經濟部中央標率局貝工消費合作社印裝 406190 五、發明説明（/ ) 1.發明背景 川崎症（Ka冊saki disease，以下簡稱KD)為日人川崎 富作於1967年在曰本確定’於此三十年間已有超過 120,000 病例發現（Yanagawa 等人，Arch Pediatr Adolesc Med 149 : 779，1995)。自 1970年發現以來在歐 美地區巳有相當多之研究，且已為最重要之小兒後天性心 臟病（Nelson等人，Textbook of Pediatrics，1996)， 與在第三世界地區（衛生條件不佳’營養不良地區）廣為發 生之風濕性心臟病互為消長（Taubert等人，J Pediatr 119:278 ’ 1991)。自從1976年以來在台灣也陸續有病例 報告’據估冲目則已有超過5，000病例（Lue，Kawasaki Disease 1995，p26) ’為台灣地區目前最重要（佔有5〇% 以上）之小兒後天性心臟病。 KD屬全身性血管發炎疾病，主要發生在五歲以下兒童， 原因尚不明。診斷主以特殊症狀（如發燒連續五天、眼結 膜炎、發疹及其它至少五大症狀）為確定（Dajani等人，Printed by the Central Standards Bureau of the Ministry of Economic Affairs, Shellfish Consumer Cooperative, 406190 V. Description of the invention (/) 1. Background of the invention Kawasaki disease (Ka book saki disease (hereinafter referred to as KD)) was established by Japanese Kawasaki Fusaku in 1967 ' More than 120,000 cases have been detected during these three decades (Yanagawa et al., Arch Pediatr Adolesc Med 149: 779, 1995). Since its discovery in 1970, there has been considerable research in Europe and the United States, and it has been the most important pediatric acquired heart disease (Nelson et al., Textbook of Pediatrics, 1996). Rheumatic heart disease, which is widespread in malnourished areas, is a growing and evolving one (Taubert et al., J Pediatr 119: 278 '1991). Cases have been reported in Taiwan since 1976. 'It is estimated that there have been more than 5,000 cases (Lue, Kawasaki Disease 1995, p26)' as the most important child in Taiwan (accounting for more than 50%). Natural heart disease. KD is a systemic vascular inflammation disease that mainly occurs in children under five years of age for unknown reasons. The diagnosis is based on special symptoms (such as fever for five consecutive days, conjunctivitis, rash, and at least five other major symptoms) (Dajani et al.,
Circulation 87:1776, 1993)。以超音波可以檢出KD對心 臟血管有侵害性（如心肌炎或心外膜炎），特別會造成冠狀 動脈瘤（coronary artery aneurysm)，也會造成猝死， 死亡率約 2%(Naoe 等人，？1^(^(：1比81〇11^5 250·· 341，1987)。KD —旦痊癒極少反復發作，在同一家庭成員 之間不傳染，可能具有種族特異性；以抗生素治療無效。 1991 年以來Newburger等人（N Engl J Med 324 : 1633， 1991)以靜脈注射高劑量免疫球蛋白（IVIG)和阿斯匹靈合 夂又度速用中國阐家榡皂（CNS ) A4圯格（210X 297公楚） 装 ^ —訂 線 一 (請先閱讀背面之注意事項再填寫本頁) 經濟部中央標準局員工消费合作社印裝 406190 五、發明说明（2) 併使用可以迅速治療此種疾病（N Engl J Med 324 : 1633，1991)，但是患有急性KD之病人在5~1〇年以後約有 25〜30%會有心臟血管内皮功能之損傷，此可能是造成一部 份成年人心肌梗塞的重要原因（Mi tani等人， Circulation 96 : 454 ， 1997)。 除了 C-反應蛋白質（Koyangi等人，Acta Paediatr 86 : 613，1997)與急性KD有關外，有許多細胞激素 (cytokines)、踵瘤壞死因子和細菌毒素也曾被提及與 KD 之發生有關（Leung 等人，Curr Opin Infect Disease 10 : 226，1997)。但是因其他急症也會產生上 述物質，作為診斷或篩檢KD其準確度皆顯然不夠，而且 也無法充分解釋病因及預測癒後《目前KD無有效之預防 方法，因此無法設定撲滅（eradication)計畫，而且也 無動物模式可供深入研究以解決KD所發生之機制性問 題。為了解決此問題，發展簡易、快速及高敏感度之生 化標記以診斷KD已是刻不容緩。 本發明利用血清中α -2結合球蛋白（a 2-haptoglobin ，Ηρα2)和脂蛋白原A-I (apolipoprotein A-I，A1)兩 種蛋白之比值以診斷川崎症，可以當為臨床醫師診斷與 治療KD預後之重要工具，具有簡單、快速、經濟、及敏 感度高之特性。 2.發明概述： 清中所含蛋白往往能反應出人體生理及病理之狀 I II^1 I -1 訂—— I 球 I (請先閱讀背面之注意事項再填寫本頁) 406190 五、發明説明（s) 態。KD目前之病因尚不明，但一般皆認為與嬰幼兒生活 之環境因子有關。此種因子或為微生物，或為化合物， 當由黏膜系統進入體内，即由免疫系統負責而進行抗原 之認識作用。因此，身體必然要對此種外來物質行急性 期反應’對KD兒童而言其臨床症狀（如高燒、發療、淋巴 腺腫大和手足指紅腫等）即為此種反應之結果。此種結果 可能因患者本身遺傳體質或當時健康情形而有各種程度 不一之症狀出現。然而，免疫反應的結果將表現在循環 系統之血球與血漿物質之變化。檢測此種變化即可探知 某種特定病原與人體免疫反應之間互動的情形。基於以 上之推理’吾人分析及比較KD患者與健康嬰幼兒血清蛋 白之成份。 