple types of antigen peptides. [0019] In the method for measuring an anti-Aldolase antibody or the method for examining SLE of the present invention, a body fluid such as serum or plasma can be used as a specimen.

 [0020] The anti-aldolase antibody measurement method of the present invention is based on an immunological technique capable of detecting an antigen-antibody reaction between the aldolase antibody and the polypeptide of the present invention. As such an immunological method, a method known per se such as latex agglutination method, Octaguchi-one method, immunochromatography method or ELISA method can be applied. An ELISA method that can process many specimens is particularly suitable. Specifically, the polypeptide of the present invention is immobilized as an antigen peptide on a solid phase, a specimen is brought into contact with the antigen peptide immobilized on the solid phase, and the antigen peptide and an anti-aldolase antibody that may be contained in the specimen. The anti-aldolase antibody can be measured by detecting the immune complex or reaction product. As a method for suppressing non-specific reaction associated with the ELISA method, a labeling substance that can be used for detection, a measuring instrument, etc., those known per se or those developed in the future can be applied.

 [0021] The present invention extends to a method for measuring an anti-aldolase antibody by using at least one polypeptide of the present invention, and a method for examining SLE using the method for measuring an anti-aldolase antibody. Sarako also extends to SLE test reagents containing at least one polypeptide of the present invention. In addition, the present invention also includes a microplate used for the ELISA method and the like, a necessary instrument such as a labeling substance, and an SLE test kit containing Z or a reagent.

 Example

 [0022] In the following, in order to deepen the understanding of the present invention, the background to the invention of the polypeptide of the present invention will be described by way of examples, and the present invention will be specifically described, but these limit the scope of the present invention. It ’s not a thing.

 [0023] (Example 1) Identification of a novel autoantigen in the serum of patients with SLE

 In SLE, vascular inflammation occurs as one of the symptoms, so HUVEC (normal human umbilical vein endothelial cell) force, which is derived from vascular endothelial cells, also extracts proteins and reacts with autoantibodies in patients with SLE Attempts were made to identify new autoantigens.

[0024] First, proteins were extracted from HUVEC, and the extracted proteins were developed as spots on the gel by 2D-PAGE (see Fig. 1). The developed protein was transferred to a PVDF membrane and reacted with the extracted protein from the patient's serum, and the antigen-antibody reaction pattern between the antibody present in the patient's serum and the developed protein was examined by Western plotting ( (See Figure 2). Compared to the Western plot pattern in healthy human serum (see Fig. 3), positive spots that were specifically found in patient serum were used as autoantigens. We also examined the Western plot pattern in samples containing anti-aldolase A antibodies (see Figure 4).

 [0025] 1) Protein extraction method from HUVEC

 HUVEC was purchased from CHAMBREX and cultured using the attached medium. After washing the proliferated HUVEC with PBS (-), peel it off with a cell scraper and collect the cells by centrifugation. Proteins were extracted from HUVEC for 2D-PAGE using a complete mammalian proteome extraction kit (Calbiochem). The extracted protein was quantified using urine serum albumin (BSA) as a standard using a protein quantification kit (RC-DC Protein Assay kit (Bio-Rad Laboratories)).

 [0026] 2) 2D—PAGE technique

 Extract 50 μg of extracted protein into 7Μ urea (urea), 2M thiourea, 4% CHAP ¾ (3— [(3— cholamidopropyl) — dimethylammonio] — 1-propane sulfonate) slow ¾r 揿, 2 mM TBP (tributyl phosphine), 0.202% bromophenol blue (BPB), and electrolyte (0.2% biolyte 3-10 (Bio-Rad)) were dissolved in a swelling solution and subjected to isoelectric focusing (IEF).

 [0027] The conditions of IEF are as follows. An 11 cm strip (ReadyStrip ™ IPG strip s pH3-10NL) was used for IEF. The strip gel was swollen with a swelling solution containing protein for 12 hours by applying a voltage of 50 V, and the protein was incorporated into the gel. After swelling, the voltage was increased to 250 V in 2 hours and then to 8,000 V in 1 hour, and 45, OOOVh was energized. The strip gel after IEF was reduced alkylated by the following procedure.

[0028] First, shake with an equilibration buffer (50 mM Tris—HCl, 6 M urea, 20% vZv glycerol, 2% SDS, pH 8.8) containing 20 mg Zml DTT, and then 25 mg / ml The mixture was shaken for 13 minutes with an equilibration buffer containing doacetamide. Subsequently, 2D electrophoresis was performed using 10% Bis-Tris Criterion ™ XT Precast gel (Bio-Rad Laboratories). Electrophoresis. The developed protein was stained with MS (Mass Spectrometry) silver dye kit (Wako Pure Chemical Industries).

 [0029] 3) Western blotting

 The HUVEC protein developed on the gel by 2D-PAGE was electrotransferred to the PVDF membrane using the above technique, and a new autoantigen that reacts with autoantibodies was searched using SLE patient serum. In order to prevent a non-specific reaction of serum, the PVDF membrane to which the protein was transferred was blocked by shaking with 5% skim milk ZPBST (PBS + 0.1% Tween 20) for 1 hour at room temperature. After the PVDF membrane was washed with PBST, the PVDF membrane was incubated at room temperature for 1 hour using SLE patient serum diluted 150-fold with PBST. After washing three times with PBST for 10 minutes, the PVDF membrane was incubated at room temperature for 1 hour using HRP-labeled anti-human IgG (GE healthcare) diluted 5,000 times with PBST. After washing with PBST for 3 minutes for 10 minutes, 7 times later, photoreaction, chemiluminescence reaction system (PerkinElmerfc) [from this, autoantigen reacting with autoantibody was detected. Since proteins are developed by 2D-PAGE, autoantigens are detected as spots.

 [0030] Gel force stained with a protein corresponding to a spot specific to patient serum was cut out, recovered as a peptide by in-gel digestion with tribsin, and analyzed by mass spectrometry. Proteins were identified by performing a database search using mass analysis data. An example of analysis software is MASCOT (Matrix science). Such a method is called immunoproteomic method.

 [0031] 4) In-gel digestion method using trypsin,

 Genore digestion was performed according to the following paper (Shevchenko A et al, Anal Chem 1996, 68, 850-8.). The trypsin-digested peptide was extracted with 5% trifluoroacetic acid (TFA), 45% distilled water (DW), 50% CH CN, and lyophilized. Then 0.1% TFA, 2% CH C

 3 3

The sample was dissolved in N, 98% DW and used as a sample for mass spectrometry.

[0032] 5) Mass spectrometry

Mass spectrometry was performed by an LC / MS analysis system that combined liquid chromatography (LC) and mass spectrometer (MS). LC is a reverse phase HPLC system using Magic 2002 ca pillary HPCL (Michrom BioResources) and the column is C—18 RP column (length 1 5 cm, id 200 mm; GL Sciences Inc.).

 [0033] Peptides were also eluted with the following solvent A and solvent B in a gradient of 5 to 65% in 30 minutes (solvent A: 0.1% formic acid containing 2:98 acetonitrile with Z distillation. Water; Solvent B: 95: 5 acetonitrile / distilled water with 0.1% formic acid). Peptides ionized via the nanospray ion source were debated using an LCQ ion trap mass spectrometer (ThermoElectron). Data were obtained with an MSZMS scan followed by a full MS scan followed by the strongest peak. MS / MS spectra were searched using the MASCOT search program (Matrix Science) against the human protein Swiss—Prot database.

 [0034] By the above-described immunoproteomics method, aldolase A was identified as a novel self-antigen of HUVEC protein extract SLE. In addition, SS-BZLa, GAPDH (glyceraldehydes-3-phosphate dehyd rogenase), hnRNP A2 / B 1 (heterogeneous nuclear rioonucleoprotein A2 / B1), "7 nexin A2 (annexin A2) (See Fig. 2.) This indicates that it is useful as a screening for self-antigens.

 (Example 2) Usefulness of anti-aldolase antibody as a marker for SLE disease

 Anti-aldolase A antibody strength against aldolase A In order to confirm whether it is useful as a disease marker for SLE, we tried to establish a detection method for anti-aldolase A antibody. For aldolase A, the gene was cloned and the recombinant protein was expressed and purified in large quantities using the E. coli expression system. The reactivity of the serum was reconfirmed by Western blotting with the recombinant protein of aldolase A and patient serum.

[0036] The aldolase A gene was amplified by PCR using a primer specific for aldolase A against a cDNA library of HEK293 (human embryonic kidney cells). The PCR product was cloned into the Ndel site of PET28, an E. coli expression vector. DNA sequence analysis confirmed that the gene sequence was correct. The recombinant protein was expressed as a protein with 6 X His fused to the N-terminus to facilitate purification. pET28 Ald-A was transformed into BL21 (DE3) codon plus RIL (Stratagene), and a large amount of recombinant aldolase A was expressed. LB culture containing transformed Escherichia coli containing 50 gZml kanamycin After culturing in the ground, the turbidity reached 600 nm force SO.6 and 0.4 mM IPTG (inducer of j8-galactosidase activity) was added, and expression was induced at 25 ° C. for 2 hours. The Escherichia coli in which expression was induced was collected by centrifugation, suspended in PBS + 1% Triton X100 + 1% protease inhibitor (Protease inhibitor cocktail (Nacalai Testa)), and disrupted by sonication. The E. coli disruption solution was separated into a supernatant and a precipitate by a high-speed centrifuge. Recombinant aldolase A was purified from the supernatant. First, Ni cephalose monofat (GE healthcare) equilibrated with PBS + lmM imidazole and the centrifugation supernatant were incubated at 4 ° C for 1 hour, transferred to a column, washed with PBS + 30 mM imidazole, Elute with PBS + 250 mM imidazole. The eluted protein was dialyzed against PBS, and the centrifuged supernatant was stored at -85 ° C. The purified protein was quantified using a protein quantification kit (Bio-Rad Laboratories) using BSA as a standard.

 Example 3 Confirmation of anti-aldolase A antibody epitope

 As described in the background art section, in Patent Document 1 and Patent Document 2, in RA, an anti-aldolase A antibody can serve as a marker, and the recognition site of the anti-aldolase A antibody is the N-terminal amino acid side of aldolase A ( 1st to 38th). Therefore, a region that can be an epitope for an anti-aldolase A antibody that can be a marker of SLE in the present invention was confirmed.

 [0038] Normal aldolase A (full-length aldolase A) is composed of 363 amino acids shown in SEQ ID NO: 1 in the sequence listing. Therefore, the amino acid region at positions 274 to 363, the amino acid region at positions 184 to 363, and the amino acid region at positions 94 to 363 of aldolase A were deleted. Three deletion mutants, aldolase AC-dels 1-3, were artificially produced (see Fig. 5). The epitope region for anti-aldolase A antibody in the serum of SLE patients was confirmed by Western blotting using full-length aldolase A and three types of deletion mutants as antigens.

 [0039] 1) Preparation of three types of deletion mutants of aldolase A C-del 1-3

In order to create three types of deletion mutants, aldolase A C-del 1 to 3, primers were prepared for each region, PCR was performed using pET28 AW-A as a saddle, and each deletion mutant was selected. Amplified. These products were cloned into the Ndel site of pET28. D The gene sequence was confirmed to be correct by NA sequence analysis. The recombinant protein was expressed as a 6 X His fusion protein at the N-terminus to facilitate purification. The respective expression vectors were named pET28Ald-AC-del1, pET28Ald-AC-del2, and pET28Ald-AC-del3. Each of the three expression vectors was transformed into BL21 (DE3) codon plus RIL (Stratagene) to express a large amount of recombinant aldolase A C-del 1-3. The transformed Escherichia coli was cultured in LB medium containing 50 gZml kanamycin, and after reaching a turbidity of 6 OOnm 6, expression was induced at 0.4 mM IPTG at 25 ° C. for 2 hours. The Escherichia coli that induced the expression was recovered by centrifugation, suspended in PBS + 1% Triton X100 + 1% Protease Inhibitor Leitester), and disrupted by ultrasonic disruption. The E. coli disruption solution was separated into a supernatant and a precipitate by a high-speed centrifuge. Recombinant aldolase A C-del 1-3 was purified from the precipitate (Inclusion body). The inclusion bodies were washed with PBS + 4% Triton X100 and then centrifuged. Next, the inclusion body was washed with distilled water and centrifuged. Extract proteins from inclusion bodies with PBS + 8M urea + 10mM DTT, dilute DTT 10 times with PBS, then incubate with Ni Sepharose scab (GE healthcare) equilibrated with PBS + lmM imidazole for 1 hour at room temperature Then, the recombinant protein was adsorbed on coconut. The protein-adsorbed coagulum was transferred to a column, washed with PBS + 8M urea + 30 mM imidazole, eluted with PBS + 8M urea + 250 mM imidazole and stored at 85 ° C. The eluted protein was quantified using a protein quantification kit (Bio-Rad Laboratories) using BSA as a standard.

2) Western blotting

For Western blotting, mix 2 ng of full-length aldolase A and aldolase A C-del 1 to 3 and use a gel (10% Bis-Tris Criterion ™ XT Precast gel (Bio-Rad Laboratories)) Electrophoresis was performed. Next, electrotransfer to PVDF membrane, blocked with 5% skim milk for 1 hour, cut into strips one lane at a time, and diluted with PBST 1,000 times. Commercially available aldolase A antibody (Santa Cruz), or 150 times with PBST The diluted serum was used for 1 hour incubation at room temperature. After washing with PBST for 10 minutes 3 times, the PVDF membrane was incubated at room temperature using HRP-labeled anti-goat antibody (Santa Cruz) or HRP-labeled anti-human IgG (GE healthcare) diluted 5,000 times with PBST. Incubated for 1 hour. 10 minutes with PBST After washing 3 times, the reacted protein was detected by a fluorescence reaction system (PerkinElmer).

 [0041] 3) Western blotting results

 In SLE patients, bands were observed for full-length aldolase 8 and aldolase A C-del 1 and C-del 2, whereas no band reaction was observed for aldolase A C-del 3. That is, the region that can be an epitope of anti-aldolase antibody observed in SLE patients should be included in the region from position 94 to position 183 (SEQ ID NO: 2) of the amino acid sequence shown in SEQ ID NO: 1 indicating aldolase A. (See Fig. 6).

 [0042] On the other hand, RA patients were similarly examined as a disease control. As a result, bands were observed in all of full-length aldolase A and aldolase A C-del 1 to 3. In other words, the region that can be an epitope of anti-aldolase antibody observed in RA patients is considered to be included in the region from position 1 to position 93 in the amino acid sequence shown in SEQ ID NO: 1 indicating aldolase A ( (See Figure 6.) This confirms that the epitope for anti-aldolase antibodies observed in SLE patients is different from the epitope for anti-aldolase antibodies observed in RA patients.

 [Example 4] Confirmation by ELISA

 The autoantigen positive rate for aldolase A was analyzed using the serum of 40 patients by ELISA, and its efficacy as a disease marker was verified. As a result, anti-aldolase A antibody was detected in about 30% of patients (see FIG. 7). It was also found that anti-aldolase antibody was positive in 64.7% of SLE patients with more severe SLE patients with nephropathy (see Figure 8). On the other hand, as a result of conducting the same analysis using the serum of 19 healthy individuals, the anti-aldolase antibody positive rate was 0% (see FIGS. 7 and 8).

 [0044] Further, as a disease control group, 49 patients with RA who were autoimmune diseases and 11 patients with polymyositis (PM) were similarly analyzed. As a result, the positive rates of anti-aldolase antibody were 8.2% and 18.2%, respectively (see Figures 7 and 8).

[0045] Full-length aldolase A expressed and purified in large quantities in the E. coli expression system is diluted to 10 µgZml with PBS, added 100 1 each to a 96-well plate (MaxiSorp Nunc), sealed, and incubated at 4 ° C. Incubate to immobilize the antigen on the plate. Next, after washing the well with PBS, 1% 100 1 PBS containing BSA was added, sealed, and shaken at room temperature for 2 hours for blocking. Samples were 40 SLE patients, 49 RA patients, 11 epilepsy patients, and 19 healthy individuals. When reacting serum with antigen, in order to absorb antibodies in the serum against E. coli protein, serum is diluted 500 times with PBST containing BL21 (DE3) codon plus RIL protein (0.1 mg / ml). Dilute and shake at room temperature for 1 hour. After blocking, the plate was washed with PBS, diluted serum was added in increments of 1001, sealed, shaken at room temperature for 1 hour, and incubated.

 [0046] Subsequently, the wells were washed 5 times with 200 μl PBST, and 111 μ-labeled anti-human IgG (GE healthcare) diluted 5,000 times with PBST was added 100 μl at a time, and sealed at room temperature. Shake for 1 hour and incubate. The wells were washed 5 times with 200 μl PBST. Add 100 μl of TMB + (Dako) to the well, seal and color at room temperature by shaking, cover 100 μl of stop solution (KPL), and absorb at 450 nm with a microplate reader. (Immuno-mini NJ-2300 microplate reader (Nalge Nunc International, Tokyo, Japan)). Absorbance Z of specimen (average value of absorbance of healthy person + 3 X standard deviation of healthy person) X 100 was calculated and graphed. From this equation, 100 binding units were taken as the cutoff value.

 [0047] From the above, regarding aldolase A, it was suggested that SLE patients and RA patients have different autoantigens. Therefore, it was confirmed that SLE patients are anti-aldolase A antibodies that recognize epitopes different from RA patients.

 Industrial applicability

[0048] As described above, when the anti-aldolase A antibody is measured and tested for SLE, the test method can be performed with higher sensitivity and specificity than the conventional test method. . Aldolase A can also be applied to ELISA methods. Furthermore, by conducting a conventional test for anti-Sm antibody and Z or anti-dsDNA antibody, a method for testing SLE with higher sensitivity and specificity can be provided. As a result, many types of specimens can be processed, and early diagnosis of SLE becomes possible. Furthermore, by enabling early diagnosis of SLE, an appropriate treatment method for SLE can be provided early.

Claims

The scope of the claims  [1] The N-terminal force of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing is also the amino acid sequence shown in the region of positions 94 to 183 (SEQ ID NO: 2), or the amino acid sequence shown in SEQ ID NO: 2 1 Anti-aldolase A antibody, characterized in that the region strength of the amino acid sequence in which one or more amino acids are substituted, deleted, added or inserted is selected, and the amino acid sequence strength includes at least 4 amino acids. A polypeptide having antigenicity against.  [2] A method for measuring an anti-aldolase A antibody, comprising using at least one of the polypeptides according to claim 1.  [3] In the measurement method, at least one of the polypeptides according to claim 1 is immobilized on a solid phase as an antigen peptide, and the immobilized antigen peptide and anti-aldolase A present in the specimen are immobilized. The method for measuring an anti-aldolase A antibody according to claim 2, wherein the anti-aldolase A antibody is measured by detecting a reaction product with the antibody.  [4] A method for examining systemic lupus erythematosus (SLE) using the method for measuring an anti-aldolase A antibody according to claim 2 or 3.  [5] A method for examining SLE, comprising detecting an antibody against one or more polypeptides according to claim 1 as a disease marker for SLE.  [6] SEQ ID NO: 2 in the sequence listing, or a polypeptide comprising an amino acid sequence in which one or more amino acids of the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, added or inserted as an antigen peptide As a method for SLE examination by detecting a reaction product between the immobilized antigen peptide and an anti-aldolase A antibody present in a sample.  [7] A method for detecting SLE by immobilizing full-length aldolase A as an antigen peptide on a solid phase and detecting a reaction product between the immobilized antigen peptide and an anti-aldolase A antibody present in a sample.  [8] In addition to the SLE test method according to any one of claims 4 to 7, an SLE test method further comprising measuring an anti-Sm antibody and a Z or anti-dsDNA antibody. [9] SEQ ID NO: 2 in the Sequence Listing, or a polypeptide comprising an amino acid sequence in which one or more amino acids in the amino acid sequence shown in SEQ ID NO: 2 are substituted, deleted, added or inserted Or an SLE test comprising at least one polypeptide according to claim 1. Vaginal reagent.