TWI357979B - Methods of diagnosing myasthenia gravis and kits t - Google Patents

Description

1357979 九、發明說明： 【發明所屬之技術領域】 本發明係有關於免疫學的範疇，包括免疫耐受性的發 展及自體免疫疾病的病理。本發明亦關於提供鑑定器官專 一性(organ-specific)自體免疫疾病’重症肌無力症，的改良 方法。 【先前技術】 自體免疫疾病是由於功能或活性異常的免疫細胞，造 成T細胞不適當的活化，使T細胞攻擊本身組織，因而促 使細胞激素（cytokines)及/或自體抗體（autoantibody)產 生’導致疾病發生與病程發展（Cotran et al.，Pathologic basis of disease 21 l-212(6th ed. 1999); Scofied，“Autoantibodies as predictors of disease，” 363:1544-1546(2004)) ° 自體 免疫係指失去自體耐受性，然而目前其機制仍不明確。自 體免疫疾病可分為全身性，會影響複數個器官或組織；或 為局部性，僅影響單一器官、器官系統或組織。 自體免疫疾病的主要特徵是產生對抗自體蛋白質之高 親和性（high affinity)自體抗體（Robinson et al.， 6tAutoantigen microarrays for multiples characterization of autoantibody responses，，， Nature Med. 8(3): 295-301(2002))。目前已知或懷疑某些自體抗體參與特定疾 病之細胞或組織損壞的發生，但大體而言，其在致病性與 疾病潛在病因之關係仍屬未知《不過，自體抗體對特定疾 i * 1357979 病如重症肌無力症之專一性致病性顯示其可應用在增進診 斷、分類及治療自體免疫疾病。因為通常在疾病的症狀發 . 生前’血清中的自體抗體便已存在許久，所以若能快速且 , 準確的偵測與特定自體免疫病症相關的自體抗體，將能在 . 發病前給予預防性的治療’甚至完全阻止自體免疫疾病的 發生。 重症肌無力症(MG)是一種神經肌肉失調症。其是因為 免疫失調而導致骨路肌之神經肌肉接點（neuromuscular junctions)的乙臨膽驗受體（acetylcholine receptor)喪失 (Cotran etal.，·?Μ；?"α(1289))。全世界估計每 1〇〇,〇〇〇 人便有 約3至超過20的人為重症肌無力症患者（Cotran et al.， supra·, Somnier, ^Increasing incidence of late-onset anti-AChR antibody-seropositive myasthenia gravis,5, Neurol, 65:928-930(2005))。而在美國則每l〇〇,〇〇〇人中，估計有約 14人為重症肌無力症患者，每年約有.36,000案例經診斷患 • 有重症肌無力症（Howard, “Myasthenia gravis: a summary,” available at http://www.myasthenia.org/information/summary.htm, last accessed October 23,2005 )。 早發性(early onset)重症肌無力症係定義為發生在40 或50歲前，好發於女性，而晚發性(late onset)重症肌無力 症在男性與女性發生的機率則相同（Cotran et al.,似 (noting that MG is most commonly observed in women “when arising before age 40 years’’））； Somnier, supra (4tthe 7 1357979 dichotomy between early-and late-onset MG was defined at age 50”）。女性的病程發展通常發生於約20至40歲，而男 性則發生於約50至60歲，雖然有時會發生的較早’但在 . 兩性通常皆較晚發生。（Cotran et al.，swpra) 在臨床上，約有2/3的重症肌無力症患者首先出現偶發 性肌(occasional muscle)無力的症狀，其大部份是發生在眼 外肌（extraocular muscles)上(Howard，此初期症狀 到後期會嚴重至眼瞼下垂及/或複視的情況發生，因而求助 於藥物的治療。約有10 %的重症肌無力症患者之肌無力是 鲁 褐限在動眼肌（ocular muscle)上(Howard，仙；7〇〇。到了後 期’許多患者會發展至經常性的肌無力症狀如每周、每天 或更頻繁。一般重症肌無力症會影響到肌肉，如顏面的控 制、咀嚼、說話、吞嚥及呼吸；在目前新的治療方法前， 常會因呼吸衰竭而導致死亡。在改進治療方法及介入重症 看護後，超過95%的患者在診斷後將可存活約5年或更多 的時間。其中約有65%的重症肌無力症患者會伴隨胸腺增 生（thymic hyperplasia)，而約15 %的患者會伴隨胸腺瘤 籲 (thymoma)的發生。 重症肌無力症患者最後都需長時間使用乙醯膽酯酶 · (cholinesterase)抑制劑及重症看護，上述治療方式可直接減 - 緩自體免疫的反應，或改變其對乙醯膽鹼受體的影響，.以 及可長時間維持周圍神經肌肉接點的功能（“Treatment of autoiummune myasthenia gravis，’’A^wro/.61:1652-1661(2003))。重症肌無力症患者通 8 1357979 常會施以抗乙醯膽酯酶劑，如乙醯膽酯酶抑制劑。治療目 的一般係以高劑量的皮質醇（corticosteroids)來降低免疫反 應，並常伴隨靜脈注射免疫球蛋白 （intravenous immunoglobulin)或血漿除去術(plasmapheresis)。在缓和免 疫反應後，可逐漸減少皮質醇的劑量，並合併使用類固醇 助減劑（steroid-sparing agent)如硫《坐嗓吟（azathioprine)或霉 紛酸酯（mycophenolate)等。在一些病例中，也可施以胸腺 切除來治療。 约有80-85%的重症肌無力症患者是以自體抗體直接攻 擊本身的於驗性乙酿膽驗受體（nicotinic acetylcholine receptor(AChR))為特徵（Richman et al., supra; Roxanis et al., “Thymic myoid cells and germinal center formation in myasthenia gravis; possible roles in pathogenesis，” J. TVewro/mwwwi?/· 125:185-197(2002))。這些自體抗體會破壞 乙醯膽鹼受體及降低成熟神經肌肉接點之肌肉終板 (muscle end-plate)的功能，如此將導致神經肌肉訊息傳導的 破壞，而產生一般重症肌無力症的肌無力症狀(Hoch et al·， “Auto-antibodies to the receptor tyrosine kinase MsSK in patients with myasthenia gravis without acetylcholine receptor antibodies,” 7(3):365-368(2001 ))。而在 病狀程度相似的患者上，其anti-AChR .的血清抗體》農度差 異極大，因此不能利用其來預測每一病患的嚴重性 (Howards，swpra) ° 且並非每一重症肌無力症的患者皆會產生抗AChR的 9 1357979 抗體(anti-AChR)(Roxanis et al.，•swprfl)。在某些病例中，並 無法偵測到anti-AChR抗體的存在，直到發病後數月或更 久才可偵測到。若比較一般型肌無力（general MG)與眼肌型 肌無力（ocular MG) ’ anti-AChR抗體較常存在於一般型肌 無力的患者上：約74%的一般型肌無力患者可偵測到 anti-AChR抗體，而54%的眼肌型肌無力患者能偵測到 anti-AChR 抗體（Howard，swpra)。且約有 10-20% — 般型肌 無力患者偵測不到anti-AChR抗體的存在，而這些無 anti-AChR抗體的患者中，約有90%的人卻具有可辨識肌 肉受體酷·胺酸激酶（muscle-specific receptor tyrosine kinase, MuSK)的自體抗體（Hoch et al.，swpra; Roxanis et al.， 仙;^)。在形成突觸時，MuSK可促使赂胺酸聚集AChRs， 並會表現於成热的神經肌肉接點(Hoch et al.，ϊΐφΓβ)。 因此，仍需要一種更好的重症無力症檢測方法。 【發明内容】 本發發提供一種檢測神經肌肉自體免疫疾病重症肌無 力症(MG)的方法。本發明至少一部份是基於重症肌無力症 患者在初期時，有專一性辨識熱休克蛋白6〇(hsP60)、熱 休克蛋白90 (HSP90)的自體抗體之發現，且上述二蛋白質 並未在先前重症肌無力症的病因學及病理發展學上提及。 因此，本發明提供一種個體檢測自體抗體的方法，包括採 取一來自該個體的生物樣本’以及檢測該生物樣本中至少 一種自體抗體的量，其中該自體抗體係專一性的與擇自以 下至少一種蛋白質鍵結:一熱休克蛋白6〇作印6〇(序列識別 1357979 號：1))、熱休克蛋白90 cx型（hsp90a(序列識別號:2))、及熱 休克蛋白90β型（hsp9〇P (序列識別號:3))。 • 自體抗體之量有許多定性及定量的測定方法，本發明 . 在一實施例中，以西方墨點法來偵測自體抗體之量。在另 - 一實施例中’以酵素聯結免疫吸附法(ELISA)來偵自體抗體 _ 之量。 本發明亦提供一種執行本發明方法之檢測套組。上述 φ 套組合併一種或複數種以上的方法來偵測至少一種的自體 抗體之上述自體抗體係專一性的與重症肌無力症特徵 陡自體抗原鍵結。在一實施例中，上述套組包括偵測至少 一種自體抗體之量的所有試劑，該自體抗體係專一性的與 擇自以下至少一種蛋白質鍵結:熱休克蛋白60(HSP60(序列 識別號.1))、熱休克蛋白9〇 a型（HSp9〇a(序列識別號:2)) 或熱休克蛋白9〇β型（HSP9〇p(序列識別號:3))。 明顯讓2明之上述和其他目的、特徵、和優點能更 • 下文特舉較佳實施例，並配合所附圖示， 細說明如下： 作咩1357979 IX. DESCRIPTION OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to the field of immunology, including the development of immune tolerance and the pathology of autoimmune diseases. The present invention also relates to an improved method for identifying an organ-specific autoimmune disease ' myasthenia gravis. [Prior Art] An autoimmune disease is an immune cell that is abnormal in function or activity, causing inappropriate activation of T cells, causing T cells to attack their own tissues, thereby promoting the production of cytokines and/or autoantibodies. 'Causes disease development and disease progression (Cotran et al., Pathologic basis of disease 21 l-212 (6th ed. 1999); Scofied, "Autoantibodies as predictors of disease," 363: 1544-1546 (2004)) ° Autologous Immune system refers to the loss of autologous tolerance, but the mechanism is still unclear. Autoimmune diseases can be classified as systemic and affect multiple organs or tissues; or local, affecting only a single organ, organ system or tissue. The main feature of autoimmune diseases is the production of high-affinity autoantibodies against autologous proteins (Robinson et al., 6t Autoantigen microarrays for multiples characterization of autoantibody responses,,, Nature Med. 8(3): 295 -301 (2002)). It is currently known or suspected that certain autoantibodies are involved in the destruction of cells or tissues of a particular disease, but in general, the relationship between pathogenicity and the underlying cause of the disease remains unknown. However, autoantibodies are specific to the disease. * 1357979 The specific pathogenicity of diseases such as myasthenia gravis indicates that it can be used to improve diagnosis, classification and treatment of autoimmune diseases. Because it usually occurs in the symptoms of the disease. The autoantibodies in the serum have existed for a long time. Therefore, if the autoantibodies related to specific autoimmune diseases can be detected quickly and accurately, they will be given before the onset of the disease. Prophylactic treatment 'even completely prevents the occurrence of autoimmune diseases. Myasthenia gravis (MG) is a neuromuscular disorder. This is due to the loss of acetylcholine receptors in neuromuscular junctions due to immune dysfunction (Cotran et al., Μ Μ; ? " α (1289)). It is estimated that every 3 〇〇 people in the world have about 3 to more than 20 people with myasthenia gravis (Cotran et al., supra·, Somnier, ^Increasing incidence of late-onset anti-AChR antibody-seropositive Myasthenia gravis, 5, Neurol, 65: 928-930 (2005)). In the United States, about 14 people are estimated to have myasthenia gravis, and about 36,000 cases are diagnosed each year. • There is myasthenia gravis (Howard, “Myasthenia gravis: a summary ," available at http://www.myasthenia.org/information/summary.htm, last accessed October 23, 2005). Early onset myasthenia gravis is defined as occurring in women before 40 or 50 years of age, and late onset myasthenia gravis is the same in both men and women (Cotran) Et al., (noting that MG is most commonly observed in women "when emergence before age 40 years'')); Somnier, supra (4tthe 7 1357979 dichotomy between early-and late-onset MG was defined at age 50") . Women's disease progression usually occurs between about 20 and 40 years of age, while men occur between about 50 and 60 years of age, although sometimes it happens earlier, but at the same time, both sexes usually occur later. (Cotran et al., swpra) Clinically, about two-thirds of patients with myasthenia gravis have first symptoms of occasional muscle weakness, most of which occur in extraocular muscles. On (Howard, this early symptom will be severe to the eyelid drooping and / or diplopia, and therefore resort to drug treatment. About 10% of myasthenia gravis patients with muscle weakness is Luma limited to the eye muscle (ocular muscle) (Howard, sen; 7 〇〇. In the later stages, many patients will develop symptoms of frequent muscle weakness such as weekly, daily or more frequent. General myasthenia gravis affects muscles, such as facial Control, chew, talk, swallow, and breath; often lead to death from respiratory failure before new treatments. More than 95% of patients will survive for about 5 years after diagnosis and improved care. Or more time. About 65% of patients with myasthenia gravis will be accompanied by thymic hyperplasia, and about 15% of patients will be accompanied by thymoma. Patients with myasthenia gravis need to use cholinesterase inhibitors and intensive care for a long time. These treatments can directly reduce or slow down the autoimmune response, or change their response to acetylcholine receptors. Effects, and the ability to maintain peripheral nerve-muscle junctions for a long time ("Treatment of autoiummune myasthenia gravis,''A^wro/.61:1652-1661 (2003)). Patients with myasthenia gravis pass 8 1357979 often An anti-acetylcholinesterase agent, such as an acetylcholinesterase inhibitor, is generally treated with high doses of corticosteroids to reduce the immune response, often accompanied by intravenous immunoglobulin or plasma. Plasmapheresis. After alleviating the immune response, the dose of cortisol can be gradually reduced, and a steroid-sparing agent such as sulfur "azathioprine" or mycophenolate can be used in combination. Etc. In some cases, thymectomy can also be used for treatment. About 80-85% of patients with myasthenia gravis are directly attacking themselves with autoantibodies. The nicotinic acetylcholine receptor (AChR) is characterized (Richman et al., supra; Roxanis et al., "Thymic myoid cells and germinal center formation in myasthenia gravis; possible roles in pathogenesis," J. TVewro /mwwwi?/· 125:185-197 (2002)). These autoantibodies destroy the acetylcholine receptor and reduce the function of the muscle end-plate of the mature neuromuscular junction, which will lead to the destruction of neuromuscular signal transmission and the general myasthenia gravis. Symptoms of muscle weakness (Hoch et al., "Auto-antibodies to the receptor tyrosine kinase MsSK in patients with myasthenia gravis without acetylcholine receptor antibodies," 7(3): 365-368 (2001)). In patients with similar degrees of disease, the anti-AChR serum antibody varies widely, so it cannot be used to predict the severity of each patient (Howards, swpra) ° and not every myasthenia gravis A patient with anti-AChR 9 13357979 antibody (anti-AChR) (Roxanis et al., • swprfl). In some cases, the presence of anti-AChR antibodies cannot be detected until several months or more after onset. If general MG and ocular MG are used, 'anti-AChR antibodies are more common in patients with general myasthenia gravis: about 74% of patients with general myasthenia gravis can detect Anti-AChR antibodies, and 54% of patients with ocular myasthenia gravis can detect anti-AChR antibodies (Howard, swpra). And about 10-20% of patients with general myasthenia gravis cannot detect the presence of anti-AChR antibodies, and about 90% of these anti-AChR antibodies have identifiable muscle receptors. Autoantibody of muscle-specific receptor tyrosine kinase (Musta) (Hoch et al., swpra; Roxanis et al., sin; ^). At the onset of synapses, MuSK promotes the accumulation of AChRs by citrulline and is expressed in the thermogenic neuromuscular junction (Hoch et al., ϊΐφΓβ). Therefore, there is still a need for a better method for detecting severe ailments. SUMMARY OF THE INVENTION The present invention provides a method for detecting neuromuscular autoimmune disease myasthenia gravis (MG). At least a part of the present invention is based on the discovery of autoantibodies of heat shock protein 6 (hsP60) and heat shock protein 90 (HSP90) in patients with myasthenia gravis at the initial stage, and the above two proteins are not It is mentioned in the etiology and pathological development of previous myasthenia gravis. Accordingly, the present invention provides a method for an individual to detect an autoantibody comprising: taking a biological sample from the individual' and detecting the amount of at least one autoantibody in the biological sample, wherein the autoantibody is specific to the system At least one of the following protein linkages: a heat shock protein 6 〇 印 6〇 (sequence recognition 1357979: 1)), heat shock protein 90 cx type (hsp90a (SEQ ID NO: 2)), and heat shock protein 90β type (hsp9〇P (sequence identification number: 3)). • The amount of autoantibody has a number of qualitative and quantitative assays, the present invention. In one embodiment, the amount of autoantibody is detected by Western blotting. In another embodiment, the amount of autoantibody is detected by enzyme-linked immunosorbent assay (ELISA). The invention also provides a test kit for performing the method of the invention. The above φ sets are combined with one or more methods to detect at least one autoantibody of the above autoantibody system specificity and myasthenia gravis characterized by steep autoantigen binding. In one embodiment, the kit comprises all reagents for detecting the amount of at least one autoantibody specific to at least one of the following proteins: heat shock protein 60 (HSP60 (sequence recognition) No. 1)), heat shock protein 9〇a type (HSp9〇a (SEQ ID NO: 2)) or heat shock protein 9〇β type (HSP9〇p (SEQ ID NO: 3)). The above and other objects, features, and advantages of the present invention will be apparent from the following description.