KD之臨床診斷採行美國疾病管制中心標準（M〇rens等 人’ Pediatrics 65 : 21，1980)，而正常對照則採自門 經濟部中央搮準局負工消費合作社印裝 (請先閱讀背面之注意事項再填寫本頁) 診複診（己無疾病症狀）病人jk清樣本；另外，疾病對照 組來自發燒而症狀與KD相似之肺炎住院患者。各組血清 蛋白樣本溶入樣本緩衝液後即行單向膠體電泳蛋白分 離》圖一顯示前階段實驗結果，發現肋患声在急性期及 患病癒後恢復期之蛋白樣式有顯著不同；其中以具分子 量20kDa (P20)和25kDa (P25)之蛋白質尤其顯著，而具 40kDa (P40)區域之蛋白質帶也有很大不同（p2〇，p25， 和P40經證實為α_2結合球蛋白，脂蛋白原A-I，和β—結 合球蛋白及α-酸性醣蛋白，見下文）。因單向電泳所示之 一蛋白質帶可能為多種胜职次單元所構成，為了更進一步 本纸張尺度適用中國國豕橾準（CNS ) Α4说格（21〇 X 297公缝） 經濟部中央樣準局貝工消費合作杜印装 406190 a? _____B7 _五、發明説明（4) 解析其成份，我們執行雙向膠體電泳分析。圖二為典型 KD患者和正常兒童血清蛋白成分比較；以相等蛋白質量 分析和比較之結果，明確顯示幾種蛋白質有大量增加， 如P20和P40之同型物（isoforms);然而，也有減少之物 種，如P25。為了解各多胜职之身份，吾人將其轉移至轉 潰膜上並且進行胺端序列定序分析。定序結果顯示P20之 兩種同型物皆為〇：2_結合球蛋白（a2-haptoglobin，Hp α2)，另單向電泳膠體上所見之卩4〇為泠-結合球蛋白（石 -haptoglobin，Ηρβ)和 α -酸性醣蛋白（a -acid glycoprotein，AGP)綜合之結果，P25則為脂蛋白原八-Kapolipoprotein A-I，A1)。以上結果也經血清蛋白 圖譜（Anderson等人，The Plasma Proteins，V〇l IV P221 ’ 1984)和西方免疫吸潰分析之確定》 雙向蛋白質膠體電泳、胺端序列分析、及免疫吸潰分 析結果提供吾人兩項重要研究方向，一即急性期之](D病 人血清蛋白變化為何？及此種變化與臨床症狀是否有關？ 以單向膠體電泳分析結果指出，取自16個KD病人和1〇個 正常人（健康對照組）之蛋白質樣式確有顯著之不同，而 且病人血清中Ηρα2有5. 2倍之增加，而A1則降低34%(圖 一及表一）。 另外，為解決KD血清蛋白變化之專一性問題，我們選 取與KD主要症狀相似之急性肺炎病患（η= 1 〇)當為疾病對 照組。圖一及表一之結果，另顯示雖肺炎病人血清中Ηρ α 2的平均表現量是正常人之1.6倍，A1則較正常組低 尺度速用中國國家標垄（CNS ) A4規格（210X297公f ) (請先聞讀背面之注意事項再填寫本頁) 装' 訂 .丨球 406190 五、發明説明（J ) 5%，但兩者在統計上差異皆不顯著。另一方面’若比較 KD與肺炎患者血清中HP0:2和A1之量’則發現兩者皆有顯 著差異（表一，P<〇. 〇01)。以血清標記的開發而言，因Hp α2屬急性期蛋白（acute phase protein)之一類，有多 表一川崎症患者與其對照組臨床特性及各項生化測定值* 臨床特性及測定值 對照組 健康 肺炎 川崎症組 樣本數 10 10 16 性別（男/女） 8/2 8/2 9/7 月齡 31.10±4.98* 25.70 ±4.47lb 16.20±10.86b 發燒曰數 — 8.30±9.71 7.27±2.02 冠狀動脈 (正常/異常） 一 — 11/5 血小板(xl000/ram3)* 303.30±43.62b 343.70±27.00b 651.47*31.71* 白血球(xl〇〇〇/im3)冰 9.65±1.03b 14.48±2.70 Λ 16.89±1.221 Hp〇2(吸光值） 33.7645.6911 52.40±11.03s 176.26±18.35a AK吸光值） 206.10±8.56* 195.61±7.18* 135.71±8.48b Hpcu/Al 0.17±0.03b 0.28±0.06b 1.36±0.15a 測定結果以平均值±標準誤差(meaiuS. E.)表^ *表示療程中之最高觀測值 a’b同一列中具相同上標字母者差異不顯著 I 策-----M訂 i tn 11 ! I: - II 1 - .. (請先閱讀背面之注意事項再填寫本頁) / 經濟部中央標準局員工消費合作社印裝 種刺激原可造成其量之增加，代表KD疾病本質之特定性 恐有不足，所以需要加入其他輔助因子（Uen()等人，Circulation 87: 1776, 1993). Ultrasound can be used to detect KD's invasiveness to cardiac blood vessels (such as myocarditis or epicarditis), especially coronary artery aneurysm, and sudden death, with a mortality rate of about 2% (Naoe et al., 1 ^ (^ (: 1 to 81〇11 ^ 5 250 ·· 341, 1987). KD-Once cured, there are very few recurrent episodes, it is not contagious between members of the same family, and may be race-specific; treatment with antibiotics is not effective. Since 1991, Newburger et al. (N Engl J Med 324: 1633, 1991) have used the high-dose immunoglobulin (IVIG) and aspirin combination intravenously to rapidly use the Chinese Aquarium Soap (CNS) A4 grid. (210X 297 Gongchu) Equipment ^ — Thread 1 (Please read the precautions on the back before filling out this page) Printed by the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs 406190 5. Description of the invention (2) and the use can quickly treat this Disease (N Engl J Med 324: 1633, 1991), but about 25 to 30% of patients with acute KD will have cardiac vascular endothelial function damage after 5 to 10 years, which may cause some adulthood Important causes of human myocardial infarction (Mi tani et al. , Circulation 96: 454, 1997). In addition to C-reactive proteins (Koyangi et al., Acta Paediatr 86: 613, 1997) are associated with acute KD, many cytokines, tumor necrosis factors and bacterial toxins have also been It has been mentioned that it is related to the occurrence of KD (Leung et al., Curr Opin Infect Disease 10: 226, 1997). However, the above substances can also be produced due to other emergencies. The accuracy of KD as a diagnosis or screening is obviously insufficient, and it is also impossible. Fully explain the cause and predict that "there is currently no effective prevention method for KD, so there is no eradication plan, and there is no animal model available for in-depth research to solve the mechanism of KD. To solve this problem, It is urgent to develop simple, rapid and highly sensitive biochemical markers for diagnosing KD. The present invention utilizes two types of α-2 binding globulin (a 2-haptoglobin (Ηρα2)) and apolipoprotein AI (A1) in serum Protein ratio for the diagnosis of Kawasaki disease can be used as an important tool for clinicians to diagnose and treat KD prognosis, which is simple, fast, economical, and High sensitivity. 2. Summary of the invention: The protein contained in Qing can often reflect the physiology and pathology of human body. I II ^ 1 I -1 Order-I ball I (Please read the precautions on the back before filling this page ) 406190 V. Description of invention (s) state. The current cause of KD is unknown, but it is generally believed to be related to environmental factors in the lives of infants and young children. Such factors are either microorganisms or compounds. When they enter the body through the mucosal system, they are responsible for the recognition of antigens by the immune system. Therefore, the body must have an acute phase reaction to this foreign substance. For KD children, the clinical symptoms (such as high fever, hair treatment, lymphadenopathy, redness and swelling of the hands and feet, etc.) are the result of this reaction. Such results may have varying degrees of symptoms due to the patient's own genetic constitution or current health conditions. However, the results of the immune response will be manifested by changes in the blood cell and plasma substances of the circulatory system. Detecting such changes can detect the interaction between a particular pathogen and the body's immune response. Based on the above reasoning, I analyzed and compared the composition of serum proteins of KD patients and healthy infants. The clinical diagnosis of KD adopts the US Centers for Disease Control (Morrens et al. 'Pediatrics 65:21, 1980), while the normal control is printed by the Consumer Work Cooperative of the Central Bureau of Standards, Ministry of Economic Affairs (please read the back first) Please note that this page is to be filled out again.) Patients who have been diagnosed and returned to the clinic (have no symptoms of the disease) have cleared samples of jk. In addition, the disease control group came from hospitalized patients with fever and symptoms similar to KD. The serum protein samples of each group are separated by sample gel solution and then subjected to unidirectional gel electrophoresis. