Use of reagents for detecting anti-dlat autoantibodies in the manufacture of a product for detecting and / or diagnosing immune-mediated necrotizing myopathy

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a reagent for detecting anti-DLAT autoantibody in preparation of a product for detecting and / or diagnosing immune-mediated necrotizing myopathy. Experiments prove that DLAT can be used as a recognition antigen of an immune-mediated necrotizing myopathy related autoantibody, DLAT is a target point of the immune-mediated necrotizing myopathy autoantibody, the reactivity of the anti-DLAT autoantibody with the related antigen in dermatomyositis is 0%, the reactivity of the anti-DLAT autoantibody with other antigens HMGCR and SRP of the immune-mediated necrotizing myopathy is 0%, the anti-DLAT autoantibody is a new biological marker of the immune-mediated necrotizing myopathy, and the immune-mediated necrotizing myopathy can be detected and / or diagnosed.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of reagents for detecting anti-DLAT autoantibodies in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy. Background Technology

[0002] Idiopathic inflammatory myopathy (IIM) is a group of autoimmune-mediated connective tissue diseases, typically characterized by chronic muscle inflammation, such as muscle weakness, decreased muscle endurance, and myalgia. Based on clinical and histopathological manifestations, IIM is mainly classified into clinical subtypes such as dermatomyositis (DM), polymyositis (PM), anti-synthetase syndrome (ASSD), immune-mediated necrotizing myopathy (IMNM), inclusion body myositis (IBM), and juvenile idiopathic inflammatory myopathy (JIIM). Autoimmunity is considered to play a key role in the pathogenesis of myositis, and autoantibodies have been detected in more than 50% of IIM patients. Studies have shown that these autoantibodies recognize components of the cell nucleus and cytoplasm, and are traditionally classified into two categories: myositis-associated autoantibodies (MAAs) and myositis-specific autoantibodies (MSAs). MAAs include antibodies against PM-Scl100 (EXOSC9), PM-Scl75 (EXOSC10), KU70 (XRCC6), RO-52, cN1A (NT5C1A), Th / To, fibrillarin, and NOR90 (UBTF); MSAs include antibodies against NXP-2 (MO... RC3), SAE1, SAE2, SRP54, Jo-1(HARS), PL-7(TARS), PL-12(AARS / ALARS), EJ(GARS), OJ(IARS), OJcomplex, KS(NA RS), Ha / tyrS (YARS), Mi-2α (CHD3), Mi-2β (CHD4), TIF1γ (TRIM33), MDA-5 (IFIH1), HMGCR, and ZO (FARSA, FARSB)) antibodies.

[0003] Immune-mediated necrotizing myopathy (IMM) is a rare disease first described in 2004. Compared to other autoimmune myopathies, IMN is more likely to present with severe muscle weakness and irreversible muscle damage, resulting in a poor clinical prognosis. In 2016, the ENMC expert group proposed classifying IMN into anti-SRP, anti-HMGCR, and antibody-negative types (IMM patients with undetectable MSAs in serum are classified as antibody-negative IMNM patients). The new classification and diagnostic criteria highlight the importance of autoantibodies; the diagnosis of antibody-positive IMNM no longer relies on muscle biopsy pathology results. Furthermore, muscle biopsy is an invasive procedure and not patient-friendly. Therefore, the development of novel and highly specific biomarkers is of great value for the diagnosis and prognosis prediction of IMN.

[0004] DLAT belongs to the E2 subunit of the pyruvate dehydrogenase complex (PDC). This complex consists of three enzymes: pyruvate dehydrogenase (PDHA1; E1), dihydrolipoyl acetyltransferase (DLAT; E2), and dihydrolipoyl phosphatase (PDP1; E3), as well as some cofactors. Literature reports that anti-DLAT autoantibodies are relatively specific antibodies for primary biliary cholangitis (PCC), detectable in over 90% of PCC patients. There are also reports suggesting that anti-DLAT autoantibodies are associated with other connective tissue diseases (SLE, SSc). Currently, there are no reports on the presence of anti-DLAT autoantibodies in immune-mediated necrotizing myopathy. Summary of the Invention

[0005] The purpose of this invention is to provide the application of reagents for detecting anti-DLAT autoantibodies in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy, enriching the autoantibody profile of immune-mediated necrotizing myopathy, distinguishing immune-mediated necrotizing myopathy from other idiopathic inflammatory myopathy, specifically detecting and / or diagnosing immune-mediated necrotizing myopathy, and improving the accuracy of detection and / or diagnosis of immune-mediated necrotizing myopathy.

[0006] This invention provides the use of reagents for detecting anti-DLAT autoantibodies in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy.

[0007] This invention provides the use of reagents for detecting anti-DLAT autoantibodies in products that differentiate immune-mediated necrotizing myopathy from other idiopathic inflammatory myopathy, including dermatomyositis.

[0008] Preferably, the reagent for detecting anti-DLAT autoantibodies comprises peptides and / or biological materials capable of expressing the peptides; the biological materials include carriers, cells, or tissues;

[0009] The polypeptide is an immunogenic polypeptide that can bind to anti-DLAT autoantibodies.

[0010] Preferably, the vector includes pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors;

[0011] The cells include one or more of HEK293 cells, HeLa cells, and CHO cells;

[0012] The tissues include mammalian tissues.

[0013] Preferably, the polypeptide comprises a DLAT protein; the amino acid sequence of the DLAT protein comprises any one of a) to c).

[0014] a) The amino acid sequence shown in SEQ ID NO:1;

[0015] b) An amino acid sequence that is ≥70% and <100% identical to the amino acid sequence shown in SEQ ID NO:1, and has the function of binding to anti-DLAT autoantibodies;

[0016] c) The amino acid sequence in a) or b) is modified or mutated and has an amino acid sequence that can bind to anti-DLAT autoantibodies.

[0017] Preferably, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 includes any one of (I) to (III):

[0018] I) The nucleotide sequence shown in SEQ ID NO:2;

[0019] II) A nucleotide sequence that is ≥70% and <100% identical to the nucleotide sequence shown in SEQ ID NO:2, and that can express amino acids that bind to anti-DLAT autoantibodies;

[0020] The nucleotide sequence in III), I), or II) is modified or mutated, and can express a nucleotide sequence of amino acids that bind to anti-DLAT autoantibodies.

[0021] The present invention also provides a kit for detecting and / or diagnosing immune-mediated necrotizing myopathy, comprising reagents for detecting anti-DLAT autoantibodies and labeled antibodies.

[0022] Preferably, the reagent for detecting anti-DLAT autoantibodies comprises peptides and / or biological materials capable of expressing the peptides; the biological materials include carriers, cells, or tissues; and the peptides are immunogenic peptides capable of binding to anti-DLAT autoantibodies.

[0023] The labeled antibodies include horseradish peroxidase-labeled antibodies, alkaline phosphatase-labeled antibodies, biotin-labeled antibodies, FITC-labeled antibodies, and Alexa Fluor dye-labeled antibodies.

[0024] Preferably, the kit further includes a solid support and / or a buffer solution.

[0025] Preferably, the solid support includes a polyethylene plate, a membrane, a glass slide, magnetic beads, chromatographic packing material, a microfluidic channel, or a polyacrylamide gel.

[0026] Beneficial effects:

[0027] This invention provides the application of reagents for detecting anti-DLAT autoantibodies in the preparation of products for the detection and / or diagnosis of immune-mediated necrotizing myopathy. This invention experimentally demonstrates that DLAT can serve as a recognition antigen for autoantibodies associated with immune-mediated necrotizing myopathy, and that DLAT is a target for these autoantibodies. The anti-DLAT autoantibody showed 0% reactivity with related antigens in dermatomyositis and 0% reactivity with other immune-mediated necrotizing myopathy antigens such as HMGCR and SRP. Therefore, the anti-DLAT autoantibody is a novel biomarker for immune-mediated necrotizing myopathy, enabling its detection and / or diagnosis.

[0028] Furthermore, this invention establishes a kit for detecting and / or diagnosing immune-mediated necrotizing myopathy, using reagents for detecting anti-DLAT autoantibodies as the main component. This kit is capable of qualitative or quantitative analysis of anti-DLAT autoantibodies, is easy to operate, and improves the accuracy of detection and / or diagnosis of immune-mediated necrotizing myopathy. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0030] Figure 1 Fluorescence patterns of six signal modes in serum staining of 293T cell smears;

[0031] Figure 2Fluorescence patterns of seven signal modes in serum staining of Hep2 cell smears;

[0032] Figure 3 The results of Western blotting for identifying the presence of a certain type of autoantibody in a sample from an antibody-negative IMNM patient.

[0033] Figure 4 The result of IP capturing antigens that can bind to patient-specific antibodies;

[0034] Figure 5 To validate the results of antibody-negative IMNM patient samples and healthy subject samples using the CBA method;

[0035] Figure 6 To verify the presence of DLAT protein in the antigen captured from antibody-negative IMNM patient samples by Western blotting. Detailed Implementation

[0036] This invention provides the use of reagents for detecting anti-DLAT autoantibodies in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy.

[0037] This invention provides the use of reagents for detecting anti-DLAT autoantibodies in products that differentiate immune-mediated necrotizing myopathy from other idiopathic inflammatory myopathy, including dermatomyositis.

[0038] As one implementation method, the product of this invention includes a reagent kit.

[0039] As one embodiment, the reagent for detecting anti-DLAT autoantibodies according to the present invention includes peptides and / or biological materials capable of expressing the peptides; the biological materials include carriers, cells or tissues; and the peptides are immunogenic peptides capable of binding to anti-DLAT autoantibodies.

[0040] In one embodiment, the present invention inserts the gene encoding the polypeptide into a starting vector, transfers the obtained recombinant vector into an expression system for expression, and obtains a vector expressing the polypeptide after purification.

[0041] In one embodiment, the vectors described in this invention include pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors. In one embodiment, the expression system described in this invention preferably includes a prokaryotic expression system, a yeast expression system, a baculovirus expression system, or a mammalian cell expression system. In one embodiment, the purification described in this invention includes affinity chromatography, molecular sieve chromatography, ion exchange chromatography, or hydrophobic chromatography. This invention does not impose strict requirements on the specific purification method; conventional methods are acceptable. In one embodiment, the tissue described in this invention includes mammalian tissue. In one embodiment, the mammals described in this invention include one or more of humans, sheep, horses, bovine primates, and mice. In one embodiment, the tissue described in this invention includes muscle tissue. In one embodiment, the cells described in this invention include one or more of HEK293, HeLa, 293T cells, and CHO cells.

[0042] In one embodiment, the polypeptide of the present invention comprises a DLAT protein; the amino acid sequence of the DLAT protein comprises any one of a) to c) : a) the amino acid sequence shown in SEQ ID NO:1; b) an amino acid sequence with ≥70% and <100% identity to the amino acid sequence shown in SEQ ID NO:1, and having the function of binding to anti-DLAT autoantibodies; c) an amino acid sequence in a) or b) modified or mutated, and having the function of binding to anti-DLAT autoantibodies. In one embodiment, the ≥70% and <100% identity to the amino acid sequence shown in SEQ ID NO:1 can be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence shown in SEQ ID NO:1.

[0043] In one embodiment, the modifications described in this invention include one or more of glycosylation, phosphorylation, acetylation, methylation, and ubiquitination. In another embodiment, the mutations described in this invention include substitution, deletion, or addition of at least one amino acid.

[0044] In one embodiment, the polypeptide of the present invention further includes a tag protein attached to the N-terminus or C-terminus of the DLAT protein. In one embodiment, the tag protein of the present invention includes one or more of the following: His tag, thioredoxin, maltose-binding protein, glutathione S-transferase, flag tag, myc tag, and strep tag. The tag protein of the present invention promotes the purification, immobilization, precipitation, or identification of the polypeptide.

[0045] In one embodiment, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 includes any one of I) to III): I) the nucleotide sequence shown in SEQ ID NO:2; II) a nucleotide sequence with ≥70% and <100% identity to the nucleotide sequence shown in SEQ ID NO:2, and capable of expressing amino acids that bind to anti-DLAT autoantibodies; III) a nucleotide sequence in I) or II) modified or mutated, and capable of expressing amino acids that bind to anti-DLAT autoantibodies. In one embodiment, the ≥70% and <100% identity to the nucleotide sequence shown in SEQ ID NO:2 can be 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID NO:1.

[0046] The sequence information of SEQ ID NO:1 and SEQ ID NO:2 of this invention is as follows:

[0047] SEQ ID NO:1:MWRVCARRAQNVAPWAGLEARWTALQEVPGTPRVTSR 。

[0048]

[0049] This invention uses serum from antibody-negative IMNM patients and healthy individuals to incubate 293T and Hep2 cell slides. It was found that, compared to healthy serum, the serum of antibody-negative IMNM patients may contain a specific antibody. Immunoprecipitation and mass spectrometry were used to screen for signals on the patient serum that identified autoantibodies recognizing the DLAT antigen. Further collection of serum from myositis patients and healthy individuals was used as a control sample. Immunofluorescence staining analysis of DLAT-overexpressing cell slides revealed that anti-DLAT autoantibodies can be used to differentiate immune-mediated necrotizing myopathy from other inflammatory myopathies. DLAT can serve as one of the recognition antigens for autoantibodies in immune-mediated necrotizing myopathy, and reagents for detecting anti-DLAT autoantibodies can facilitate the auxiliary diagnosis of immune-mediated necrotizing myopathy.

[0050] The present invention also provides a kit for detecting and / or diagnosing immune-mediated necrotizing myopathy, comprising reagents for detecting anti-DLAT autoantibodies and labeled antibodies.

[0051] In one embodiment, the reagent for detecting anti-DLAT autoantibodies according to the present invention includes peptides and / or biological materials capable of expressing said peptides; said biological materials include carriers, cells, or tissues; said peptides are immunogenic peptides capable of binding to anti-DLAT autoantibodies. Descriptions of peptides, carriers, cells, and tissues have been previously described and will not be repeated here.

[0052] In one embodiment, the labeled antibodies of this invention include horseradish peroxidase-labeled antibodies, alkaline phosphatase-labeled antibodies, biotin-labeled antibodies, FITC-labeled antibodies, and Alexa Fluor dye-labeled antibodies. In another embodiment, the kit of this invention further includes a solid-phase support and / or a buffer solution. In one embodiment, the solid-phase support of this invention includes a polyethylene plate, membrane, glass slide, magnetic beads, chromatographic packing material, microfluidic channel, or polyacrylamide gel. In another embodiment, the membrane of this invention includes a nylon membrane, nitrocellulose membrane, or PVDF membrane. This invention does not have strict requirements on the specific type of buffer solution; the appropriate buffer solution can be selected according to the specific detection method of the kit.

[0053] This invention uses reagents for detecting anti-DLAT autoantibodies as the main component to establish a kit for detecting and / or diagnosing immune-mediated necrotizing myopathy. It can qualitatively or quantitatively analyze anti-DLAT autoantibodies, thereby detecting and / or diagnosing immune-mediated necrotizing myopathy, and is particularly useful for auxiliary diagnosis. When using the kit described in this invention, there are no strict requirements on the detection method used; any method well-known to this invention can be used, such as cell-based immunofluorescence (CBA), tissue-based immunofluorescence (TBA), enzyme-linked immunosorbent assay (ELISA), immunogold immunoassay, Western blotting, immunospot assay, membrane strip assay, chemiluminescence, radioimmunoassay, liquid chromatography-array assay, lateral chromatography, or flow cytometry.

[0054] To further illustrate the present invention, the application of the reagent for detecting anti-DLAT autoantibodies provided by the present invention in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0055] The samples studied in this invention were serum samples from a group of well-characterized idiopathic inflammatory myopathy patients (17 antibody-negative IMNM patients) at Qilu Hospital of Shandong Province and serum samples from 50 healthy individuals. The following autoantibodies were detected in the samples from the 17 antibody-negative IMNM patients: anti-NXP-2 (MORC3), SAE1, SAE2, SRP54, Jo-1 (HARS), PL-7 (TARS), PL-12 (AARS / ALARS), EJ (GARS), OJ (IARS), and OJ. complex, KS(NARS), Ha / tyrS(YARS), cN1A(NT5C1A), PM-Scl100(EX0SC9), PM-Scl75(EXOSC10), KU70(XRCC6), RO-52, Th / To, Fibrillarin, NOR90(UBTF), Mi-2α(CHD3), Mi-2β(C HD4), TIF1γ (TRIM33), MDA-5 (IFIH1), HMGCR, ZO (FARSA, FARSB), PM-Scl100 (EX0SC9), PM-Scl75 (EXOSC10), KU70 (XRCC6), cN1A (NT5C1A), Th / To, Fibrillarin and NOR90 (UBTF) antibodies.

[0056] The clinical characteristics of antibody-negative IMNM patients 1, 2, 3, and 7 in this embodiment of the invention are as follows:

[0057] Patient 1: Female, age of onset 41 years; no history of statin or hormone use; no rash (Heliotrope rash or Gottron's papules); asymmetric muscle weakness (weakest muscle strength score 1), affecting neck muscles and swallowing function, but not the eyelids; the patient is comatose and requires bed rest and intubation for maintenance; no arthralgia / Raynaud's syndrome / idiopathic pulmonary fibrosis / tumor; cardiac abnormalities, multiple premature atrial contractions, arrhythmia; no electromyography or MRI data; muscle biopsy showed muscle fiber necrosis, no muscle or perifascicular atrophy, no endothelial edema, HE staining showed no inflammatory cell infiltration, immunohistochemical staining showed no MHC1, MHC2 expression in the myocyte membrane, and MAC deposition in the capillary wall. Anti-RO-52 antibody was positive, and all other autoantibodies were negative. The clinical diagnosis was IMNM.

[0058] Patient 2: Male, age of onset 55 years; no history of statin or hormone use; asymmetric muscle weakness (weakest muscle strength score 4+), mildly affecting proximal upper arm muscles (score 4+), iliopsoas, quadriceps femoris, and biceps femoris, with mild impairment of swallowing function; arthralgia and Raynaud's syndrome, no idiopathic pulmonary fibrosis or tumor; normal cardiac function; electromyography showed myogenic electrophysiological changes, no neurogenic changes; MRI showed muscle group involvement; muscle biopsy showed myofiber necrosis, no muscle or perifascicular atrophy, and no endothelial edema; HE staining showed no inflammatory cell infiltration; immunohistochemical staining showed no MHC1 or MHC2 expression in the myocyte membrane, no CD8+ cell infiltration, and no MAC. Anti-RO-52 antibody was positive, and all other autoantibodies were negative. The clinical diagnosis was IMNM.

[0059] Patient 3, female, age of onset 46 years; no history of statin or hormone use; no rash (Heliotrope rash or Gottron's papules); symmetrical iliopsoas and deltoid muscle weakness (weakest muscle strength score 3), without involvement of limb muscles, neck muscles, facial muscles (score 5), or swallowing function; no eyelid muscles involved; no muscle weakness / arthralgia / Raynaud's syndrome / idiopathic pulmonary fibrosis / tumor; normal cardiac function; electromyography showed myogenic electrophysiological changes, extensive damage, especially proximal, without neurogenic involvement; MRI showed fatty degeneration of the posterior thigh and mild fascial edema in some posterior thigh muscles; muscle biopsy showed no muscle fiber necrosis, no muscle or perifascial atrophy, and no endothelial edema; HE staining showed no inflammatory cell infiltration; immunohistochemical staining showed no MHC1 or MHC2 expression in the myocyte membrane, no CD8+ cell infiltration, and no MAC. All antibody tests were negative, and the clinical diagnosis was autoantibody-negative IMNM.

[0060] Patient 7: Female, age of onset 67 years; no history of statin use, but had received hormone and intravenous immunoglobulin therapy, and plasma exchange therapy; no rash (Heliotrope rash or Gottron's papules); symmetrical muscle weakness (weakest muscle strength score 2), affecting neck muscles and proximal muscles of the limbs (scores 3-0, especially the lower limbs), facial muscles (score 4), and swallowing function; no arthralgia / Raynaud's syndrome / idiopathic pulmonary fibrosis, but was diagnosed with sigmoid colon malignancy four months after onset; cardiac abnormalities. The patient presented with atrial fibrillation; electromyography showed myogenic and neurogenic electrophysiological changes, and magnetic resonance imaging showed diffuse atrophy and inflammation of the affected muscle groups; muscle biopsy showed no myofiber necrosis, muscle and perifascicular atrophy, or endothelial edema; HE staining showed no inflammatory cell infiltration, and immunohistochemical staining showed diffuse expression of MHC1 in the myocyte membrane, no CD8+ cell infiltration, and a small amount of MAC in the capillary wall; all antibody tests were negative, and the clinical diagnosis was autoantibody-negative IMNM.

[0061] Example 1

[0062] Cellular indirect immunofluorescence assay was used to screen antibody-negative IMNM patient samples and healthy subject samples.

[0063] 1.293T and Hep2 cell crawling slices

[0064] Using 10% FBS-DMEM high-glucose medium, 293T cells and Hep2 cells were seeded on the bottom of a 6cm×6cm slab in a 37℃, 5% CO2 cell culture incubator. When the cell density reached 30%–40%, the cells were fixed with 2% paraformaldehyde, washed with 1×PBS, dried, and stored at -20℃ for later use.

[0065] 2. Serum staining

[0066] Serum from 17 antibody-negative IMNM patients and 50 normal individuals were used as test samples. The serum was diluted with Hank's buffer at a volume ratio of 1:50 and incubated on 293T cell and Hep2 cell slides at room temperature for 1 hour. After washing three times with Hank's buffer, the samples were incubated with FITC-labeled anti-human IgG secondary antibody diluted 1:200 with Hank's buffer for 1 hour. After washing three times with Hank's buffer, the samples were observed under a microscope.

[0067] Based on the signal patterns observed on 293T cell smears, six patterns were identified, with corresponding staining results as follows: Figure 1 As shown; where, Figure 1From left to right, the patterns are: Pattern 1 (few coarse granules in the nucleus), Pattern 2 (coarse granules in the cytoplasm), Pattern 3 (fine granules in the cytoplasm), Pattern 4 (multiple granules in the nucleus), Pattern 5 (strong signal in the nucleus), and Pattern 6 (faint diffuse staining in the cell body, considered negative based on its abundant presence in normal serum). The staining results of the test samples on 293T cells are shown in Table 1.

[0068] Based on the signal pattern of serum staining from Hep2 cell smears, seven patterns were identified, with the corresponding staining results as follows: Figure 2 As shown; where, Figure 2 From left to right, the patterns are: Pattern 1 (few coarse granules in the nucleus), Pattern 2 (fine granules in the cytoplasm), Pattern 3 (obvious diffuse staining in the cytoplasm), Pattern 4 (multiple granules in the nucleus), Pattern 5 (strong signal in the nucleus), Pattern 6 (faint diffuse staining in the nucleus, considered negative based on its abundance in normal serum), and Pattern 7 (relatively dispersed coarse granule staining in the cytoplasm). The staining results of the test samples on Hep2 cells are shown in Table 2.

[0069] Table 1. Staining results of the test samples on 293T cells.

[0070]

[0071] Note: Serum samples from one healthy individual were not captured in the image.

[0072] Table 2. Staining results of the test samples on Hep2 cells.

[0073]

[0074] Note: Serum samples from 11 healthy individuals were not captured in images.

[0075] As shown in Tables 1 and 2, a relatively large number of antibody-negative IMNM patients exhibited Pattern II staining on 293T cells. Serum samples from 6 antibody-negative IMNM patients showed Pattern II staining on 293T cells, and serum samples from 5 antibody-negative IMNM patients also showed Pattern II staining on Hep2 cells. Furthermore, the serum samples from the 5 antibody-negative IMNM patients in Table 2 showed Pattern II staining on 293T cells. Therefore, the presence of a specific antibody in the Pattern II-like antibody-negative IMNM patient samples on 293T cells and Hep2 cells suggests the possible presence of a specific antibody.

[0076] Example 2

[0077] Western blotting determined that pattern 2 signals in serum samples from antibody-negative IMNM patients represented a specific class of antibodies.

[0078] 1. Extraction of total protein and determination of protein content in 293T and Hep2 cells

[0079] (1) Using 10% FBS-DMEM high glucose medium, 293T cells and Hep2 cells were cultured in a 37℃, 5% CO2 cell culture incubator. When the cells were confluent, the culture medium was discarded, and the cells were washed twice with PBS pre-cooled at 4℃. 500 μL of cell lysis buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris-HCl at pH 7.5, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 5% glycerol) was added to each dish and incubated on ice for 10 min.

[0080] (2) Use a cell scraper to scrape the cells off and transfer them to a 1.5 mL centrifuge tube. Place the tube on ice and sonicate at 10% power for 1 min (Ningbo Xinzhi: JY92-IIN). Centrifuge at 12000 rpm and 4℃ for 20 min. Collect the supernatant, add protease inhibitors, aliquot, and store at -20℃ for later use.

[0081] (3) Protein concentration was determined using the BCA protein concentration assay kit (Beyotime).

[0082] 2. Western blotting detection

[0083] (1) Gel preparation: Use a gradient gel generation system (Zhongke Tairui) to prepare 8-15% gradient gel, which can be used after the gel solidifies;

[0084] (2) Sample loading, electrophoresis and transfer: Take the Hep2 cells and 293T cell proteins prepared in step 1 for sample loading, with a sample loading amount of 20 μg per well. After electrophoresis, perform wet transfer at 300 mA for 90 min. After transfer, block with 5% skim milk powder at room temperature for 1 h. Cut the membrane strip into 10 sets for later use.

[0085] (3) Antibody incubation and color development: Serum from patients with IMNM showing pattern II-like autoantibody negativity on 293T cells and Hep2 cells as determined in Example 1, serum from 2 healthy subjects, and serum from 2 RO52 autoantibody positive subjects were diluted 600-fold using TBST. The diluted serum was used as the primary antibody and incubated overnight at 4°C to each membrane strip. The next day, the membrane strips were washed 3 times with TBST for 5 min each time. HRP-labeled goat anti-human secondary antibody (manufacturer: Jackson, catalog number: 109-035-098, dilution ratio 1:5000) was added and incubated at room temperature for 1 h. The membrane strips were washed 3 times with TBST for 5 min each time. ECL chemiluminescence solution was added for color development and photography. The results are as follows. Figure 3 As shown; where Figure 3The sample loaded in lane 1 was 293T cell lysis protein; the sample loaded in lane 2 was Hep2 cell lysis protein. The marker was purchased from Thermo Fisher Scientific, catalog number 26616.

[0086] according to Figure 3 It can be seen that the positive control RO52 antibody-positive serum sample has a distinct band at around 52KD. Compared with the serum samples of the positive control group and healthy subjects, the samples of patients 1, 2, and 3 have a distinct band at 70KD, while the samples of patients 4, 5, and 6 do not have a band at 70KD. The strong bands in the samples of patients 1, 2, and 3 are in the same position, suggesting that these three patients may have the same autoantibodies in their samples.

[0087] Example 3

[0088] Immunoprecipitation (IP) was used to capture antigens recognized by autoantibodies in serum samples from antibody-negative IMNM patients.

[0089] The serum used in this embodiment was from patient 1, patient 2, patient 3, and 3 healthy subjects. The specific operating steps are as follows:

[0090] (1) Take 6 dishes of 293T cells and Hep2 cells, discard the supernatant, add 2% paraformaldehyde to fix at room temperature for 10 min, and wash twice with 1×Hank's.

[0091] (2) Serum incubation: Dilute the serum 200 times with 1×Hank's (i.e., 25μL serum + 5mL 1×Hank's) and add it to the cells in step (1). Incubate overnight at 4°C and discard the supernatant the next day.

[0092] (3) Add 500 μL LIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris, 1% Triton X-100, 0.1% SDS; pH 7.5), lyse the cells on ice for 10 min, scrape the cells off with a cell scraper and collect them, add protease inhibitor, centrifuge at 6000 rpm for 5 min, and collect the supernatant;

[0093] (4) Add 500 μL of RIPA lysis buffer (150 mM NaCl, 1 mM EDTA, 100 mM Tris, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate; pH 7.5) to the precipitate, lyse on ice for 10 min, sonicate at 1% power for 1 min, centrifuge at 12000 rpm for 5 min, collect the supernatant, mix it with the supernatant collected in step (3), and determine the protein concentration;

[0094] (5) Magnetic bead treatment (Bimake Protein A / G magnetic beads): Take 50 μL of magnetic beads for each reaction, wash twice with magnetic bead binding / washing buffer (50 mM Tris, 150 mM NaCl, 0.1% Triton X-100) for 5 min each time, and add 1% BSA to block for 2 h.

[0095] (6) Discard the blocking BSA, add the mixed supernatant (i.e. antigen-antibody complex) collected in step (4), and incubate overnight at 4°C by rotation.

[0096] (7) Elution: Discard the supernatant, wash three times with magnetic bead binding / washing buffer, 5 min each time; add 100 μL of glycine with pH 2.5, rotate at room temperature for 20 min, then place in a 70℃ metal bath and boil for 10 min. Collect the eluent, neutralize with 3M NaOH, and this is the IP eluted sample.

[0097] (8) SDS-PAGE: Prepare 8-15% gradient gel using a gradient gel generation system. Take 20 μL of each IP elution sample obtained above, add loading buffer, and then load the sample.

[0098] (9) Silver staining: Silver staining was performed using the THERMO Pierce™ Silver Stain Kit. The target bands were observed, and the results are as follows: Figure 4 As shown; where A represents antigen proteins captured from 293T cell lysates of patient and healthy subject samples; and B represents antigen proteins captured from Hep2 cell lysates of patient and healthy subject samples. According to... Figure 4 It can be seen that serum samples from patients 1, 2, and 3 captured antigen proteins in 293T cell lysates. Compared with serum samples from three healthy subjects, the serum samples from patients 1 and 2 showed distinct protein bands at the 70KD position, which is consistent with the Western blotting results. No distinct bands were observed in the patient 3 sample. The results of antigen protein capture in Hep2 cell lysates from serum samples from patients 1, 2, and 3 are consistent with the results of antigen protein capture in 293T cell lysates.

[0099] Example 4

[0100] Mass spectrometry analysis

[0101] Using a clean blade, the 70KD band of the IP gel from Patient 1, Patient 2, and Healthy Subject 1 and Healthy Subject 2 samples in Example 3 was cut and mass spectrometry analysis was performed by Beijing Novogene. The target protein was selected according to the following principles: (1) The abundance values ​​of the two experimental samples in the protein label were sorted, and the protein with higher abundance in both experimental groups was selected; (2) Starting with the protein with high abundance, the protein with a large difference in abundance value between the experimental group and the control group was selected. If the control group has a value, the mean values ​​of the experimental group and the control group were calculated first, and then the ratio between the experimental group and the control group was calculated; if the control group has no value, the lowest value in this table was assigned to the control group, and then the ratio between the experimental group and the control group was calculated. When the ratio between the two groups of most proteins is close to 1, the protein with a ratio greater than 10 or 100 was selected; (3) The molecular weight was selected to be close to 70KD, or an integer multiple of 70KD, or the molecular weight of the complex that may be formed was close to 70KD. For myositis antibodies, enzyme proteins were preferred.

[0102] Results: Based on the above analysis, DLAT protein, also known as PDC-E2 or PDCE2, was selected. It had the highest abundance in the mass spectrometry results and is a subunit of the pyruvate dehydrogenase complex in the mitochondrial respiratory chain. Its amino acid sequence is shown in SEQ ID NO:1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:2.

[0103] Example 5

[0104] CBA method confirmed the presence of anti-DLAT antibodies in serum samples from antibody-negative IMNM patients.

[0105] 1. Construction of recombinant vectors

[0106] Shanghai Sangon Biotech was commissioned to synthesize and clone the CDS segment of the DLAT protein encoding gene and the other four genes selected based on mass spectrometry results into the pcDNA3.4 plasmid vector, resulting in five eukaryotic cell overexpression vectors.

[0107] 2. Cell transfection

[0108] (1) 293T cell culture: DMEM high glucose medium and FBS were mixed at a volume ratio of 9:1 to prepare 10% FBS-DMEM high glucose medium. When the cells were fully covered, they were passaged at a ratio of 1:5 to 1:6 and cultured overnight in a cell culture incubator at 37°C and 5% CO2.

[0109] (2) When the cell density is 30% to 40%, the pcDNA3.4-DLAT recombinant vector and four other recombinant vectors are transferred into the cells.

[0110] 3. Fixing the climbing plate

[0111] (1) Washing: Wash the cells that have grown for 48 hours twice with PBS;

[0112] (2) Fixation: Add acetone and fix for 5 min;

[0113] (3) Washing: The acetone-fixed slides were washed twice with PBS and dried for later use.

[0114] 4. Serum staining of subjects

[0115] (1) Washing of cell smears: Wash the cell smears obtained in step 3 with PBST;

[0116] (2) Sample incubation: The serum of patients 1, 2, 3 and 3 healthy subjects was diluted at a volume ratio of 1:10 and added to the slide, and incubated at room temperature for 1 hour;

[0117] (3) Washing: Wash 3 times with PBST, 5 min each time;

[0118] (4) Secondary antibody incubation: Add FITC-labeled secondary antibody and incubate at room temperature for 60 min;

[0119] (5) Washing: Wash 3 times with PBST, 5 min each time;

[0120] (6) Observe the results under a microscope, take pictures, and the results are as follows: Figure 5 As shown.

[0121] according to Figure 5 It can be seen that the serum samples from patients 1, 2, and 3 reacted with cells overexpressing DLAT antigen, producing coarse granular or patchy signals; the samples from healthy subjects 1, 2, and 3 did not react with cells overexpressing DLAT antigen (the results are not shown because the serum samples from patients 1-3 and healthy subjects 1-3 did not react with cells overexpressing the other four antigens). Therefore, the novel autoantibodies present in the serum samples of patients 1, 2, and 3 are autoantibodies that recognize DLAT antigen.

[0122] Example 6

[0123] 1. Association between serum anti-DLAT autoantibodies and IMNM in patients with myositis

[0124] To investigate whether anti-DLAT antibodies are serum-specific for IMNM, serum samples from 17 DM patients, 9 IMNM autoantibody-positive serum samples (5 anti-HMGCR positive, 4 anti-SRP antibody positive), 19 psoriasis patients, 14 ANCA-associated vasculitis patients, 73 rheumatoid arthritis patients, 47 connective tissue diseases, 63 systemic lupus erythematosus patients, 16 anti-SAA antibody-positive autoimmune diseases, 3 antiphospholipid antibody syndrome patients, 2 systemic sclerosis patients, 3 autoimmune hepatitis patients, 96 antibody-negative IMNM patients, and 100 healthy subjects were analyzed by immunofluorescence staining using DLAT-overexpressing cell smears prepared in Example 5. The results are shown in Table 3.

[0125] Table 3. Detection rate of anti-DLAT autoantibodies in different samples

[0126]

[0127]

[0128] As shown in Table 3, the sera of DM patients did not show reactivity with DLAT. A relatively high frequency of DLAT autoantibodies was confirmed in the independent IMNM sera cohort, with 15.63% of sera reacting with DLAT (5 strongly positive, 8 moderately positive, and 2 weakly positive samples), meaning that 15.63% of antibody-negative IMNM patients were positive for anti-DLAT antibodies. No reactivity with DLAT was shown in the IMNM autoantibody-positive sera cohort (positive for anti-HMGCR and anti-SRP antibodies). Except for one patient in the rheumatoid arthritis cohort, no reactivity with DLAT was observed in the other disease cohorts. The sera of 100 healthy subjects also did not contain anti-DLAT autoantibodies. According to literature reports, anti-HMGCR type IMNM patients account for 26% of IMNM patients, and anti-SRP type IMNM patients account for 39% of IMNM patients (Allenbach Y, Mammen AL, Benveniste O, et al. 224th ENMC International Workshop: Clinico-sero-pathological classification of immune-mediated necrotizing myopathies Zandvoort, The Netherlands, 14–16 October 2016 [J]. Neuromuscular disorders, 2018, 28(1): 87-99.). Anti-DLAT antibodies were also detected at a high frequency of 15.63% in antibody-negative IMNM patients.

[0129] 2. Sensitivity and specificity analysis of anti-DLAT autoantibodies

[0130] ROC curves are a method commonly used in clinical medicine and epidemiological studies to evaluate diagnostic accuracy and determine critical value points. Since the indirect immunofluorescence method used in step 1 to detect DLAT antibodies is a qualitative method, the correlation data analysis results between the presence of anti-DLAT autoantibodies in human body fluids and whether or not IMNM exists are shown in Tables 4 and 5.

[0131] Table 4. Clinical evaluation results of anti-DLAT antibodies

[0132]

[0133] Table 5. Analysis of various indicators in the clinical evaluation test results of anti-DLAT antibodies.

[0134]

[0135]

[0136] As shown in Tables 4 and 5, anti-DLAT antibodies have a specificity of 99.72% and a sensitivity of 14.29% when used to diagnose IMNM. A positive anti-DLAT antibody result indicates high reliability in diagnosing IMNM. Therefore, anti-DLAT autoantibodies can be used to diagnose IMNM and differentiate it from other inflammatory myopathies. The presence of anti-DLAT autoantibodies is a novel biomarker for IMNM.

[0137] Example 7

[0138] Western blotting of Hep2 protein was used to verify the presence of DLAT antibodies in patient samples.

[0139] To verify the presence of DLAT autoantibodies in the DLAT autoantibody-positive patient samples screened in Example 6 and to confirm that DLAT antigens could be captured from cell lysates, following the steps in Example 3, serum samples from patients 1, 2, and 3 of Example 3 were taken. One of the five patient serum samples screened in Example 6 (patient 7) and two randomly selected healthy subjects (healthy subject 4 and healthy subject 5) were also taken. Antigen proteins were captured from Hep2 cell lysates and verified by Western blotting. The verification steps are as follows:

[0140] (1) Gel preparation: Use a gradient gel generation system (Zhongke Tairui) to prepare 8-15% gradient gel, which can be used after the gel solidifies;

[0141] (2) Sample loading, electrophoresis and transfer: Take the antigen proteins captured from Hep2 cell lysate by patients 1, 2 and 3 in Example 3, and the antigen proteins captured from Hep2 cell lysate by serum samples from patients 7 and healthy subjects 4-5 selected in Example 6 (prepared according to the steps in Example 3), load the samples, perform wet transfer after electrophoresis, and transfer the membrane at 300mA for 90min; after the transfer, block with 5% skim milk powder at room temperature for 1h, and set aside for later use.

[0142] (3) Antibody incubation and color development: Commercial DLAT antibody (sc-271534, Santa Cruz) was diluted 1:1000 with TBST as the primary antibody and incubated overnight at 4°C. The next day, the antibody was washed three times with TBST for 5 minutes each time. HRP-labeled goat anti-mouse IgG secondary antibody (manufacturer: Jackson, catalog number: 115-035-166, dilution ratio: 1:5000) was added and incubated at room temperature for 1 hour. The antibody was washed three times with TBST for 5 minutes each time. ECL chemiluminescence solution was added for color development and photographing. The results are as follows: Figure 6 As shown.

[0143] according to Figure 6 It can be seen that DLAT signals were detected in the IP elution samples of the four anti-DLAT antibody-positive patients described in this invention; no DLAT signals were found in the IP elution samples of the two healthy subjects.

[0144] As can be seen from the above, anti-DLAT autoantibodies can be detected in IMNM patient samples. Anti-DLAT autoantibodies are a novel biomarker for IMNM and have an auxiliary diagnostic role in IMNM.

[0145] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of reagents for detecting anti-DLAT autoantibodies in the preparation of products for detecting and / or diagnosing immune-mediated necrotizing myopathy.

2. The use of reagents for detecting anti-DLAT autoantibodies in products that differentiate immune-mediated necrotizing myopathy from other idiopathic inflammatory myopathy, including dermatomyositis.

3. The application according to claim 1 or 2, characterized in that, The reagent for detecting anti-DLAT autoantibodies includes peptides and / or biological materials capable of expressing the peptides; the biological materials include carriers, cells, or tissues. The polypeptide is an immunogenic polypeptide that can bind to anti-DLAT autoantibodies.

4. The application according to claim 3, characterized in that, The vectors include pTriEx series vectors, pCDNA3 series vectors, pET series vectors, or pBac series vectors; The cells include one or more of HEK293 cells, HeLa cells, and CHO cells; The tissues include mammalian tissues.

5. The application according to claim 3, characterized in that, The polypeptide includes DLAT protein; the amino acid sequence of the DLAT protein is the amino acid sequence shown in SEQ ID NO:

1.

6. The application according to claim 5, characterized in that, The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:1 is the nucleotide sequence shown in SEQ ID NO:2.

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