*〜々工、J I檢測自體抗體的方法 本發明$ — / a 法，包 一形態係提供於一個體檢測自體抗旁 本中 的與擇自下 θ杜· 一 * 一 60(hSp60(庠列1至少—種蛋白質鍵結：一熱休. 列識別號：1))、熱休克蛋白90 a型（hs] 本中至小知取一來自個體的生物樣本；以及檢測該」 種的自體抗體之量，其中該自體抗體係j 1357979 列識別號:2))、及熱休克蛋白90 β型（hsp9〇p (序列識別 號:3))。 自體免疫是一種疾病或失調的症狀，其特徵為免疫功 能異常’且通常伴隨自體抗體的產生。自體抗體為一種任 何形式的免疫球蛋白（immun〇gl〇buiin protein，也或抗 體）’如 IgG!、IgG2、IgG3、IgG4、IgA!、IgA2、IgD、IgE 或IgM等’其可專一性的與來自能產生Ig蛋白或抗體之相 同組織之蛋白的目標抗原鍵結。以自體抗體所為特徵的自 體免疫失調’包括如艾迪生氏症（自體抗體攻擊21-羥化 酶）；乳糜瀉（自體抗體攻擊組織轉榖胺醯胺酵素 (transglutaminase));第1型胰島素依賴型糖尿病（自體抗體 攻擊GAD-65及/或胰島素）；格雷氏病/曱狀腺亢進（自體抗 體攻擊促曱狀腺激素受體（thyroid-stimulating-hormone receptor));橋本氏曱狀腺炎（自體抗體攻擊甲狀腺過氧化腌 (thyroid peroxidase)及/或曱狀腺球蛋白（thyroglobulin));重 症肌無力症（自體.抗體攻擊菸鹼性乙醯膽鹼受體及/或肌肉 受體酪胺酸激酶）；古柏氏症候群（自體抗體攻擊腎小球基 底膜的第IV型膠原蛋白）；天疱瘡（自體抗體攻擊細胞黏附 因子（desmogleins));惡性貧血（自體抗體攻擊H/K ATPase);原發型膽汁性肝硬化（自體抗體攻擊E2PDS);白 斑症（自體抗體攻擊酪胺酸及/或SOX-10);多發性硬化症 (自體抗體攻擊髓構脂驗性蛋白（myelin basic protein)及/或 聽碌脂寡樹狀蛋白（myelin oligodendritic glycoprotein); 紅斑性狼瘡（自體抗體攻擊剪接體snRNA(spliceosomal 12 1357979 snRNA)，Ro/La 核醣粒（Ro/La ribonuclear particle)，組蛋 白（histone)及/或自然的DNA);乾燥综合症（自體抗體攻擊 Ro/La 核醣粒(Ro/La ribonuclear particle)及/或蕈毒鹼受體 (muscarinic receptor));類風濕性關節炎（自體抗體攻擊抗環 瓜胺酸胜肽（citrillunated cyclic peptide)及 / 或 IgM)等 (Scofield，at 1545)。*~Completion, JI method for detecting autoantibody The present invention is a method of $-/a, and a morphological form is provided for the detection of autologous anti-parallel in a body and is selected from the lower θDu·一*60(hSp60(庠1, at least one type of protein linkage: a heat break. Column identification number: 1)), heat shock protein 90 a type (hs) to obtain a biological sample from an individual; and detect the species The amount of autoantibody, wherein the autoantibody system j 1357979 column identification number: 2)), and heat shock protein 90 β type (hsp9〇p (SEQ ID NO: 3)). Autoimmune is a symptom of a disease or disorder characterized by an abnormal immune function' and usually accompanied by the production of autoantibodies. Autoantibodies are any form of immunoglobulin (immun〇gl〇buiin protein, or antibody) such as IgG!, IgG2, IgG3, IgG4, IgA!, IgA2, IgD, IgE or IgM, etc. Binding to a target antigen from a protein that produces the same tissue as the Ig protein or antibody. Autoimmune disorders characterized by autoantibodies include, for example, Addison's disease (autoantibody attack 21-hydroxylase); celiac disease (autologous antibody attack tissue transglutaminase); Type 1 insulin-dependent diabetes mellitus (autologous antibody attacks GAD-65 and/or insulin); Gracie's disease/thymoid hyperthyroidism (thyroid-stimulating-hormone receptor); Hashimoto's thyroid gland (autoantibodies attack thyroid peroxidase and/or thyroglobulin); myasthenia gravis (autologous. antibody attack nicotinic acetylcholine) And/or muscle receptor tyrosine kinase); Guber's syndrome (autologous antibody attacks type IV collagen of the glomerular basement membrane); pemphigus (autologous antibody attacks cell adhesion factor (desmogleins)); malignant Anemia (autologous antibody attack H/K ATPase); primary biliary cirrhosis (autologous antibody attack E2PDS); leukoplakia (autologous antibody attack tyrosine and/or SOX-10); multiple sclerosis ( Autologous antibody Myelin basic protein) and/or myelin oligodendritic glycoprotein; erythematosus (autologous antibody attack splice RNA snRNA (spliceosomal 12 1357979 snRNA), Ro/La ribonuclear particle) , histone (histone) and/or natural DNA); dry syndrome (autologous antibodies attack Ro/La ribonuclear particle and/or muscarinic receptor); rheumatoid Arthritis (self-antibody attack citrillunated cyclic peptide and/or IgM), etc. (Scofield, at 1545).

本發明中所提及之個體為動物，包括人類或非人類的 動物，如狗、貓、小鼠、大鼠、牛、綿羊、豬、山羊、'或 非人之靈長類，特別是實驗動物、經濟類動物及家畜。2 η施例中，哺乳類動物為人，在另一實施例中，哺乳 動物為喃齒類動物，如小鼠或大鼠。 ' 生物樣本為從個體之細胞、組織或器官所採取的任 之來源依待診斷個體所㈣ =而異，生物樣本在取得後可立刻接受分析或經貯存 再刀析。生物樣本若經貯存，則須置於適當的貯 以保持平衡並保持在4〇c、·2(Γ(：、_ π 、、’ 、Κ 氮或液離奢、由.— C或低溫液（如液態 ΐίΐ 實例中，生物樣本可為血液、血清 :實二本可為羊水或乳汁。在其 懸吁物。在另—實施射，生物樣本也 H、，’田胞 淋巴^液、、滑液、淚液或其他臨床上液 的狀態(如一穩定的抗原/抗體複合體)。上述敘述 13 1357979 於其他地方，例如，一抗原鍵結結構（antigen_binding d〇man) 為一特疋的抗原決疋位(ePit〇Pe) ’其可在複數個分子中發 現。因此’只要抗體可與這些蛋白質上所具有的抗原決定 位結合，抗體即可專一性的與該蛋白質鍵結。專一性鍵結 的特徵為選擇性的反應，通常包括與低至中度反應物量做 高親和性的鍵結。非專一性鍵結通常為一低選擇性的反 應，且是與中至高度反應物量做低親和性的鍵結。一般來 說，當鍵結至少在低於ΙΟ、·1、U^M·1、ηΓμ·1或1〇8μ·】 時，為專一性鍵結。如有需要可改變鍵結條件來減少非專 一性鍵結，但實質上並不影響專一性鍵結，此條件為本領 域所習知’且可選擇熟悉此技術領域者所採用一般技術為 適當鍵結條件。如先前的文獻及常用的技術為選擇適合的 鍵結條件。上述條件通常為設定一抗體濃度、溶液中的離 子強度、溫度、鍵結時間、非直接作用的分子（如封阻劑， 如血清蛋白、牛奶酪蛋白）之濃度等（Morgan et al., “The Matrix Effects on Kinetic Rate Constants of Antibody-Antigen interactions Reflects Solvent Viscosity,J. /mwwwo/. A/ei/z. 217:51-60(1998);及 Zhuang et al.， “Measurement of Association Rate Constant of Antibody-Antigen Interaction in Solution Based on Enzyme-Linked Immunosorbent Assay, “《/· Biosci. Bioeng. 92(4):330-336(2001)) ° A.自體抗體的偵測 本發明用定性及定量的方法來偵測自體抗體之量。定 1357979 * · 性方法為單純的偵測生物樣本中是否存在特定的自體抗 體。定量方法除了可知生物樣本中是否存在有特定的自體 • 抗體，也可計算出自體抗體的濃度。上述定性及定量方法 . 皆為習知技術。例如，西方墨點法、免疫墨點法 (immun〇bl〇tting)、免疫螢光法（immunofluorescence)、酵素 聯結免疫吸附法(ELISA)及其他可比較的技術。 1.西方墨點法 西方墨點法首先為一電泳步驟，其利用聚丙烯醯胺膠 體(polyacrylamide gel)，依蛋白質的大小及帶電荷，將有興 趣的蛋白質分離出來，並將蛋白質轉潰到一帶電荷的膜 上。轉/貝元全後開始進行雜交程序，先以封阻(block)步驟 將膜上未反應的鍵結位置填滿，以避免抗體的非專一性吸 附，之後加入一次抗體以與一或複數個目標抗原鍵結，最 後加入二次抗體，經過適當的時間反應後，其中所形成的 抗原-抗體複合體以射線照像（radi〇graphic)、發色物質 φ (chrorn〇genic)或化學呈色物質（chemiluminescem means)來 偵測。二次抗體通常可專一性的辨識所有同類型免疫球蛋 白之恆定區（Fc)。二次抗體可來自於多種的哺乳類動物， • 包括兔子、绵羊、山羊、小鼠及大鼠，但不限定必為上述 . 動物。 為了以西方墨點法來偵測可專一性辨識mg特徵性之 自體抗原的自體抗體之量，將純化或重組之一或複數個重 症肌無力症特徵性自體抗原，先以習知技術進行十二烧基 磺酸鈉-聚丙烯醯胺膠體電泳（SDS-PAGE)(參照Sambrook 15 1357979 et aL, 3Molecular cloning: A laboratory manual A8.40-A8.45(2001))(介紹許多蛋白質膠體電泳的試劑及方 法)後’將膠體中的蛋白質轉潰到硝化纖維(nitr〇cellul〇se)、 尼龍(nylon)、氟化氣亞乙稀(PVDF)，或適用於一般固定及 西方墨點法的其他膜或濾紙，上述固定及西方墨點法的標 . 準技術與習知技術相同。上述轉潰方法可為濕式（immersi〇n)' 或半乾式(semi-dry)的轉潰法’也可為其他習知技術。 . 此外，純化或重組之一或複數個自體抗原可存在於硝 化纖維(nitrocellulose)、尼龍(nylon)、氟化氟亞乙稀(pvDF) 鲁 等其他的膜或其他適用於固定及西方墨點法的其他膜或濾 紙上。若無法得到以純化或重組的目標自體抗原，也可以 習知標準技術從溶胞產物或研磨組織獲得自體抗原。 接著將蛋白質固定於膜或濾紙上，以避免在後續西方 墨點法之多次的雜交、清洗、及染色步驟中流失目標蛋白。 固疋的方法可由’熱、紫外線交聯反應（cross_linking with ultraviolet light)或其他習知方法達成（參照Sambrook et al., 3 Molecular cloning: A laboratory manual A8.52-A8.55(介紹籲 許多免疫墨點及偵測抗原/抗體複合體的方法)）。 並以缓衝溶液（如磷酸緩衝液（phosphate_buffer * s a 1 m e，P B S)等其他類似溶液）來封阻經固定的膜或濾紙上之 . 非專一性抗體鍵結位置，上述緩衝溶液中含有封阻劑，如 0.5 %(w/v)的低脂奶粉或5 %(w/v)的牛血白清蛋白（BSA) 等。封阻之後，加入一次抗體與膜或濾紙反應，在本發明 即是與取自診斷為自體免疫失調之個體的生物樣本反應。 16 1357979 在與一次抗體反應完成後，清洗上述膜或濾紙，並以 二次抗體辨識自體抗體/自體抗原複合體，上述二次抗體上 - 標記有呈色物質（chromogenic)、螢光物質（fluorogenic)或化 . 學冷光物質（chemiluminescent means)。上述之自體抗體/自 體抗原複合體可以比色法（如山葵過氧化酶（horseradish peroxidase) and TMB)或自動射線照像法(如驗性填酸酶）偵 測。若利用比色法或化學發冷法，則此顏色或螢光的量可 以冷光{貞測儀（liminometer)、分光光度計（spectrophotometer) * 或其他類似儀器來測量。若為自動射線照像法，則抗體鍵 結之量可以光密度或其他類似儀器計測量曝光的X光片得 知（參照 Sambrook et al·, 3Molecular Cloning: A laboratory manual A8.52-A8.5 5(介紹許多免疫墨點及偵測抗原/抗體複 合體的方法)）。 二次抗體可用於定性或定量分析上，其可為單株或多 株抗體，可在二次抗體上以習知的技術標記上一配位子（如 φ 生物素）或可偵測的標誌(如螢光或酵素）。較適合的標記包 括螢光物質（fluorophores)、呈色物質（chromophores)、電密 度試劑（electron-dense reagents)(如金或銀）、酵素及可專一 性與他物質鍵結的配位子。酵素如山葵過氧化酶或鹼性磷 酸酶通常是偵測其活性。例如，山葵過氧化酶可使四曱基 聯本胺（TMB)轉變成藍色並以分光分度計定量。其它較適 合的配位子及/或可偵測的標誌包括生物素、卵白素 (avidin)、或鏈黴抗生素（streptavidin)、IgG、蛋白質A及眾 多已知之附加受器-配位子組合。以習知技術進行的其他置 17 1357979 換及可能性皆為顯而易見，且皆為本發明之一形態。 在一貫施例中，以西方墨點法偵測至少一種可專一性 與一自體抗原鍵結的自體抗體之量’上述自體抗原可專一 性的與擇自下列蛋白質鍵結：熱休克蛋白60(HSP 60)、熱 休克蛋白90 α型（HSP90o〇、或熱休克蛋白90 β型（HSP90p)。 在標準的西方墨點步驟，如遠西方墨點法（far western blots) ’及其他定量墨點法皆有各種變形。熟悉此技藝人士 可依想要偵_的自體抗體、所使用自體抗原、自體抗原及/ 或一級自體抗體的來源，及任何其他實驗相關參數，選擇 適合的方法。以習知技術進行的其他置換及可能性皆為顯 而易見，且皆為本發明之一形態。 2.酵素聯結免疫吸附法(ELISA) 某些酵素聯結免疫吸附法係用於定性，其可簡單的分 析是否具有特定抗原或抗體，但酵素聯結免疫吸附法也可 用於定量上’能精確的計算抗原或抗體的濃度。因為其可 輕易的完成及自動讀取，所以酵素聯結免疫吸附法一般常 用於大量地篩選特定結合專一性的抗原或抗體。 酵素聯結免疫吸附法一開始為抗原包覆步驟，一種或 複數種目標抗原吸附於微量滴定盤(microtiter plate)的孔洞 中（參照 Kirkegaard & Perry laboratories, Inc.，Technical Guide for ELISA 9-13(2003) ， available at http://www.kpl. com/home.cfm#(last accessed November 3,2005)(介紹ELISA的各種微量滴定盤和多種將蛋白質包 覆於孔洞中的方法及試劑）。一般抗原常用的吸附缓衝液包 1357979 括 50 mM 碳酸鹽，ΡΗ=9·6 ; 10 mM Tris-HCl, pH=9.6 ; ι〇 mMPBS，pH=7,2。上述緩衝液適合用於許多蛋白質。若上 述一種或複數種抗原無法被吸附於微量滴定盤的孔洞表 面’也可使用一般商品化的微量滴定盤，其表面經過修部， 可利用各種化學方法使蛋白質以共價鍵方式鍵結於表面 上。在蛋白質的吸附過程中，時間及溫度皆為重要的影響 因子。 將目標抗原包覆於微量滴定盤上後，以封阻缓衝液清 洗微量滴定盤來去除非專一性的鍵結及降低偽陽性的發生 at 13-14)(介紹封阻的方法及試劑）。一般常用的封阻試 劑不是蛋白質溶液，如BSA(於PBS濃度介於約1 %至5 %(w/v)之間，pH=7.0)、脫脂奶粉、酪蛋白（脫脂奶粉的主要 蛋白質）、就是酷·蛋白酸(caseinate)(溶解性較酷蛋白高，以 氫氧化鈉做部份切割）、一般的血清（濃度介於約1 %至5 %(v/v)之間）及明膠（濃度約介於1 %至5 %(w/v)之間）或無 離子的清潔劑如Tween-20 TM及Triton X-100TM。 一般會選擇不影響各反應成分間低親和***互作用的 緩衝液做為清洗緩衝液(w ash buffer)，因各反應成分會影響 偵測專一性抗原-抗體作用的能力（zW. at 14-15(介紹清洗微 量滴定盤的各種方法及試劑））。清洗溶液通常具有生理緩 衝液(physiological buffer)以避免抗原及抗體變性，且可維 持酵素的活性。常使用在自然生理相同pH之緩衝液，如 PBS、Tris-saline、十一烧基σ米嗤鹽（imidazole-buffered saline) 等。在特定的分析實驗中，應依特定的偵測試驗所採方法 19 1357979 而選擇特定的緩衝液。同時，清洗緩衝液也應含有非離子 的清潔劑如Tween-20 TM及Triton X-100™等，濃度在〇 〇! 0/〇 至0·05〇/〇(ν/ν)之間’以避免反應成分間低親和性及非專_ 性的鍵結。 封阻程序後，清洗微量滴定盤的孔洞，加入一次抗體 -以與吸附於孔洞的抗原反應，本發明之一次抗體為取自診 斷為自體免疫失調個體的生物樣本。在一次抗體作用完成 後，清洗孔洞並加入已標記呈色物質（如山葵過氧化酶及 ΤΜΒ)、螢光物質或化學冷光物質（如鹼性磷酸酶）的二次抗 籲 體來偵測自體抗原-自體抗體複合體(W at 15-21(介紹抗體 的製備及使用，與一般常用的於偵測物質。最後利用冷 光俄測儀（luminometer)、分光光度計（spectrophotometer)等 其他類似儀器來計數顏色與螢光的量。 標準酵素免疫分析方法有種種變形，如競爭型 ELISA、三明治ELISA等。熟悉此技藝人士可依想偵測的 抗體、所使用的抗原、抗原及/或一次抗體的來源、及實驗 上相關的參數而選用適合的方法。以習知技術進行的其他 籲 置換及可能性皆為顯而易見，且皆為本發明之一形態。 B.臨床上的重症肌無力症 ·The individuals referred to in the present invention are animals, including human or non-human animals, such as dogs, cats, mice, rats, cows, sheep, pigs, goats, 'or non-human primates, especially experiments. Animals, economic animals and livestock. In the η embodiment, the mammal is human, and in another embodiment, the mammal is a serrata, such as a mouse or a rat. ' Biological samples are taken from the cells, tissues or organs of the individual depending on the individual (4) = the biological sample can be analyzed immediately after storage or stored. If the biological sample is stored, it should be placed in an appropriate storage to maintain balance and remain at 4〇c, ·2 (Γ(:, _ π , , ' , Κ 或 or liquid luxuriant, by - C or cryogenic liquid (In the case of liquid ΐίΐ, the biological sample can be blood or serum: the actual two can be amniotic fluid or milk. In its suspension, in another, the biological sample is also H,, 'Tianji lymphatic fluid, The state of synovial fluid, tears, or other clinical fluids (such as a stable antigen/antibody complex). The above description 13 1357979 elsewhere, for example, an antigen binding structure (antigen_binding d〇man) is a special antigen疋 position (ePit〇Pe) 'It can be found in a plurality of molecules. Therefore, as long as the antibody binds to the epitopes on these proteins, the antibody can specifically bind to the protein. Specific binding Characterized by a selective reaction, usually involving a high affinity bond with a low to moderate amount of reactants. A non-specific bond is usually a low selectivity reaction and is low affinity with moderate to high reactant amounts. Sexual bond. Generally speaking, when The junction is at least below ΙΟ,·1, U^M·1, ηΓμ·1 or 1〇8μ·], which is a specific bond. If necessary, the bonding condition can be changed to reduce the non-specific bond, but It does not affect the specific bond in nature, and the conditions are well known in the art' and the general techniques employed by those skilled in the art can be selected as suitable bonding conditions. For example, the prior literature and commonly used techniques are to select suitable keys. The above conditions are usually set to an antibody concentration, ionic strength in solution, temperature, bonding time, concentration of molecules that are not directly acting (such as blocking agents, such as serum proteins, bovine cheese protein), etc. (Morgan et al "The Matrix Effects on Kinetic Rate Constants of Antibody-Antigen interactions Reflects Solvent Viscosity, J. /mwwwo/. A/ei/z. 217:51-60 (1998); and Zhuang et al., "Measurement of Association Rate Constant of Antibody-Antigen Interaction in Solution Based on Enzyme-Linked Immunosorbent Assay, ""·· Biosci. Bioeng. 92(4): 330-336 (2001)) ° A. Detection of Autoantibodies And quantitative methods The amount of antibodies to detect autologous. 1357979 * * set of methods for the presence or absence of specific autologous antibodies simple detection in biological samples. The quantitative method calculates the concentration of autoantibodies in addition to whether a specific autologous antibody is present in the biological sample. The above qualitative and quantitative methods are all known techniques. For example, Western blotting, immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and other comparable techniques. 1. Western ink point method Western ink point method is first an electrophoresis step, which uses polyacrylamide gel, according to the size and charge of the protein, separates the protein of interest and breaks the protein to On a charged film. After the transversion/bein element is completed, the hybridization procedure is started. The unreacted bonding position on the membrane is filled in a blocking step to avoid non-specific adsorption of the antibody, and then the antibody is added once with one or more The target antigen is bonded, and finally the secondary antibody is added. After an appropriate time reaction, the antigen-antibody complex formed therein is radiant, chromophoric, or chemically colored. The substance (chemiluminescem means) is detected. Secondary antibodies typically uniquely recognize the constant regions (Fc) of all immunoglobulins of the same type. Secondary antibodies can be derived from a variety of mammals, including rabbits, sheep, goats, mice, and rats, but are not necessarily limited to the above. Animals. In order to detect the amount of autoantibodies that can uniquely identify the characteristic autoantigen of mg by Western blotting, one or more of the characteristic autoantigens of myasthenia gravis will be purified or recombined. Techniques were carried out on sodium dodecyl sulfonate-polypropylene guanamine colloidal electrophoresis (SDS-PAGE) (see Sambrook 15 1357979 et aL, 3Molecular cloning: A laboratory manual A8.40-A8.45 (2001)) (introduction of many proteins) Colloidal electrophoresis reagents and methods) after 'crushing the protein in the colloid to nitrocellulose (nitr〇cellul〇se), nylon (nylon), fluorinated ethylene ethylene (PVDF), or suitable for general fixed and western ink Other membranes or filter papers of the dot method, the above-mentioned fixed and western ink dot method standards are the same as the conventional techniques. The above-described tumbling method may be a wet immersion or a semi-dry tumbling method, and may be other conventional techniques. In addition, one or more of the autoantigens may be present in other membranes such as nitrocellulose, nylon, fluorinated fluoroethylene (pvDF) or other suitable for stationary and western inks. Point on other membranes or filter paper. If a target autoantigen is purified or recombined, standard methods can also be used to obtain autoantigens from lysates or milled tissues. The protein is then immobilized on a membrane or filter paper to avoid loss of the target protein during multiple hybridization, washing, and staining steps following subsequent Western blotting. The method of solidification can be achieved by 'hot_linking with ultraviolet light' or other conventional methods (refer to Sambrook et al., 3 Molecular cloning: A laboratory manual A8.52-A8.55 (introduction of many immunizations) Ink dots and methods for detecting antigen/antibody complexes)). And a buffer solution (such as phosphate buffer (phosphate_buffer * sa 1 me, PBS) and other similar solutions) to block the fixed membrane or filter paper. Non-specific antibody binding position, the above buffer solution contains a seal Resistors, such as 0.5% (w/v) low-fat milk powder or 5% (w/v) bovine serum albumin (BSA). After blocking, the antibody is added to react with the membrane or filter paper, and in the present invention, it is reacted with a biological sample taken from an individual diagnosed as an autoimmune disorder. 16 1357979 After the reaction with the primary antibody is completed, the membrane or filter paper is washed, and the autoantibody/autoantigen complex is identified by a secondary antibody, which is labeled with a chromogenic substance and a fluorescent substance. (fluorogenic) or chemical. Chemiluminescent means. The autoantibody/autoantigen complex described above can be detected by a colorimetric method such as horseradish peroxidase and TMB or an automatic radiography such as an immunolase. If colorimetric or chemical chilling is used, the amount of this color or fluorescence can be measured by luminescence {liminometer, spectrophotometer* or other similar instrument. In the case of automatic radiography, the amount of antibody binding can be known by optical density or other similar instrumentation exposure X-rays (see Sambrook et al., 3Molecular Cloning: A laboratory manual A8.52-A8.5). 5 (introducing many methods of immunizing ink dots and detecting antigen/antibody complexes)). Secondary antibodies can be used for qualitative or quantitative analysis, which can be single or multiple antibodies, and a ligand (such as φ biotin) or a detectable marker can be labeled on a secondary antibody by conventional techniques. (such as fluorescent or enzyme). Suitable labels include fluorophores, chromophores, electron-dense reagents (such as gold or silver), enzymes, and ligands that are specifically bonded to other substances. Enzymes such as wasabi peroxidase or alkaline phosphatase are usually tested for activity. For example, wasabi peroxidase converts tetradecylamine (TMB) to blue and quantified by spectrophotometry. Other suitable ligands and/or detectable markers include biotin, avidin, or streptavidin, IgG, protein A, and a number of additional receptor-coordinator combinations known. Other possibilities and advantages of the prior art are all obvious and are all aspects of the invention. In a consistent example, Western blotting is used to detect at least one autoantibody bound to an autoantigen antigen. The above autoantigen is specific and linked to the following proteins: heat shock Protein 60 (HSP 60), heat shock protein 90 alpha (HSP90o, or heat shock protein 90 beta (HSP90p). In standard Western blotting steps, such as far western blots' and others There are various variations in the quantitative dot method. Those skilled in the art can rely on the source of the autoantibody, the autoantigen, the autoantigen and/or the primary autoantibody used, and any other experimentally relevant parameters. Selecting suitable methods. Other substitutions and possibilities by conventional techniques are obvious and are one of the forms of the invention. 2. Enzyme-linked immunosorbent assay (ELISA) Some enzyme-linked immunosorbent assays are used for qualitative analysis. It can be easily analyzed for specific antigens or antibodies, but the enzyme-linked immunosorbent assay can also be used to quantitatively calculate the antigen or antibody concentration accurately because it can be easily and automatically read. Therefore, enzyme-linked immunosorbent assays are commonly used to screen a large number of specific binding-specific antigens or antibodies. Enzyme-linked immunosorbent assay is initially an antigen-coating step, and one or more target antigens are adsorbed to a microtiter plate. In the hole (see Kirkegaard & Perry laboratories, Inc., Technical Guide for ELISA 9-13 (2003), available at http://www.kpl. com/home.cfm# (last accessed November 3, 2005) ( Introduce various microtiter plates of ELISA and various methods and reagents for coating proteins in pores. General adsorption buffers commonly used for antigens 1357979 include 50 mM carbonate, ΡΗ=9·6; 10 mM Tris-HCl, pH =9.6; ι〇mMPBS, pH=7, 2. The above buffer is suitable for many proteins. If one or more of the above antigens cannot be adsorbed on the pore surface of the microtiter plate, a commercially available microtiter plate can also be used. The surface is repaired, and various chemical methods can be used to bond proteins to the surface by covalent bonding. Time and temperature are important in the adsorption process of proteins. Impact factor. After coating the target antigen on the microtiter plate, wash the microtiter plate with blocking buffer to remove non-specific bond and reduce the occurrence of false positives. 13-13) (Introduction of blocking method and reagent The commonly used blocking reagents are not protein solutions, such as BSA (between about 1% to 5% (w/v) in PBS, pH=7.0), skim milk powder, casein (main protein of skim milk powder) ) is cool casein (higher solubility protein, partial cut with sodium hydroxide), general serum (concentration between about 1% and 5% (v/v)) and Gelatin (concentration between about 1% and 5% (w/v)) or ion-free detergents such as Tween-20TM and Triton X-100TM. Buffers that do not affect the interaction of low affinity between the various reaction components are generally selected as the wash buffer, as each reaction component affects the ability to detect specific antigen-antibody effects (zW. at 14- 15 (Introducing various methods and reagents for cleaning microtiter plates)). The cleaning solution usually has a physiological buffer to avoid denaturation of the antigen and the antibody, and maintains the activity of the enzyme. Buffers of the same natural pH as natural physiology, such as PBS, Tris-saline, imidazole-buffered saline, and the like, are often used. In a specific analytical experiment, the specific buffer should be selected according to the method 17 1357979 of the specific detection test. At the same time, the cleaning buffer should also contain non-ionic detergents such as Tween-20 TM and Triton X-100TM at concentrations ranging from 〇! 0/〇 to 0·05〇/〇(ν/ν). Avoid low-affinity and non-specific bonding between the reaction components. After the blocking procedure, the pores of the microtiter plate are washed, and the primary antibody is added to react with the antigen adsorbed to the pore. The primary antibody of the present invention is a biological sample taken from an individual diagnosed as an autoimmune disorder. After the primary antibody is completed, the pores are cleaned and a secondary anti-calling body marked with a coloring substance (such as wasabi peroxidase and hydrazine), a fluorescent substance or a chemically luminescent substance (such as alkaline phosphatase) is detected. Body antigen-autoantibody complex (W at 15-21 (introducing the preparation and use of antibodies, and commonly used in detecting substances. Finally using luminometers, spectrophotometers, etc.) The instrument counts the amount of color and fluorescence. Standard enzyme immunoassay methods have various variants, such as competitive ELISA, sandwich ELISA, etc. Familiar with the antibodies, antigens, antigens and/or once used by those skilled in the art. Appropriate methods are selected for the source of the antibody and experimentally relevant parameters. Other substitutions and possibilities by conventional techniques are obvious and are one of the forms of the invention. B. Clinical myasthenia gravis ·

Osserman及Genkins等人將成人的重症肌無力症分為 4 大類（Osserman，K.E.及 Genkins,G.，的 “Studies in myasthenia gravis: review of a twenty-year experience in over 1200 patients,Mt. Sinai J. Med. 38(6):497-537(1971); “Myasthenia Gravis,” available at http://www.neuroland.com 20 1357979 /nm/myas_gra.htm (last accessed December 1,2005))。接近 20%的病人診斷為第i型，30%為第n a型，20%為第ΠΒ 型’ 11%為第ΠΙ型及9%為第IV型。其餘的10%的病人一般 為兒童型（Osserman and Genkins, swpra at 502) 〇 第I型MG又稱為眼無力型重症肌無力症，症狀一般 侷限於單眼或雙眼的肌肉。其特徵為眼瞼下垂及複視，但 若2年内上述症狀無發展至其他肌肉，則此型重症肌無力 症通常不會愈來愈嚴重。此型的重症肌無力症患者只有 55%的病人具有可專一性辨識 AChR的自體抗體 (Osserman, K.E. and Genkins, G., supra at 501; ''Myasthenia Gravis,” available at http://www.neuroland.com/nm/myas_gra.htm (last accessed December 1,2005)) ° 第Π a型MG又稱為輕度全身性重症肌無力症，其症狀 傾向於發展緩慢’有時開始於動眼肌但逐漸地發展至骨路 肌及延髓肌。但呼吸系統的肌肉通常不會被影響到。此型 患者約有80%的病人具有可專一性辨識AChR的自體抗 體。第nb型MG又稱為中度全身性重症肌無力症，其症 狀發展緩慢’一般開始於動眼肌且逐漸地發展至骨絡肌及 延髓肌。此型的症狀通常較第Ha型MG嚴重，且通常會 影響到呼吸系統的肌肉（Osserman, K.E. and Genkins，G., supra, at 501-02, Myasthenia Gravis,55 available at http: //www .neuroland. com/nm/myas__gra.htm(last accessedOsserman and Genkins et al. classify adult myasthenia gravis into four categories (Osserman, KE and Genkins, G., "Studies in myasthenia gravis: review of a twenty-year experience in over 1200 patients, Mt. Sinai J. Med. 38(6): 497-537 (1971); “Myasthenia Gravis,” available at http://www.neuroland.com 20 1357979 /nm/myas_gra.htm (last accessed December 1, 2005)). Close to 20 % of patients were diagnosed with type i, 30% for na, 20% for type '11% for type III and 9% for type 4. The remaining 10% were generally children (Osserman and Genkins, swpra at 502) 〇Type I MG is also known as ocular myasthenia gravis, and the symptoms are generally limited to the muscles of one or both eyes. It is characterized by drooping and diplopia, but if the symptoms do not develop within 2 years As for other muscles, this type of myasthenia gravis usually does not become more and more serious. Only 55% of patients with this type of myasthenia gravis have autoantibodies that can specifically recognize AChR (Osserman, KE and Genkins, G ., supra at 501; ''Myasthenia Gravis," available at http://www.neu Roland.com/nm/myas_gra.htm (last accessed December 1,2005)) ° Dijon MG is also known as mild systemic myasthenia gravis, whose symptoms tend to develop slowly 'sometimes starting with the eye muscles but Gradually develop to the bone path muscle and the bulbar muscle. But the muscles of the respiratory system are usually not affected. About 80% of patients with this type of patient have autoantibodies that specifically recognize AChR. The nb-type MG, also known as moderate systemic myasthenia gravis, develops slowly with symptoms that generally begin with the ocular muscles and gradually develop into the osseous and medullary muscles. Symptoms of this type are usually more severe than those of the Ha-type MG and usually affect the muscles of the respiratory system (Osserman, KE and Genkins, G., supra, at 501-02, Myasthenia Gravis, 55 available at http: //www. Neuroland. com/nm/myas__gra.htm (last accessed

December 1，2005)) o 21 1357979 在第ΠΙ型MG又為稱急性猛爆型重症肌無力症，此型 既嚴重又急性，症狀會快速的發展到骨骼肌及延髓肌，且 更快地影響到呼吸系統的肌肉。第m型的重症肌無力症一 般會發展成胸腺瘤。整個疾病的病程通常在6個月内。第 ΠΙ型MG死亡率高。此型的病人幾乎100%都具有可專一性 辨識 AChR 的自體抗體（Osserman，K.E. G.，仰;at 502; “Myasthenia Gravis,” available at http://neuroland.com/nm/myas_gra.htm (last accessedDecember 1,2005)) o 21 1357979 In type I MG, it is called acute blasting myasthenia gravis, which is both severe and acute. The symptoms develop rapidly into skeletal and bulbar muscles and affect it more quickly. To the muscles of the respiratory system. The m-type myasthenia gravis usually develops into a thymoma. The course of the disease is usually within 6 months. The first type of MG has a high mortality rate. Almost 100% of patients with this type have autologous antibodies that specifically recognize AChR (Osserman, KEG, Yang; at 502; "Myasthenia Gravis," available at http://neuroland.com/nm/myas_gra.htm (last Accessed

December 1, 2005)) ° 第IV型MG又稱為晚發性嚴重型重症肌無力症，一般 在第I及似Π型症狀發生後2年以上才發展至此型MG。 此型的重症肌無力症病人約有89%的病人具有可專一性辨 識 AChR 的自體抗體（Osserman，K.E. and Genkins，G” supra at 502; “Myasthenia Gravis，，， available at http://neuroland.com/nm/myas_gra.htm (last accessedDecember 1, 2005)) ° Type IV MG, also known as late-onset severe myasthenia gravis, usually develops to this type of MG more than 2 years after the onset of I and sputum-like symptoms. About 89% of patients with myasthenia gravis have an autoantibody that specifically recognizes AChR (Osserman, KE and Genkins, G" supra at 502; "Myasthenia Gravis,,, available at http://neuroland .com/nm/myas_gra.htm (last accessed

December 1, 2005)) ° 其中至少2種的胸腺病變會發生於重症肌無力症的病 人上。約50%的MG病人具有不同程度的胸腺增生。約i5〇/0 的病人會演變為胸腺瘤。在一些病例中，重症肌無力症病 人可以胸腺切除來成功冶癒’且超過5〇%重症肌無力症患 者可改善其症狀’但對年老患者的效果較差（〇ssennan，K E and Genkins，G.，at 515-23)。 C.與重症肌無力症有關的自體抗體 目前已有多種可鑑別特定自體免疫失調症之候選自體 22December 1, 2005)) ° At least two of the thymic lesions occur in patients with myasthenia gravis. About 50% of MG patients have varying degrees of thymic hyperplasia. A patient with an i5〇/0 will evolve into a thymoma. In some cases, patients with myasthenia gravis can be cured by thymectomy and more than 5% of patients with myasthenia gravis can improve their symptoms' but are less effective for older patients (〇ssennan, KE and Genkins, G ., at 515-23). C. Autoantibodies associated with myasthenia gravis There are a number of candidate autopsies that can identify specific autoimmune disorders.

抗原的方法，包括 A US-2005/0124076-A1 ^ v記載於美國專利公開No. 該公開專利併入本案之^於細5年6月9日之方法， 與自體免失調㈣& 依照上述方法，從健康個體 體。將純化的免疫球蛋白肌無力症）的血清樣本中純化抗 中，做為-親和性料。W共價鍵的方式鍵結於層析介質 或細胞類型，將上述物質2相關種系來源之器官、組織 失將m二 容胞後淬取蛋白質成分。 的Μ /蛋質樣本通過具有健康個體之免疫球蛋白 心目’與官柱中免疫球蛋白結合之蛋白質會流出管 、’再將上述流出的蛋白質通過具有自MG患者分離出之 免疫球蛋自的個管柱，則所有停留於第二管柱的蛋白質皆 為候選的自體抗原，洗提出來後可用於後續之:分析，如質 譜儀(mass spectrometry)以鑑定各種分離出的蛋白質。 由上述方法，可分離出HSP60及HSP90二種新的重症 肌無力症患者之自體抗原。由ELISA分析的結果發現，在 重症肌無力症的早期（第I及Π a期），並無專一性鍵結 AChR的自體抗體，但卻有專一性鍵結HSP60及HSP90的 自體抗體。由上述自體抗體可提供重症肌無力症一新的檢 測方法，因一般認為可專一性鍵結AChR自體抗體會伴隨 重症肌無力症，但在重症肌無力症患者的早期並無上述自 體抗體的存在（參照第5圖）。 此外，也可以其他方法來分離重症肌無力症之候選自 體抗原’如分析cDNA表現基因庫或相減噬菌體呈現系 統°以分離自]ViG病因及疾病發展相關器官、組織或細胞 23 1357979 種類（如成熟神經肌肉接點的肌肉終板）的mRNA以嗟菌體 呈現系統(phage display)或其他的蛋白質表現資料庫來分 離可能的抗原。將欲了解的自體免疫失調個體分離之血清 之免疫球蛋白與上述資料庫進行分析，並將結果與健康者 之免疫球蛋白做比較。 在各種標準篩選自體抗原步驟中，仍各有差異。就先 前習知方法中選擇一適合的技術來研究自體免疫疫疾病， 並可由分析生物樣本來得知可能的自體抗原及其他實驗的 相關參數。以習知技術進行的其他置換及可能性皆為顯而 易見’且皆為本發明之一形態。 1.熱休克蛋白（Heat-Shock Protein) 在環境溫度突然升高或其他逆境時，生物體會因此反 應合成一小組保留性蛋白質，稱為熱休克蛋白。在的演化 上，自然的熱休克反應及各種熱休克蛋白的胺基酸序列皆 非常相似。例如，真核與原核的熱休克蛋白7〇(HSP7〇)胺 基酸序列有約 50 %相同（parseu et al.，“The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged protein,Ann. Rev. Genet. 27:437-496(1993))。在逆境耐受性中，各種不同的熱休克 蛋白在保護生物體上扮演著不同角色。 a.熱休克蛋白 60(The 60 kD Heat-Shock Protein) 熱休克蛋白60(HSP60)首先發現於粒線體基質，上述蛋 白佔粒線體基質所有蛋白質的l%(Parsell et al., swpra at 465)。人類HSP60(胺基酸序列：1)與大腸桿菌的GroEL在 24 1357979 胺基酸序列上約有60%相同。且上述二種蛋白質有相同的 寡聚體結構（oligomeric structure)，可構成一 7元環 (seven-membered ring)，有時則可形成雙7元環（two seven-membered rings)的雙環（double doughnut)結構。在溫 度上升時，HSP60的表現量會增加，且其具有ATPase的 功能，與HSP60鍵結的ATP寡聚體上做一適當的結構改變。 HSP60不同於其他的熱休克蛋白，其在正常的溫度 下’同樣也具有功能(/d at 465-66)，且可在所有的溫度中 表現。HSP60與未摺疊或變性的蛋白質有高度的親和性， 可促使它們進行適當的摺疊。蛋白質在與HSP60鍵結後， 變性的結構可轉變為二級結構，並抑制其他複合體的產生。 b.熱休克蛋白 90(The 90 kD Heat-Shock Proetin) 熱休克蛋白90(shp90)彳艮早就發現存在於真核生物的細 胞質及細胞核中，但在無胞器的生物上仍未發現(parsell et al” swrpa at 470)。HSP90在細胞質中具有二種同型結構 (isoform) ’分別為HSP90ct，受誘導表現，主要結構(序列 辨識號：2)及HSP90p ’可持續性表現，次要結構（序列辨 識號·· 3)(Sreedhar et al.，“HSP90 isoform: functions， expression and clinical importance，，， FEBS Letters 56.2:11-1.5(2004))。現階段難以生化的方式來分辨α型及β 型，且目前皆是使用二者的混合來進行H S Ρ 9 〇的相關研究。 人類的HSP90a與大腸桿菌的HtpG在胺基酸序列上有 約40%相同（parsell，仰;να·)。HtpG在正常溫度時可維持一 定的濃度’而在高溫時可被大量誘導產生，但其並不是生 25 1357979 物所必需的蛋白質，因在將其基因移除後，並不會影響大 腸桿菌在正常溫度的生長，且只稍微影響其在高溫的生 長。相較之下，HSP90是真核細胞中眾多蛋白質的其中之 一 ’在非逆境時，其佔所有細胞蛋白質的1_2%。若將真核 生物的HSP90基因移除，會導致生物體的死亡，表示真核 的HSP90具有原核生物所不具有的一新功能（Sreedhar et al·，—ra; Parsell et al.，似；。 HSP90可與細胞中許多的蛋白質反應，包括酪蛋白激 酶Π (casein kinase Π )、受血紅素控制之eIF-2a填酸酶 (heme-regulated eIF-2a kinase)及固醇類荷爾蒙受體如雌激 素（estrogen)、黃體素(progesterone)、雄性素（androgen)、糖 皮質類固醇(glucocorticoid)、戴奥辛（dioxin)受體，src族的 致癌路胺酸激酵素（oncogenic tyrosine kinases of the src family)、鈣調理素（calmodulin)、肌動蛋白（actin)及微管蛋 白（加1)111111)等（？&犷56116{已1.，抓;?^3&1471)。經由實驗發現， 在細胞的分化及發育上’ HSP90扮演多功能的角色，如調 控肌肉發育、早期胚胎發育至計畫性細胞死亡(programmed cell death)或細胞凋亡（apoptosis)(Sreedhar et al., supra，at 12)。此外’也已證實HSP90會在複數種的腫瘤中表現，如 胰臟癌、乳癌及血癌（M at 13)等。且其中HSP9〇p轉錄的 能力在紅斑性狼瘡患者中較高，而紅斑性狼瘡為一種自體 免疫疾病，其原因為自體抗體攻擊自身的剪接體snRNA， Ro/La 核醣體粒（Ro/La ribonuclear particle)，組蛋白及 (histone)及/或自然的DNA等。 26 1357979 n '&測自體免疫的檢測套組 本發明之檢測方法可由一檢測套組來完成，上述檢測 • 套組包括一或複數種上述偵測自體抗體的技術，上述自體 抗體係專一性的與至少一種重症肌無力症的自體抗原鍵 結°在一實施例中，上述套組以西方墨點法分析上述自體 抗體。上述套組包括複數個測試工具，其中包含混合本發 明新發現之二種自體抗HSP60及HSP90a/HSP90p。 在—實施例中’上述檢測套組更包括專一於每個自體 •抗原的對照抗體(control antibodies)，二次抗體.以直接或間 接的方式與山葵過氧化酶或任何試劑結合，上述試劑可呈 現自體^原-自體抗體複合體，且上述套組包括西方墨點法 封阻’月洗、雜合及偵測等過程中所有需要的試管、容器 或反應器、緩衝液及試劑。上述對照抗體可以緩衝液、溶 劑或乾燥粉末等形式來提供。若對照抗體為緩衝液或溶劑 的^式’則可預先混合，但若為粉末的形式，則必須在使 用則加人溶液回溶。此外’檢測套組還包括其它成份 裝、說明書或可幫助翻自體抗體的物質。 在另貫施例中，上述套組可以ELISA來價測自體抗 體本發明t形態係為上述套組，包括複數個微量滴定 -盤’包覆有本發明新發現的二種新自體抗原聊6 HSP90a/HSP90p 〇 在-實施例巾’上述套組更包括各自體抗原的對照抗 ，(control antibodies) ’二次抗體以直接或間接的方式與山 葵過氧化酶或任何習知試劑結合.，上述試劑可呈現自體抗 27 1357979 • . t自體複合體，且上述套組包括ELISA中封阻、清 經财^價測等過程所有所需的試管、容器或反應器、 太^T。上述對照抗體可以緩衝液、溶劑或乾燥粉 乂來h供。若對照抗體為緩衝液或溶劑形式，則可 $先混合，但若為粉未形式，則必須在使用前加入溶液回 /谷此外，檢測套組還包括其它的成份、包裝、說明書或 可幫助偵測自體抗體的物質。 〆 實施例 實施例1 :以西方墨點法偵測自體抗體 血清樣本取自中華民國台灣新光醫院中209位重症肌 無力症患者。可將這些患者依其胸腺的病理分為四大類： 其中37位病人有不同程度的胸腺萎縮(thymic atr〇phy); 121 位的病人發展成胸腺瘤(thymoma) ; 40位病人有不同程度 的胸腺增生（thymic hyperplasia);及11位病人的胸腺有未 知病變。此外，76位病人為笫I型MG，81位為第na型 MG ’ 38位為第nb型MG，14位為第皿或第IV型MG。陰 性對照組(negative control)的血清樣本為來自台中榮民總 醫院的54位患者，他們為另一種自體免疫疾病的膜狀腎 絲球腎炎（Membranous glomerulonephritis, MGN)病人。 將HepG2/C3A細胞溶胞產物以10%的SDS-PAGE膠體 分離，其每條泳道給予Kg的細胞萃取物及而在驗證 HSP60或HSP90蛋白的每條泳道則是給予2 pg蛋白。電 泳過程中，樣本在焦集膠體時給予80 V的電壓’而進行至 在分離膠體時給予120 V的電壓。分離的蛋白質可利用 28 1357979 • · TE70系列半乾式轉潰單位轉潰器（TE70 Series Semi-dry Transfer Unit)(Amersham Bioscience)轉潰到 PVDF 膜上。所The method of the antigen, including A US-2005/0124076-A1 ^ v is described in the U.S. Patent Publication No. This patent is incorporated into the present application, the method of the present invention, and the self-dissociation (4) & Methods from healthy individuals. The serum samples of the purified immunoglobulin myasthenia gravis were purified as anti-affinities. The method of W covalent bond is bonded to the chromatographic medium or cell type, and the organ and tissue derived from the above-mentioned substance 2 are lost, and the protein component is extracted. The sputum/egg sample is passed through the immunoglobulin with healthy individuals, and the protein bound to the immunoglobulin in the official column will flow out of the tube, and then the above-mentioned efflux protein will pass through the immunoglobulin isolated from the MG patient. For each column, all proteins remaining in the second column are candidate autoantigens, which can be used for subsequent analysis: analysis, such as mass spectrometry, to identify various isolated proteins. By the above method, autoantigens of two new patients with myasthenia gravis, HSP60 and HSP90, can be isolated. As a result of ELISA analysis, in the early stage of myasthenia gravis (stage I and Π a), there was no autoantibody that specifically binds AChR, but there is an autoantibody that specifically binds HSP60 and HSP90. The above autologous antibody can provide a new method for detecting myasthenia gravis, because it is generally believed that the specific binding of AChR autoantibodies is accompanied by myasthenia gravis, but in the early stage of patients with myasthenia gravis, there is no such autologous body. The presence of antibodies (see Figure 5). In addition, other methods can be used to isolate candidate autoantigens for myasthenia gravis, such as analysis of cDNA expression gene pools or subtractive phage display systems, to isolate the organs, tissues, or cells of the [ViG etiology and disease development] 23 1357979 species ( mRNAs such as the muscle endplates of mature neuromuscular junctions are separated by phage display or other protein expression databases to isolate possible antigens. The immunoglobulin of the serum from which the autoimmune disorder is to be isolated is analyzed with the above-mentioned database, and the result is compared with the immunoglobulin of the healthy person. There are still differences in the various criteria for screening autoantigens. A suitable technique is selected in the prior methods to study autoimmune diseases, and biological samples can be analyzed to know possible autoantigens and other experimental parameters. Other substitutions and possibilities made by the prior art are readily apparent and are all aspects of the invention. 1. Heat-Shock Protein When the ambient temperature suddenly rises or other adverse conditions, the organism reacts to synthesize a small group of retained proteins called heat shock proteins. In the evolution, the natural heat shock response and the amino acid sequences of various heat shock proteins are very similar. For example, the eukaryotic and prokaryotic heat shock protein 7〇 (HSP7〇) amino acid sequence is about 50% identical (parseu et al., “The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged protein, Ann. Rev. Genet. 27:437-496 (1993). In stress tolerance, various heat shock proteins play different roles in protecting organisms. a. Heat shock protein 60 (The 60 kD Heat -Shock Protein) Heat shock protein 60 (HSP60) was first discovered in the mitochondrial matrix, which accounts for 1% of all proteins in the mitochondrial matrix (Parsell et al., swpra at 465). Human HSP60 (amino acid sequence: 1) GroEL with E. coli is about 60% identical in the amino acid sequence of 24 1357979. And the above two proteins have the same oligomeric structure, which can constitute a seven-membered ring. Sometimes, a double doughnut structure of two seven-membered rings can be formed. When the temperature rises, the amount of HSP60 increases, and it has the function of ATPase, and ATP bonded to HSP60. Make an appropriate knot on the oligomer HSP60 differs from other heat shock proteins in that it also functions at normal temperatures (/d at 465-66) and can be expressed at all temperatures. HSP60 has unfolded or denatured proteins. The high affinity allows them to fold properly. After the protein is bound to HSP60, the denatured structure can be converted into a secondary structure and inhibit the production of other complexes. b. Heat shock protein 90 (The 90 kD Heat -Shock Proetin) Heat shock protein 90 (shp90) has long been found in the cytoplasm and nucleus of eukaryotes, but has not been found in acellular organisms (parsell et al "swrpa at 470). HSP90 has two isoforms in the cytoplasm, 'HSP90ct, induced expression, main structure (SEQ ID NO: 2) and HSP90p 'sustainability, secondary structure (sequence identification number·· 3) Sreedhar et al., "HSP90 isoform: functions, expression and clinical importance,,, FEBS Letters 56.2:11-1.5 (2004).) At this stage, it is difficult to biochemically distinguish between α-type and β-type, and currently use two. A mixture of HS Ρ 9 。 is studied. Human HSP90a and E. coli HtpG are about 40% identical in amino acid sequence (parsell, MF; να·). HtpG can maintain a certain temperature at normal temperature. Concentration' can be induced in large quantities at high temperatures, but it is not a protein essential for the production of 25 1357979, because the removal of its gene does not affect the growth of E. coli at normal temperature, and only slightly affects Its growth at high temperatures. In contrast, HSP90 is one of the many proteins in eukaryotic cells. In non-adversity, it accounts for 1_2% of all cellular proteins. If the eukaryotic HSP90 gene is used Removal, leading to the death of organisms, indicates that eukaryotic HSP90 has a new function not found in prokaryotes (Sreedhar et al., -ra; Parsell et al., like; HSP90 can interact with many proteins in cells Reactions, including casein kinase 、, heme-regulated eIF-2a kinase, and sterol-like hormone receptors such as estrogen, lutein ( Progesterone), androgen, glucocorticoid, dioxin receptor, src family oncogenic tyrosine kinases of the src family, calmodulin, muscle Actin and tubulin (plus 1) 111111), etc. (?&犷56116{已1.,抓;?^3&1471). It was found through experiments that 'HSP90 plays a role in cell differentiation and development. Multi-functional roles such as regulation of muscle development, early embryonic development to programmed cell death or apoptosis (Sreedhar et al., supra, at 12). In addition, HSP90 has also been shown to be present in a variety of tumors, such as pancreatic cancer, breast cancer, and blood cancer (M at 13). And the ability of HSP9〇p to transcribe is higher in patients with lupus erythematosus, an autoimmune disease caused by autoantibodies attacking their own splice RNA, Ro/La ribosomes (Ro/ La ribonuclear particle), histone and/or natural DNA. 26 1357979 n '& Test kit for measuring autoimmunity The detection method of the present invention can be carried out by a test kit comprising one or more of the above-mentioned techniques for detecting autoantibodies, said autoantibody System-specific autoantigen binding to at least one myasthenia gravis. In one embodiment, the above kit analyzes the autoantibody described above by Western blotting. The kit includes a plurality of test tools comprising two of the auto-anti-HSP60 and HSP90a/HSP90p newly discovered in the present invention. In the embodiment, the above detection kit further comprises a control antibody, a secondary antibody specific to each autoantigen, in a direct or indirect manner, in combination with wasabi peroxidase or any reagent, the above reagent The autologous-pro-antibody complex can be presented, and the above kit includes Western blotting to block all required tubes, containers or reactors, buffers and reagents during the month of monthly washing, hybridization and detection. . The above control antibody may be provided in the form of a buffer, a solvent or a dry powder. If the control antibody is in the form of a buffer or a solvent, it may be premixed, but if it is in the form of a powder, it must be added to the solution to dissolve it. In addition, the test kit also includes other components, instructions, or substances that can help reverse the autoantibodies. In another embodiment, the above kit can be used to measure autologous antibodies by ELISA. The t-form of the present invention is the above kit, and includes a plurality of microtiter-plates coated with two new autoantigens newly discovered by the present invention. Chat 6 HSP90a / HSP90p - - Example towel 'The above kits include control antigens of their respective body antigens, (control antibodies) 'secondary antibodies in a direct or indirect way combined with wasabi peroxidase or any of the conventional reagents. The above reagent may present an autologous anti-27 1357979 • t self-complex, and the above kit includes all the required test tubes, containers or reactors in the ELISA, such as blocking, clearing, and the like, too . The above control antibody may be supplied as a buffer, a solvent or a dry powder. If the control antibody is in the form of a buffer or a solvent, it can be mixed first, but if it is in powder form, it must be added to the solution before use. In addition, the test kit also includes other ingredients, packaging, instructions or can help. A substance that detects autoantibodies.实施 Examples Example 1: Detection of autoantibodies by Western blotting The serum samples were taken from 209 patients with myasthenia gravis in the Shin Kong Hospital of Taiwan, Republic of China. These patients can be divided into four categories according to their thymus pathology: 37 patients have varying degrees of thymic atr〇phy; 121 patients develop thymoma; 40 patients have varying degrees of Thymic hyperplasia; and 11 patients have unknown lesions in the thymus. In addition, 76 patients were 笫I MG, 81 was na-type MG ‘38 was nb-type MG, and 14 was a third or fourth type MG. The serum samples from the negative control were 54 patients from the Taichung Veterans General Hospital who were another melanoma glomerulonephritis (MGN) patient with autoimmune disease. HepG2/C3A cell lysates were isolated as 10% SDS-PAGE colloids, each of which was given Kg cell extract and 2 pg of protein was administered in each lane of the HSP60 or HSP90 protein. During the electrophoresis, the sample was subjected to a voltage of 80 V when the colloid was collected, and a voltage of 120 V was applied when the colloid was separated. The separated protein can be used to break onto the PVDF membrane using the TE 70 Series Semi-dry Transfer Unit (Amersham Bioscience). Place

. 使用的轉潰緩衝液為Towbin Buffer(l倍的濃度為25 mMThe use of the buffer is Towbin Buffer (1-fold concentration of 25 mM)

Tris-HCl，pH=8.30，192 mM glycine 及 20%(v/v) methanol)。Tris-HCl, pH = 8.30, 192 mM glycine and 20% (v/v) methanol).

上述PVDF膜以含5%脫脂奶粉的PBST(80 mM Na2HP04,20 mM NaH2P04,100 mM NaCl，0·05%-0.1%(ν/ν) Tween-20)的缓衝液，置於室溫反應1小時或置於4°C隔夜 來進行封阻。將病人血清以1 : 1，000的比例稀釋於含5% ® 脫脂奶粉的PBST緩衝液中，並在室溫下與經封阻的PVDF 膜反應1小時，與一次抗體反應後，以PBST緩衝液清洗 上述PVDF膜。接著在室溫與二次抗體反應1小時，上述 二次抗體為標記山葵過氧化酶的抗人IgG(anti-human IgG)，以1:5,000稀釋於含5%脫脂奶粉的PBST缓衝液中。 上述二次抗體為標記山葵過氧化酶之老鼠抗人類 IgG(horseradish peroxidase-conjugated mouse anti-human IgG)的 Fcr(Jackson immunoResearcli Laboratories, Inc·)購 買自 Biosource International, of Camarillo, CA.USA。在自 體抗體鍵結上抗原後以ECL(Amersham Pharmacia Biotech) • 進行酵聯結素冷光分析。 • 第1及2圖顯示本實施例之結果，樣本取自胸腺增生 的女性患者(HF)，胸腺增生的男性患者(HM)，具有胸腺瘤 的女性患者(TF)及具有胸腺瘤的男性患者(TM)。以 '點" 表示正反應，為具有可辨識熱休克蛋白60、熱休克蛋白90 的自體抗體。由結果可證實重症肌無力症患者具有可專一 29 1357979 性鍵結熱休克蛋白60、熱休克蛋白90的自體抗體。 實施例2 :以酵素聯結免疫吸附法偵測自體抗體 以酵素聯結免疫吸附法去對實施例1之血清樣本進行 定量分析。將人類HSP60(純化自五.cW)或HSP90(純化自 S.cerevbz’fle)在 4°C 下以 0.1 M NaHC03, pH=8.6 的條件包 覆於 ELISA 微孔盤（Corning Life Sciences，New York，NY， USA)上。上述二種重組熱休克蛋白購買自Sigama_Aldrich Co。覆蓋完成之微孔盤以PBS清洗3次，每次1分鐘，且 在37°C下與200 μΐ的封阻溶液（5 mg/ml的BSA溶於PBST 中）反應1小時，再以封阻溶液清洗6次每次1。之後與稀 釋的病人血清在37°C下反應1小時至30分鐘，再以PBST 清洗6次’每次1分鐘，最後與二次抗體反應，上述二次 抗體為標記山葵過氧化酶之老鼠抗人類IgG的Fcr(Jackson immunoResearch Laboratories，Inc.)以 1 : 10,000 稀釋於含 5 mg/ml BSA的PBST緩衝液中，在室溫下反應1小時後， 在室溫下以含5 mg/ml BSA的PBST緩衝液清洗6次，並 加入100 μΐ的3,3’，5,5’-四曱基對二氨基聯苯(TMB)溶液。 上述ΤΜΒ溶液以5 0 mM的檸檬酸罐酸鹽（citrate phosphate) 製備。在加入TMB溶液後’於室溫下反應30分鐘，依照 儀器的操作手冊以 MRX Microplate Reader(Dynex Technologies)測量波長450 nm下的吸光值。 第3(HSP60)及笫4(HSP90)圖顯示，分別比較對照組與 重症肌無力症病患組在所有時期的ELISA分析結果。波長 450 nm的吸光平均值以一橫槓表示。本實施例並無區分 Γ357979 HSP90a與HSP9〇P。上述結果表示所有時期的重症肌無力 症患者皆具有可專一性鍵結HSP60及HSP90的自體抗體。 表一顯示因在第I及Da型MG患者中，皆無可專一性 鍵結AChR的自體抗體，但卻具有可專一性鍵結HSP60及 HSP90的自體抗體因此可正確的鑑定重症肌無力症患者。 以ELISA分析40位患者之可專一性鍵結HSP60及HSP90 的自體抗體，發現其中24位第I型MG患者及16位第Π a 型患者具有可專一性鍵結HSP60及HSP90的自體抗體。分 ® 析的過程已揭示於實施例2中。判斷蛋白質是否存在的基 準為：（1) OD450= 0.2(AChR) ; (2) OD45〇=0.375(HSP60); (3) OD450= 0.232(HSP90)。當中的40位病人皆無偵測到可 專一性鍵結AChR的自體抗體（表中以AChR表示），而其中 28位病人可偵測到可專一性鍵結HSP60的自體抗體(24位 第I型病人中有18位可偵測到，16位第Ha型病人中有 10位可偵測到）。33位病人可彳貞測到可專一性鍵結HSP90 的自體抗體(24位第I型病人中有21位可偵測到，16位第 ® IT a型病人中有I2位可偵測到）。 表一 病人編 號 及 MG 時 期 AChR AChR HSP60 HSP60 HSP90 HSP90 1-1 <0.2 — 0.539 + 0.609 + I -2 <0.2 — 0.593 + 0.293 + 1357979 1-3 <0.2 一 0.328 一 0.467 + 1-4 <0.2 - 0.716 + 0.498 + I -5 <0.2 一 1.063 + 1.088 + 1-6 <0.2 - 0.907 + 0.898 + 1-7 <0.2 - 0.484 + 0.376 + I -8 <0.2 - 0.724 + 0.644 + 1-9 <0.2 一 0.608 + 0.539 + I -10 <0.2 — 0.61 + 0.495 + I -11 <0.2 - 0.96 + 1.156 + I -12 <0.2 - 0.412 + 0.365 + I -13 <0.2 — 0.771 + 0.678 + 1-14 <0.2 — 0.769 + 0.743 + I -15 <0.2 - 0.321 — 0.196 一 I -16 <0.2 - 0.623 + 0.632 + I -17 <0.2 — 0.197 - 0.246 + I -18 <0.2 — 0.309 - 0.236 + 1-19 <0.2 - 0.476 + 0.398 + I -20 <0.2 - 0.708 + 0.175 一 I -21 <0.2 — 0.293 - 0.344 + I -22 <0.2 - 0.711 + 0.708 + I -23 <0.2 — 0.251 - 0.217 - I -24 <0.2 — 0.535 + 0.34 + Π a-1 <0.2 - 0.811 + 2.626 + Ha-2 <0.2 — 0.542 + 0.497 + 32 1357979 Πα-3 <0.2 — 0.306 一 0.306 + Π a-4 <0.2 一 0.498 + 0.324 + Π a-5 <0.2 一 0.406 + 0.261 + Π 3.-6 <0.2 — 0.169 — 0.162 - Π a-7 <0.2 一 0.734 + 0.75 + Π 3.-8 <0.2 一 0.313 — 0.226 一 Ha-9 <0.2 一 0.776 + 1.199 + Ha-10 <0.2 一 0.214 — 0.106 一 na-11 <0.2 — 1.201 + 0.918 + Π a-12 <0.2 一 0.193 — 0.138 — Ha-13 <0.2 一 0.351 一 0.258 + Π a-14 <0.2 — 0.552 + 0.8 + Π a-1-5 <0.2 — 0.498 + 0.642 + Π a-16 <0.2 — 1.051 + 0.783 + 笫5圖顯示本發明對於鑑定重症肌無力症的、、敏感性 (sensitive)"與專一性(specific广。、'敏感性"係為計算 有多少比例鑑定為MG的患者可偵測到專一性鍵結 HSP60(“protein_l”）或 HSP90(“protein_2”）的自體抗體 (Hennekens et al, Epidemiology in medicine 331-335(lst ed.1987))。專一性"係為計算有多少比例的非MG患者 不會偵測到專一性鍵結 HSI>6〇(“protein_r)或 HSP90(“pr〇tein_2”)的自體抗體。本實施例證實眼肌型肌無 力症（第I型）及輕度全身性（第n a型）MG患者不具有可與 AChR專一性鍵結的自體抗體，而76 %的患者具有可與 33 1357979 HSP60專一性鍵結的自體抗體，且86 %的患者具有可與 HSP90專一性鍵結的自體抗體，此外，91%的非重症肌無 力症患者不會偵測到可與HSP60專一性鍵結的自體抗體， 86 %的非重症肌無力症患者不會偵測到可與HSP90專一性 鍵結的自體抗體。 表二顯示自體抗體專一性鍵結HSP60、HSP90、 HSP60+HSP90、AChR+HSP60、或 AChR+HSP90 等的分析’ 由上述分析結果可提供比只彳貞測第I型MG患者AChR自 體抗體更好的方法。本實施例數據整理於表二，其中對73 位笫I型MG患者樣本做ELISA分析’且其判定基準已揭 示於表一。 表二 自體抗原 笫I型MG患者的敏感性(％) AChR 67.1 HSP60 78.1 HSP90 86.3 HSP60+HSP90 98.6 AChR+HSP60 91.7 AChR+HSP90 95.9 本發明以HSP60及/或HSP90提供一新型，且更敏感、 專一之鑑定重症肌無力症的方法，通常應用於重症肌無力 症的早期，因此時並不具有抗AChR自體抗體，而抗AChR 自體抗體為此疾病常用的臨床標記(clinical marker)。 雖然本發明已以較佳實施例揭露如上，然其並非用以 34 1357979 . · 限定本發明，任何熟習此技藝者，在不脫離本發明之精神 和範圍内，當可作些許之更動與潤飾，因此本發明之保護 . 範圍當視後附之申請專利範圍所界定者為準。The PVDF membrane was placed in a buffer containing 5% skim milk powder in PBST (80 mM Na2HP04, 20 mM NaH2P04, 100 mM NaCl, 0. 05%-0.1% (v/v) Tween-20) at room temperature. Blocking was performed overnight or at 4 ° C overnight. The patient's serum was diluted 1:1,000 in PBST buffer containing 5% ® skim milk powder, and reacted with the blocked PVDF membrane for 1 hour at room temperature. After reacting with the primary antibody, buffered with PBST. The above PVDF membrane was washed with liquid. Subsequently, the secondary antibody was reacted with a secondary antibody at room temperature for 1 hour, and the above secondary antibody was an anti-human IgG labeled with wasabi peroxidase, and diluted 1:5,000 in PBST buffer containing 5% skim milk powder. The above secondary antibody was purchased from Biosource International, of Camarillo, CA. USA, Fcr (Jackson immunoResearcli Laboratories, Inc.) of horseradish peroxidase-conjugated mouse anti-human IgG. After the antigen was bound to the autoantibody, ENG (Amersham Pharmacia Biotech) was used to perform cold light analysis of the yeast. • Figures 1 and 2 show the results of this example. Samples were taken from female patients with thymic hyperplasia (HF), male patients with thymic hyperplasia (HM), female patients with thymoma (TF), and male patients with thymoma. (TM). A positive reaction is indicated by 'dot", and is an autoantibody having a recognizable heat shock protein 60 and a heat shock protein 90. The results confirmed that patients with myasthenia gravis have autoantibodies that can specifically bind heat shock protein 60 and heat shock protein 90. Example 2: Detection of autoantibodies by enzyme-linked immunosorbent assay The serum samples of Example 1 were quantitatively analyzed by enzyme-linked immunosorbent assay. Human HSP60 (purified from c.cW) or HSP90 (purified from S. cerevbz'fle) was coated on ELISA microplates at 4 °C with 0.1 M NaHC03, pH = 8.6 (Corning Life Sciences, New York) , NY, USA). The above two recombinant heat shock proteins were purchased from Sigama_Aldrich Co. The completed microplate was washed 3 times with PBS for 1 minute each time, and reacted with 200 μΐ of blocking solution (5 mg/ml of BSA in PBST) for 1 hour at 37 ° C, and then blocked. The solution was washed 6 times each time. Then, it is reacted with the diluted patient serum at 37 ° C for 1 hour to 30 minutes, and then washed 6 times with PBST for 1 minute each time, and finally reacted with a secondary antibody, which is a mouse anti-marked horseradish peroxidase. Human IgG Fcr (Jackson immunoResearch Laboratories, Inc.) was diluted 1: 10,000 in PBST buffer containing 5 mg/ml BSA, reacted at room temperature for 1 hour, and contained 5 mg/ml BSA at room temperature. The PBST buffer was washed 6 times and 100 μM of 3,3',5,5'-tetradecyl-p-diaminobiphenyl (TMB) solution was added. The above hydrazine solution was prepared as 50 mM citrate phosphate. After the TMB solution was added, the reaction was carried out for 30 minutes at room temperature, and the absorbance at a wavelength of 450 nm was measured by an MRX Microplate Reader (Dynex Technologies) in accordance with the instrument's operation manual. Figures 3 (HSP60) and 笫4 (HSP90) show the results of ELISA analysis of the control group and the myasthenia gravis patients at all times. The average value of the absorbance at a wavelength of 450 nm is represented by a bar. This embodiment does not distinguish between Γ357979 HSP90a and HSP9〇P. The above results indicate that all patients with myasthenia gravis have autologous antibodies that specifically bind HSP60 and HSP90. Table 1 shows that there is no autoantibody that specifically binds AChR in patients with type I and type D MG, but it has autologous antibodies that specifically bind HSP60 and HSP90, so it can correctly identify myasthenia gravis. patient. Fourteen patients with autologous antibodies that specifically bind HSP60 and HSP90 were analyzed by ELISA. It was found that 24 patients with type I MG and 16 patients with type Π a had autologous antibodies that specifically bind HSP60 and HSP90. . The process of fractionation is disclosed in Example 2. The basis for determining the presence of protein is: (1) OD450 = 0.2 (AChR); (2) OD45 〇 = 0.375 (HSP60); (3) OD450 = 0.232 (HSP90). None of the 40 patients detected autoantibodies that specifically bind AChR (indicated by AChR), and 28 of them were able to detect autoantibodies that specifically bind HSP60 (24-bit) Eighteen of the type I patients were detectable, and 10 of the 16 Ha-type patients were detectable). Thirty-three patients were able to detect autoantibodies that specifically bind HSP90 (21 of 24 patients with type I can detect it, and 1 of 16 patients with IT a can detect it) ). Table 1 Patient No. and MG Period AChR AChR HSP60 HSP60 HSP90 HSP90 1-1 <0.2 - 0.539 + 0.609 + I -2 <0.2 - 0.593 + 0.293 + 1357979 1-3 <0.2 A 0.328 A 0.467 + 1-4 <0.2 - 0.716 + 0.498 + I -5 < 0.2 - 1.063 + 1.088 + 1-6 < 0.2 - 0.907 + 0.898 + 1-7 < 0.2 - 0.484 + 0.376 + I -8 < 0.2 - 0.724 + 0.644 + 1-9 < 0.2 - 0.608 + 0.539 + I -10 < 0.2 - 0.61 + 0.495 + I -11 < 0.2 - 0.96 + 1.156 + I -12 < 0.2 - 0.412 + 0.365 + I -13 <;0.2 — 0.771 + 0.678 + 1-14 < 0.2 — 0.769 + 0.743 + I -15 < 0.2 - 0.321 — 0.196 I - 16 < 0.2 - 0.623 + 0.632 + I -17 < 0.2 — 0.197 - 0.246 + I -18 < 0.2 — 0.309 - 0.236 + 1-19 < 0.2 - 0.476 + 0.398 + I -20 < 0.2 - 0.708 + 0.175 I - 21 < 0.2 - 0.293 - 0.344 + I -22 < 0.2 - 0.711 + 0.708 + I -23 < 0.2 - 0.251 - 0.217 - I -24 < 0.2 - 0.535 + 0.34 + Π a-1 < 0.2 - 0.811 + 2.626 + Ha-2 < 0.2 - 0.542 + 0.497 + 32 1357979 Πα-3 <0.2 — 0.306 a 0.306 + Π a-4 <0.2 - 0.498 + 0.324 + Π a-5 < 0.2 - 0.406 + 0.261 + Π 3.-6 < 0.2 - 0.169 - 0.162 - Π a-7 < 0.2 - 0.734 + 0.75 + Π 3.-8 <0.2 a 0.313 — 0.226 a Ha-9 <0.2 a 0.776 + 1.199 + Ha-10 <0.2 a 0.214 — 0.106 a na-11 <0.2 — 1.201 + 0.918 + Π a-12 &lt ; 0.2 - 0.193 - 0.138 - Ha-13 < 0.2 - 0.351 - 0.258 + Π a-14 < 0.2 - 0.552 + 0.8 + Π a-1-5 < 0.2 - 0.498 + 0.642 + Π a-16 < 0.2 - 1.051 + 0.783 + 笫 5 shows the sensitivity, sensitivity, and specificity of the present invention for identifying myasthenia gravis. 'Sensitivity' is a self-antibody that detects specific binding to HSP60 ("protein_l") or HSP90 ("protein_2") in order to calculate the proportion of patients identified as MG (Hennekens et al, Epidemiology in medicine 331-335 (lst ed. 1987)). Specificity" is to calculate the proportion of non-MG patients who do not detect autoantibodies that specifically bind HSI>6〇 ("protein_r" or HSP90 ("pr〇tein_2"). Myasthenia gravis (type I) and mild systemic (na-type) MG patients do not have autologous antibodies that can be specifically linked to AChR, while 76% of patients have specific bonds with 33 1357979 HSP60 Autoantibodies of the knot, and 86% of patients have autoantibodies that can be specifically linked to HSP90. In addition, 91% of patients with non-myasthenia gravis do not detect autologous bonds that are specific to HSP60. Antibodies, 86% of patients with non-myasthenia gravis do not detect autoantibodies that bind specifically to HSP90. Table 2 shows autoantibody-specific linkages HSP60, HSP90, HSP60+HSP90, AChR+HSP60, Or the analysis of AChR+HSP90, etc. The results of the above analysis can provide a better method than the AChR autoantibody of only type I MG patients. The data of this example are summarized in Table 2, where 73 笫I type MG Patient samples were analyzed by ELISA' and the criteria for their determination have been disclosed in Table 1. Table 2 Sensitivity (%) of antigen 笫 type I MG patients AChR 67.1 HSP60 78.1 HSP90 86.3 HSP60+HSP90 98.6 AChR+HSP60 91.7 AChR+HSP90 95.9 The present invention provides a novel, more sensitive and specific identification of severe cases with HSP60 and/or HSP90. The method of myasthenia gravis, usually applied in the early stage of myasthenia gravis, does not have an anti-AChR autoantibody, and anti-AChR autoantibody is a clinical marker commonly used for this disease. Although the present invention has The preferred embodiment is disclosed above, but it is not intended to be used in the present invention. It is to be understood that the invention may be modified and modified without departing from the spirit and scope of the invention. Protection. The scope is subject to the definition of the scope of the patent application.

35 1357979 【圖式簡單說明】 第1圖顯不：以西方墨點法分析實施例1中MG患者 之血清’結果顯示偵測到可辨識熱休克蛋白60(HSP60)的 自體抗體。以點〃表示正反應。樣本取自胸腺增生的女 性患者(HF)’胸腺增生的男性患者(HM)，具有胸腺瘤的女 性患者(TF)及具有胸腺瘤的男性患者(tm)。 第2圖顯示：以西方墨點法分析實施例1之mg患者 血清’結果顯示偵測到可辨識熱休克蛋白90(hsP90)的自 體抗體。以、、點〃表示正反應。樣本取自胸腺增生的女性 患者(HF) ’胸腺增生的男性患者(HM)，具有胸腺瘤的女性 患者(TF)及具有胸腺瘤的男性患者(TM) ^ 第3圖顯示：以酵素聯結免疫吸附法(ELISA)證實，實 施例2中’一般MG症患者之血清具有熱休克蛋白 60(HSP60)的存在。以橫槓表示od·的平均值。 第4圖顯示：以酵素聯結免疫吸附法(ELISA)證實，實 施例2中’一般MG患者之血清具有熱休克蛋白9〇(HSP90) 的存在。以橫槓表示〇D450的平均值。 第5圖顯示：本發明之鑑定重症肌無力症方法的能力， 若為重症肌無力症患者則為正反應(test-positive)，若非則 為負反應(test-negative)。正反應的患者具有可專一性辨識 HSP60(“protein_l”)及/或 HSP90(“protein_2”）的自體抗體， 負反應則無可專一性辨識HSP60(“protein_l，，）及/或 HSP90(“protein_2”）的自體抗體。35 1357979 [Simple description of the diagram] Fig. 1 shows that the serum of MG patients in Example 1 was analyzed by Western blotting. The results showed that autoantibodies recognizing heat shock protein 60 (HSP60) were detected. The positive reaction is indicated by a point. The samples were obtained from a female patient with thymic hyperplasia (HF)' thymic hyperplasia (HM), a female patient with thymoma (TF), and a male patient with thymoma (tm). Fig. 2 shows that the serum of the patient of Example 1 was analyzed by Western blotting. The results showed that an autoantibody recognizing heat shock protein 90 (hsP90) was detected. The positive reaction is indicated by . Samples were taken from female patients with thymic hyperplasia (HF) 'Male patients with thymic hyperplasia (HM), female patients with thymoma (TF) and male patients with thymoma (TM) ^ Figure 3 shows: Immunization with enzymes The adsorption method (ELISA) confirmed that the serum of the patient with general MG in Example 2 had the presence of heat shock protein 60 (HSP60). The average value of od· is represented by a horizontal bar. Fig. 4 shows that the serum of the general MG patient of Example 2 was confirmed to have the presence of heat shock protein 9 (HSP90) by enzyme-linked immunosorbent assay (ELISA). The average value of 〇D450 is represented by a bar. Fig. 5 shows the ability of the present invention to identify a method for myasthenia gravis, which is a test-positive if it is a myasthenia gravis, and a test-negative if it is not. Positively responding patients have autologous antibodies that can specifically identify HSP60 ("protein_l") and/or HSP90 ("protein_2"), while negative reactions have no specificity to identify HSP60 ("protein_l,," and / or HSP90 (" Autoantibody for protein_2").

表1顯示：以ELISA分析重症肌無力症患，發現第I 36 1357979 或na時期並無可辨識AChR的自體抗體，但具有可辨識 HSP60或HSP90的自體抗體。 表 2 顯示：分別比較（l)AChR ; (2)HSP60 ; (3)HSP60+HSP90 ； (4)HSP60+HSP90 ； (5)AChR+HSP60 ； (6)AChR+HSP90之不同鑑定方法的敏感度。結果顯示可辨 識AChR的自體抗體僅出現於第I期之眼肌型肌無力症患 者，且敏感度最低。 序列識別號1為人類熱休克蛋白60(HSP60)的胺基酸 • 序列。 序列識別號2為人類熱休克蛋白90 α型（HSP90a)的胺 基酸序列》 序列識別號3為人類熱休克蛋白90 β型(HSP9〇P)的胺 基酸序列。 【主要元件符號說明】 赢無0 37 1357979 、的年曰修(更)正本 【序歹丨】編號】 _____ <110>財團法人工業技術研究院 <120〉檢測自體抗體之方法及鑑定重症肌無力症之套組 <130> 09708.0005-00000 <160> 3 <170> Patentln version 3.3 <210> 1 <211> 573Table 1 shows that patients with myasthenia gravis were analyzed by ELISA. It was found that there was no autoantibodies to recognize AChR in the period of I 36 1357979 or na, but there were autoantibodies that could recognize HSP60 or HSP90. Table 2 shows: (1) AChR; (2) HSP60; (3) HSP60 + HSP90; (4) HSP60 + HSP90; (5) AChR + HSP60; (6) Sensitivity of different identification methods of AChR + HSP90 . The results showed that the autoantibodies recognizing AChR only appeared in the first stage of myocardium myasthenia gravis patients with the lowest sensitivity. Sequence ID 1 is the amino acid sequence of human heat shock protein 60 (HSP60). SEQ ID NO: 2 is the amino acid sequence of human heat shock protein 90 alpha (HSP90a). Sequence ID 3 is the amino acid sequence of human heat shock protein 90 beta (HSP9〇P). [Explanation of main component symbols] Winning no 0 37 1357979, the year of repair (more) original [Original] No.] _____ <110> The Institute of Industrial Technology < 120> Method and identification of autoantibodies Set of myasthenia gravis <130> 09708.0005-00000 <160> 3 <170> Patentln version 3.3 <210> 1 <211>

<212〉 PRT <213> Homo sapiens <400> 1<212> PRT <213> Homo sapiens <400> 1

Met Leu Arg Leu Pro Thr Val Phe Arg Gin Met Arg Pro Val Ser Arg 15 10 15Met Leu Arg Leu Pro Thr Val Phe Arg Gin Met Arg Pro Val Ser Arg 15 10 15

Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30Val Leu Ala Pro His Leu Thr Arg Ala Tyr Ala Lys Asp Val Lys Phe 20 25 30

Gly Ala Asp Ala Arg Ala Leu Met Leu Gin Gly Val Asp Leu Leu Ala 35 40 45Gly Ala Asp Ala Arg Ala Leu Met Leu Gin Gly Val Asp Leu Leu Ala 35 40 45

Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val lie lie 50 55 60Asp Ala Val Ala Val Thr Met Gly Pro Lys Gly Arg Thr Val lie lie 50 55 60

Glu Gin Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80Glu Gin Ser Trp Gly Ser Pro Lys Val Thr Lys Asp Gly Val Thr Val 65 70 75 80

Ala Lys Ser lie Asp Leu Lys Asp Lys Tyr Lys Asn lie Gly Ala Lys 85 90 95 1357979Ala Lys Ser lie Asp Leu Lys Asp Lys Tyr Lys Asn lie Gly Ala Lys 85 90 95 1357979

Leu Val Gin Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110Leu Val Gin Asp Val Ala Asn Asn Thr Asn Glu Glu Ala Gly Asp Gly 100 105 110

Thr Thr Thr Ala Thr Val Leu Ala Arg Ser lie Ala Lys Glu Gly Phe 115 120 125Thr Thr Thr Ala Thr Val Leu Ala Arg Ser lie Ala Lys Glu Gly Phe 115 120 125

Glu Lys lie Ser Lys Gly Ala Asn Pro Val Glu lie Arg Arg Gly Val 130 135 140Glu Lys lie Ser Lys Gly Ala Asn Pro Val Glu lie Arg Arg Gly Val 130 135 140

Met Leu Ala Val Asp Ala Val lie Ala Glu Leu Lys Lys Gin Ser Lys 145 150 155 160Met Leu Ala Val Asp Ala Val lie Ala Glu Leu Lys Lys Gin Ser Lys 145 150 155 160

Pro Val Thr Thr Pro Glu Glu lie Ala Gin Val Ala Thr lie Ser Ala 165 170 175Pro Val Thr Thr Pro Glu Glu lie Ala Gin Val Ala Thr lie Ser Ala 165 170 175

Asn Gly Asp Lys Glu lie Gly Asn lie lie Ser Asp Ala Met Lys Lys 180 185 190Asn Gly Asp Lys Glu lie Gly Asn lie lie Ser Asp Ala Met Lys Lys 180 185 190

Val Gly Arg Lys Gly Val lie Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205Val Gly Arg Lys Gly Val lie Thr Val Lys Asp Gly Lys Thr Leu Asn 195 200 205

Asp Glu Leu Glu lie lie Glu Gly Met Lys Phe Asp Arg Gly Tyr lie 210 215 220Asp Glu Leu Glu lie lie Glu Gly Met Lys Phe Asp Arg Gly Tyr lie 210 215 220

Ser Pro Tyr Phe lie Asn Thr Ser Lys Gly Gin Lys Cys Glu Phe Gin 225 230 235 240Ser Pro Tyr Phe lie Asn Thr Ser Lys Gly Gin Lys Cys Glu Phe Gin 225 230 235 240

Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys lie Ser Ser lie Gin Ser 245 250 255 lie Val Pro Ala Leu Glu lie Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270 2 1357979 lie lie Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275 280 285Asp Ala Tyr Val Leu Leu Ser Glu Lys Lys lie Ser Ser lie Gin Ser 245 250 255 lie Val Pro Ala Leu Glu lie Ala Asn Ala His Arg Lys Pro Leu Val 260 265 270 2 1357979 lie lie Ala Glu Asp Val Asp Gly Glu Ala Leu Ser Thr Leu Val Leu 275 280 285

Asn Arg Leu Lys Val Gly Leu Gin Val Val Ala Val Lys Ala Pro Gly 290 295 300Asn Arg Leu Lys Val Gly Leu Gin Val Val Ala Val Lys Ala Pro Gly 290 295 300

Phe Gly Asp Asn Arg Lys Asn Gin Leu Lys Asp Met Ala lie Ala Thr 305 310 315 320Phe Gly Asp Asn Arg Lys Asn Gin Leu Lys Asp Met Ala lie Ala Thr 305 310 315 320

Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335Gly Gly Ala Val Phe Gly Glu Glu Gly Leu Thr Leu Asn Leu Glu Asp 325 330 335

Val Gin Pro His Asp Leu Gly Lys Val Gly Glu Val lie Val Thr Lys 340 345 350Val Gin Pro His Asp Leu Gly Lys Val Gly Glu Val lie Val Thr Lys 340 345 350

Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gin lie Glu 355 360 365Asp Asp Ala Met Leu Leu Lys Gly Lys Gly Asp Lys Ala Gin lie Glu 355 360 365

Lys Arg lie Gin Glu lie lie Glu Gin Leu Asp Val Thr Thr Ser Glu 370 375 380Lys Arg lie Gin Glu lie lie Glu Gin Leu Asp Val Thr Thr Ser Glu 370 375 380

Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395 400Tyr Glu Lys Glu Lys Leu Asn Glu Arg Leu Ala Lys Leu Ser Asp Gly 385 390 395 400

Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405 410 415Val Ala Val Leu Lys Val Gly Gly Thr Ser Asp Val Glu Val Asn Glu 405 410 415

Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430Lys Lys Asp Arg Val Thr Asp Ala Leu Asn Ala Thr Arg Ala Ala Val 420 425 430

Glu Glu Gly lie Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys lie 1357979 435 440 445Glu Glu Gly lie Val Leu Gly Gly Gly Cys Ala Leu Leu Arg Cys lie 1357979 435 440 445

Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gin Lys lie Gly 450 455 4 60 lie Glu lie lie Lys Arg Thr Leu Lys lie Pro Ala Met Thr lie Ala 465 470 475 480Pro Ala Leu Asp Ser Leu Thr Pro Ala Asn Glu Asp Gin Lys lie Gly 450 455 4 60 lie Glu lie lie Lys Arg Thr Leu Lys lie Pro Ala Met Thr lie Ala 465 470 475 480

Lys Asn Ala Gly Val Glu Gly Ser Leu lie Val Glu Lys lie Met Gin 485 490 495Lys Asn Ala Gly Val Glu Gly Ser Leu lie Val Glu Lys lie Met Gin 485 490 495

Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510Ser Ser Ser Glu Val Gly Tyr Asp Ala Met Ala Gly Asp Phe Val Asn 500 505 510

Met Val Glu Lys Gly lie lie Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525Met Val Glu Lys Gly lie lie Asp Pro Thr Lys Val Val Arg Thr Ala 515 520 525

Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540Leu Leu Asp Ala Ala Gly Val Ala Ser Leu Leu Thr Thr Ala Glu Val 530 535 540

Val Val Thr Glu lie Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala 545 550 555 560Val Val Thr Glu lie Pro Lys Glu Glu Lys Asp Pro Gly Met Gly Ala 545 550 555 560

Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 570 <210> 2 <211〉732Met Gly Gly Met Gly Gly Gly Met Gly Gly Gly Met Phe 565 570 <210> 2 <211〉732

<212> PRT <213> Homo sapiens <400> 2<212> PRT <213> Homo sapiens <400> 2

Met Pro Glu Glu Thr Gin Thr Gin Asp Gin Pro Met Glu Glu Glu Glu 4 1357979 5 10 15Met Pro Glu Glu Thr Gin Thr Gin Asp Gin Pro Met Glu Glu Glu Glu 4 1357979 5 10 15

Val Glu Thr Phe Ala Phe Gin Ala Glu lie Ala Gin Leu Met Ser Leu 20 25 30 lie lie Asn Thr Phe Tyr Ser Asn Lys Glu lie Phe Leu Arg Glu Leu 35 40 45 lie Ser Asn Ser Ser Asp Ala Leu Asp Lys lie Arg Tyr Glu Ser Leu 50 55 60Val Glu Thr Phe Ala Phe Gin Ala Glu lie Ala Gin Leu Met Ser Leu 20 25 30 lie lie Asn Thr Phe Tyr Ser Asn Lys Glu lie Phe Leu Arg Glu Leu 35 40 45 lie Ser Asn Ser Ser Asp Ala Leu Asp Lys lie Arg Tyr Glu Ser Leu 50 55 60

Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His lie Asn Leu 65 70 75 80 lie Pro Asn Lys Gin Asp Arg Thr Leu Thr lie Val Asp Thr Gly lie 85 90 95Thr Asp Pro Ser Lys Leu Asp Ser Gly Lys Glu Leu His lie Asn Leu 65 70 75 80 lie Pro Asn Lys Gin Asp Arg Thr Leu Thr lie Val Asp Thr Gly lie 85 90 95

Gly Met Thr Lys Ala Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys 100 105 110Gly Met Thr Lys Ala Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys 100 105 110

Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gin Ala Gly Ala Asp lie 115 120 125Ser Gly Thr Lys Ala Phe Met Glu Ala Leu Gin Ala Gly Ala Asp lie 115 120 125

Ser Met lie Gly Gin Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val 130 135 140Ser Met lie Gly Gin Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val 130 135 140

Ala Glu Lys Val Thr Val lie Thr Lys His Asn Asp Asp Glu Gin Tyr 145 150 155 160Ala Glu Lys Val Thr Val lie Thr Lys His Asn Asp Asp Glu Gin Tyr 145 150 155 160

Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr 165 170 175 L357979Ala Trp Glu Ser Ser Ala Gly Gly Ser Phe Thr Val Arg Thr Asp Thr 165 170 175 L357979

Gly Glu Pro Met Gly Arg Gly Thr Lys Val lie Leu His Leu Lys Glu 180 185 190Gly Glu Pro Met Gly Arg Gly Thr Lys Val lie Leu His Leu Lys Glu 180 185 190

Asp Gin Thr Glu Tyr Leu Glu Glu Arg Arg lie Lys Glu lie Val Lys 195 200 205Asp Gin Thr Glu Tyr Leu Glu Glu Arg Arg lie Lys Glu lie Val Lys 195 200 205

Lys His Ser Gin Phe lie Gly Tyr Pro lie Thr Leu Phe Val Glu Lys 210 215 220Lys His Ser Gin Phe lie Gly Tyr Pro lie Thr Leu Phe Val Glu Lys 210 215 220

Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp 225 230 235 240Glu Arg Asp Lys Glu Val Ser Asp Asp Glu Ala Glu Glu Lys Glu Asp 225 230 235 240

Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro 245 250 255Lys Glu Glu Glu Lys Glu Lys Glu Glu Lys Glu Ser Glu Asp Lys Pro 245 250 255

Glu lie Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly 260 265 270Glu lie Glu Asp Val Gly Ser Asp Glu Glu Glu Glu Lys Lys Asp Gly 260 265 270

Asp Lys Lys Lys Lys Lys Lys lie Lys Glu Lys Tyr lie Asp Gin Glu 275 280 285Asp Lys Lys Lys Lys Lys Lys Ly Ly Lys Glu Lys Tyr lie Asp Gin Glu 275 280 285

Glu Leu Asn Lys Thr Lys Pro lie Trp Thr Arg Asn Pro Asp Asp lie 290 295 300Glu Leu Asn Lys Thr Lys Pro lie Trp Thr Arg Asn Pro Asp Asp lie 290 295 300

Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp 305 310 315 320Thr Asn Glu Glu Tyr Gly Glu Phe Tyr Lys Ser Leu Thr Asn Asp Trp 305 310 315 320

Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu Gly Gin Leu Glu 325 330 335Glu Asp His Leu Ala Val Lys His Phe Ser Val Glu Gly Gin Leu Glu 325 330 335

Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe 340 345 350 6 1357979Phe Arg Ala Leu Leu Phe Val Pro Arg Arg Ala Pro Phe Asp Leu Phe 340 345 350 6 1357979

Glu Asn Arg Lys Lys Lys Asn Asn He Lys Leu Tyr Val Arg Arg Val 355 360 365 Phe lie Met Asp Asn Cys Glu Glu Leu lie Pro Glu Tyr Leu Asn Phe 370 375 380Glu Asn Arg Lys Lys Lys Asn Asn He Lys Leu Tyr Val Arg Arg Val 355 360 365 Phe lie Met Asp Asn Cys Glu Glu Leu lie Pro Glu Tyr Leu Asn Phe 370 375 380

He Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn lie Ser Arg 385 390 395 400He Arg Gly Val Val Asp Ser Glu Asp Leu Pro Leu Asn lie Ser Arg 385 390 395 400

Glu Met Leu Gin Gin Ser Lys lie Leu Lys Val lie Arg Lys Asn Leu 405 410 415Glu Met Leu Gin Gin Ser Lys lie Leu Lys Val lie Arg Lys Asn Leu 405 410 415

Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu 420 425 430Val Lys Lys Cys Leu Glu Leu Phe Thr Glu Leu Ala Glu Asp Lys Glu 420 425 430

Asn Tyr Lys Lys Phe Tyr Glu Gin Phe Ser Lys Asn lie Lys Leu Gly 435 440 445 lie His Glu Asp Ser Gin Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg 450 455 460Asn Tyr Lys Lys Phe Tyr Glu Gin Phe Ser Lys Asn lie Lys Leu Gly 435 440 445 lie His Glu Asp Ser Gin Asn Arg Lys Lys Leu Ser Glu Leu Leu Arg 450 455 460

Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr 465 470 475 480Tyr Tyr Thr Ser Ala Ser Gly Asp Glu Met Val Ser Leu Lys Asp Tyr 465 470 475 480

Cys Thr Arg Met Lys Glu Asn Gin Lys His lie Tyr Tyr lie Thr Gly 485 490 495Cys Thr Arg Met Lys Glu Asn Gin Lys His lie Tyr Tyr lie Thr Gly 485 490 495

Glu Thr Lys Asp Gin Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg 500 505 510Glu Thr Lys Asp Gin Val Ala Asn Ser Ala Phe Val Glu Arg Leu Arg 500 505 510

Lys His Gly Leu Glu Val lie Tyr Met lie Glu Pro lie Asp Glu Tyr 515 520 525 1357979Lys His Gly Leu Glu Val lie Tyr Met lie Glu Pro lie Asp Glu Tyr 515 520 525 1357979

Cys Val Gin Gin Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val 530 535 540Cys Val Gin Gin Leu Lys Glu Phe Glu Gly Lys Thr Leu Val Ser Val 530 535 540

Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys 545 550 555 560Thr Lys Glu Gly Leu Glu Leu Pro Glu Asp Glu Glu Glu Lys Lys Lys 545 550 555 560

Gin Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys lie Met Lys 565 570 575Gin Glu Glu Lys Lys Thr Lys Phe Glu Asn Leu Cys Lys lie Met Lys 565 570 575

Asp lie Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu 580 585 590Asp lie Leu Glu Lys Lys Val Glu Lys Val Val Val Ser Asn Arg Leu 580 585 590

Val Thr Ser Pro Cys Cys lie Val Thr Ser Thr Tyr Gly Trp Thr Ala 595 600 605Val Thr Ser Pro Cys Cys lie Val Thr Ser Thr Tyr Gly Trp Thr Ala 595 600 605

Asn Met Glu Arg lie Met Lys Ala Gin Ala Leu Arg Asp Asn Ser Thr 610 615 620Asn Met Glu Arg lie Met Lys Ala Gin Ala Leu Arg Asp Asn Ser Thr 610 615 620

Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu lie Asn Pro Asp His 625 630 635 640Met Gly Tyr Met Ala Ala Lys Lys His Leu Glu lie Asn Pro Asp His 625 630 635 640

Ser lie lie Glu Thr Leu Arg Gin Lys Ala Glu Ala Asp Lys Asn Asp 645 650 655Ser lie lie Glu Thr Leu Arg Gin Lys Ala Glu Ala Asp Lys Asn Asp 645 650 655

Lys Ser Val Lys Asp Leu Val lie Leu Leu Tyr Glu Thr Ala Leu Leu 660 665 670Lys Ser Val Lys Asp Leu Val lie Leu Leu Tyr Glu Thr Ala Leu Leu 660 665 670

Ser Ser Gly Phe Ser Leu Glu Asp Pro Gin Thr His Ala Asn Arg lie 675 680 685Ser Ser Gly Phe Ser Leu Glu Asp Pro Gin Thr His Ala Asn Arg lie 675 680 685

Tyr Arg Met lie Lys Leu Gly Leu Gly lie Asp Glu Asp Asp Pro Thr 8 1357979 690 695 700Tyr Arg Met lie Lys Leu Gly Leu Gly lie Asp Glu Asp Asp Pro Thr 8 1357979 690 695 700

Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu 705 710 715 720Ala Asp Asp Thr Ser Ala Ala Val Thr Glu Glu Met Pro Pro Leu Glu 705 710 715 720

Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp 725 730 <210> 3 <211> 724Gly Asp Asp Asp Thr Ser Arg Met Glu Glu Val Asp 725 730 <210> 3 <211> 724

<212> PRT <213> Homo sapiens <400> 3<212> PRT <213> Homo sapiens <400> 3

Met Pro Glu Glu Val His His Gly Glu Glu Glu Val Glu Thr Phe Ala 15 10 15Met Pro Glu Glu Val His His Gly Glu Glu Glu Val Glu Thr Phe Ala 15 10 15

Phe Gin Ala Glu lie Ala Gin Leu Met Ser Leu lie lie Asn Thr Phe 20 25 30 Tyr Ser Asn Lys Glu lie Phe Leu Arg Glu Leu lie Ser Asn Ala Ser 35 40 45Phe Gin Ala Glu lie Ala Gin Leu Met Ser Leu lie lie Asn Thr Phe 20 25 30 Tyr Ser Asn Lys Glu lie Phe Leu Arg Glu Leu lie Ser Asn Ala Ser 35 40 45

Asp Ala Leu Asp Lys lie Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys 50 55 60Asp Ala Leu Asp Lys lie Arg Tyr Glu Ser Leu Thr Asp Pro Ser Lys 50 55 60

Leu Asp Ser Gly Lys Glu Leu Lys lie Asp lie lie Pro Asn Pro Gin 65 70 75 80Leu Asp Ser Gly Lys Glu Leu Lys lie Asp lie lie Pro Asn Pro Gin 65 70 75 80

Glu Arg Thr Leu Thr Leu Val Asp Thr Gly lie Gly Met Thr Lys Ala 85 90 95Glu Arg Thr Leu Thr Leu Val Asp Thr Gly lie Gly Met Thr Lys Ala 85 90 95

Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys Ser Gly Thr Lys Ala 1357979 ♦ · 100 105 110Asp Leu lie Asn Asn Leu Gly Thr lie Ala Lys Ser Gly Thr Lys Ala 1357979 ♦ · 100 105 110

Phe Met Glu Ala Leu Gin Ala Gly Ala Asp lie Ser Met lie Gly Gin 115 120 125Phe Met Glu Ala Leu Gin Ala Gly Ala Asp lie Ser Met lie Gly Gin 115 120 125

Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Val 130 135 140Phe Gly Val Gly Phe Tyr Ser Ala Tyr Leu Val Ala Glu Lys Val Val 130 135 140

Val lie Thr Lys His Asn Asp Asp Glu Gin Tyr Ala Trp Glu Ser Ser 145 150 155 160Val lie Thr Lys His Asn Asp Asp Glu Gin Tyr Ala Trp Glu Ser Ser 145 150 155 160

Ala Gly Gly Ser Phe Thr Val Arg Ala Asp His Gly Glu Pro lie Gly 165 170 175Ala Gly Gly Ser Phe Thr Val Arg Ala Asp His Gly Glu Pro lie Gly 165 170 175

Arg Gly Thr Lys Val lie Leu His Leu Lys Glu Asp Gin Thr Glu Tyr 180 185 190Arg Gly Thr Lys Val lie Leu His Leu Lys Glu Asp Gin Thr Glu Tyr 180 185 190

Leu Glu Glu Arg Arg Val Lys Glu Val Val Lys Lys His Ser Gin Phe 195 200 205 lie Gly Tyr Pro lie Thr Leu Tyr Leu Glu Lys Glu Arg Glu Lys Glu 210 215 220 lie Ser Asp Asp Glu Ala Glu Glu Glu Lys Gly Glu Lys Glu Glu Glu 225 230 235 240Leu Glu Glu Arg Arg Val Lys Glu Val Val Lys Lys His Ser Gin Phe 195 200 205 lie Gly Tyr Pro lie Thr Leu Tyr Leu Glu Lys Glu Arg Glu Lys Glu 210 215 220 lie Ser Asp Asp Glu Ala Glu Glu Glu Lys Gly Glu Lys Glu Glu Glu 225 230 235 240

Asp Lys Asp Asp Glu Glu Lys Pro Lys lie Glu Asp Val Gly Ser Asp 245 250 255Asp Lys Asp Asp Glu Glu Lys Pro Lys lie Glu Asp Val Gly Ser Asp 245 250 255

Glu Glu Asp Asp Ser Gly Lys Asp Lys Lys Lys Lys Thr Lys Lys lie 260 265 270 10 1357979Glu Glu Asp Asp Ser Gly Lys Asp Lys Lys Lys Lys Thr Lys Lys lie 260 265 270 10 1357979

Lys Glu Lys Tyr lie Asp Gin Glu Glu Leu Asn Lys Thr Lys Pro lie 275 280 285Lys Glu Lys Tyr lie Asp Gin Glu Glu Leu Asn Lys Thr Lys Pro lie 275 280 285

Trp Thr Arg Asn Pro Asp Asp lie Thr Gin Glu Glu Tyr Gly Glu Phe 290 295 300Trp Thr Arg Asn Pro Asp Asp lie Thr Gin Glu Glu Tyr Gly Glu Phe 290 295 300

Tyr Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His 305 310 315 320Tyr Lys Ser Leu Thr Asn Asp Trp Glu Asp His Leu Ala Val Lys His 305 310 315 320

Phe Ser Val Glu Gly Gin Leu Glu Phe Arg Ala Leu Leu Phe lie Pro 325 330 335Phe Ser Val Glu Gly Gin Leu Glu Phe Arg Ala Leu Leu Phe lie Pro 325 330 335

Arg Arg Ala Pro Phe Asp Leu Phe Glu Asn Lys Lys Lys Lys Asn Asn 340 345 350 lie Lys Leu Tyr Val Arg Arg Val Phe lie Met Asp Ser Cys Asp Glu 355 360 365Arg Arg Ala Pro Phe Asp Leu Phe Glu Asn Lys Lys Lys Lys Asn Asn 340 345 350 lie Lys Leu Tyr Val Arg Arg Val Phe lie Met Asp Ser Cys Asp Glu 355 360 365

Leu lie Pro Glu Tyr Leu Asn Phe lie Arg Gly Val Val Asp Ser Glu 370 375 380Leu lie Pro Glu Tyr Leu Asn Phe lie Arg Gly Val Val Asp Ser Glu 370 375 380

Asp Leu Pro Leu Asn lie Ser Arg Glu Met Leu Gin Gin Ser Lys lie 385 390 395 400Asp Leu Pro Leu Asn lie Ser Arg Glu Met Leu Gin Gin Ser Lys lie 385 390 395 400

Leu Lys Val lie Arg Lys Asn lie Val Lys Lys Cys Leu Glu Leu Phe 405 410 415Leu Lys Val lie Arg Lys Asn lie Val Lys Lys Cys Leu Glu Leu Phe 405 410 415

Ser Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Phe Tyr Glu Ala 420 425 430Ser Glu Leu Ala Glu Asp Lys Glu Asn Tyr Lys Lys Phe Tyr Glu Ala 420 425 430

Phe Ser Lys Asn Leu Lys Leu Gly lie His Glu Asp Ser Thr Asn Arg 435 440 445 1357979Phe Ser Lys Asn Leu Lys Leu Gly lie His Glu Asp Ser Thr Asn Arg 435 440 445 1357979

Arg Arg Leu Ser Glu Leu Leu Arg Tyr His Thr Ser Gin Ser Gly Asp 450 455 460Arg Arg Leu Ser Glu Leu Leu Arg Tyr His Thr Ser Gin Ser Gly Asp 450 455 460

Glu Met Thr Ser Leu Ser Glu Tyr Val Ser Arg Met Lys Glu Thr Gin 465 470 475 480Glu Met Thr Ser Leu Ser Glu Tyr Val Ser Arg Met Lys Glu Thr Gin 465 470 475 480

Lys Ser lie Tyr Tyr lie Thr Gly Glu Ser Lys Glu Gin Val Ala Asn 485 490 495Lys Ser lie Tyr Tyr lie Thr Gly Glu Ser Lys Glu Gin Val Ala Asn 485 490 495

Ser Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr 500 505 510Ser Ala Phe Val Glu Arg Val Arg Lys Arg Gly Phe Glu Val Val Tyr 500 505 510

Met Thr Glu Pro lie -Asp Glu Tyr Cys Val Gin Gin Leu Lys Glu Phe 515 520 525Met Thr Glu Pro lie -Asp Glu Tyr Cys Val Gin Gin Leu Lys Glu Phe 515 520 525

Asp Gly Lys Ser Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro 530 535 540Asp Gly Lys Ser Leu Val Ser Val Thr Lys Glu Gly Leu Glu Leu Pro 530 535 540

Glu Asp Glu Glu Glu Lys Lys Lys Met Glu Glu Ser Lys Ala Lys Phe 545 550 555 560Glu Asp Glu Glu Glu Lys Lys Lys Met Glu Glu Ser Lys Ala Lys Phe 545 550 555 560

Glu Asn Leu Cys Lys Leu Met Lys Glu lie Leu Asp Lys Lys Val Glu 565 570 575Glu Asn Leu Cys Lys Leu Met Lys Glu lie Leu Asp Lys Lys Val Glu 565 570 575

Lys Val Thr lie Ser Asn Arg Leu Val Ser Ser Pro Cys Cys lie Val 580 585 590Lys Val Thr lie Ser Asn Arg Leu Val Ser Ser Pro Cys Cys lie Val 580 585 590

Thr Ser Thr Tyr Gly Trp Thr Ala Asn Met Glu Arg lie Met Lys Ala 595 600 605Thr Ser Thr Tyr Gly Trp Thr Ala Asn Met Glu Arg lie Met Lys Ala 595 600 605

Gin Ala Leu Arg Asp Asn Ser Thr Met Gly Tyr Met Met Ala Lys Lys 610 615 620 12 1357979Gin Ala Leu Arg Asp Asn Ser Thr Met Gly Tyr Met Met Ala Lys Lys 610 615 620 12 1357979

His Leu Glu lie Asn Pro Asp His Pro lie Val Glu Thr Leu Arg Gin 625 630 635 640His Leu Glu lie Asn Pro Asp His Pro lie Val Glu Thr Leu Arg Gin 625 630 635 640

Lys Ala Glu Ala Asp Lys Asn Asp Lys Ala Val Lys Asp Leu Val Val 645 650 655Lys Ala Glu Ala Asp Lys Asn Asp Lys Ala Val Lys Asp Leu Val Val 645 650 655

Leu Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp 660 665 670Leu Leu Phe Glu Thr Ala Leu Leu Ser Ser Gly Phe Ser Leu Glu Asp 660 665 670

Pro Gin Thr His Ser Asn Arg lie Tyr Arg Met lie Lys Leu Gly Leu 675 680 685Pro Gin Thr His Ser Asn Arg lie Tyr Arg Met lie Lys Leu Gly Leu 675 680 685

Gly lie Asp Glu Asp Glu Val Ala Ala Glu Glu Pro Asn Ala Ala Val 690 695 700Gly lie Asp Glu Asp Glu Val Ala Ala Glu Glu Pro Asn Ala Ala Val 690 695 700

Pro Asp Glu lie Pro Pro Leu Glu Gly Asp Glu Asp Ala Ser Arg Met 705 710 715 720Pro Asp Glu lie Pro Pro Leu Glu Gly Asp Glu Asp Ala Ser Arg Met 705 710 715 720

Glu Glu Val Asp 13Glu Glu Val Asp 13

Claims (1)

13579791357979 第94147646號 修正日期:99.2.6 佟if太- 「_ .........— 一塵.丨 _ _ I 十、申請專利範圍： 《ff·少月G曰修(支>^十、 1. 一種決定一人類個體罹患重症肌無力症風險的方 法，包括： (a) 提供一人類個體之一生物樣本；以及 (b) 檢測該生物樣本中熱休克蛋白60及熱休克蛋白90 自體抗體之量，其中該熱休克蛋白60自體抗體係專一性的 - 與熱休克蛋白60蛋白質（序列識別號：1)鍵結，該熱休克 蛋白90自體抗體包括熱休克蛋白90 α型及熱休克蛋白90 β • 型自體抗體，其中熱休克蛋白90α型自體抗體係專一性的 與熱休克蛋白90 α型蛋白質（序列識別號:2)鍵結，且熱休 克蛋白90 β型自體抗體係專一性的與熱休克蛋白90 β型蛋 白質（序列識別號:3)鍵結； 其中相較於非重症肌無力症之人類個體，若於該生物 樣本中存在有較高的熱休克蛋白60及熱休克蛋白90自體 抗體，表示該人類個體罹患重症肌無力症的風險較高。 2. 如申請專利範圍第1項所述之決定一人類個體罹患 • 重症肌無力症風險的方法，其中該生物樣本係擇自下列所 組成之族群：血清、血液、血聚、唾液、羊水、滑液、淚 液、***、淋巴、尿液、及汗液。 3. 如申請專利範圍第2項所述之決定一人類個體罹患 重症肌無力症風險的方法，其中該自體抗體係擇自下列一 種或以上之方法來偵側： (a) 西方墨點法；以及 (b) 酵素聯結免疫吸附法(ELISA)。 1 1357979 4. 一種決定一人類個體罹患重症肌無力症風險的套 組，包括偵測生物樣本中熱休克蛋白60及熱休克蛋白90 自體抗體之量的試劑，其中該熱休克蛋白60自體抗體係專 一性的與熱休克蛋白60蛋白質（序列識別號：1)鍵結，該 熱休克蛋白90自體抗體包括熱休克蛋白90 α型及熱休克 蛋白90 β型自體抗體，其中熱休克蛋白90 α型自體抗體係 專一性的與熱休克蛋白90 α型蛋白質（序列識別號:2)鍵 結，且熱休克蛋白90 β型自體抗體係專一性的與熱休克蛋 白90 β型蛋白質（序列識別號:3)鍵結， 其中相較於非重症肌無力症之人類個體，若於該生物 樣本中存在有較高的熱休克蛋白60及熱休克蛋白90自體 抗體，表示該人類個體罹患重症肌無力症的風險較高。 5. 如申請專利範圍第4項所述之決定一人類個體罹患 重症肌無力症風險的套組，其中該生物樣本係擇自下列所 組成之族群:血清、血液、血漿、唾液、羊水、滑液、淚液、 ***、淋巴、尿液、及汗液。 6. 如申請專利範圍第5項所述之決定一人類個體罹患 重症肌無力症風險的套組，其中該自體抗體係擇自下列至 少一種方法來偵側： (a) 西方墨點法；以及 (b) 酵素聯結免疫吸附法(ELISA)。Amendment date: 9414646: 99.2.6 佟if too - "_ ......... - a dust. 丨 _ _ I X. Patent application scope: "ff. Shaoyue G 曰 repair (support > ^10, 1. A method for determining the risk of myasthenia gravis in a human subject, comprising: (a) providing a biological sample of a human individual; and (b) detecting heat shock protein 60 and heat shock protein in the biological sample The amount of autologous antibody, wherein the heat shock protein 60 is autoantibody specific for the system - is linked to the heat shock protein 60 protein (SEQ ID NO: 1), and the heat shock protein 90 autoantibody comprises heat shock protein 90 Alpha-type and heat shock protein 90 β-type autoantibodies, in which the heat shock protein 90α-type autoantibody system is specifically linked to the heat shock protein 90 α-type protein (SEQ ID NO: 2), and the heat shock protein 90 The β-type autoantibody system is specifically associated with the heat shock protein 90 β-type protein (SEQ ID NO: 3); in contrast, in human subjects with non-myasthenia gravis, if there is a higher presence in the biological sample Heat shock protein 60 and heat shock protein 90 autoantibody, expressed Human subjects have a higher risk of myasthenia gravis. 2. A method for determining the risk of a human individual suffering from myasthenia gravis as described in the first paragraph of the patent application, wherein the biological sample is selected from the following group : serum, blood, blood, saliva, amniotic fluid, synovial fluid, tears, milk, lymph, urine, and sweat. 3. As determined in the second paragraph of the patent application, the human subject suffers from the risk of myasthenia gravis. The method wherein the autoantibody system is selected from one or more of the following methods: (a) Western blotting; and (b) enzyme-linked immunosorbent assay (ELISA). 1 1357979 4. A decision on a human individual A kit for the risk of myasthenia gravis, comprising an agent for detecting the amount of heat shock protein 60 and heat shock protein 90 autoantibodies in a biological sample, wherein the heat shock protein 60 is autoantibody specific to the heat shock protein 60 protein (SEQ ID NO: 1) linkage, the heat shock protein 90 autoantibody includes heat shock protein 90 α type and heat shock protein 90 β type autoantibody, wherein heat shock egg 90 alpha type autoantibody system specificity binding to heat shock protein 90 alpha type protein (SEQ ID NO: 2), and heat shock protein 90 beta type autoantibody system specificity with heat shock protein 90 beta protein (Sequence ID: 3) linkage, wherein compared to non-asthematic myasthenia, if there is a higher heat shock protein 60 and heat shock protein 90 autoantibody in the biological sample, the human Individuals are at higher risk for myasthenia gravis. 5. As determined in claim 4 of the patent application, a human individual is at risk of myasthenia gravis, which is selected from the following groups: Serum, blood, plasma, saliva, amniotic fluid, synovial fluid, tears, milk, lymph, urine, and sweat. 6. The method of claim 5, wherein the human subject suffers from a risk of myasthenia gravis, wherein the autoantibody system is selected from at least one of the following methods: (a) Western blotting; And (b) an enzyme-linked immunosorbent assay (ELISA).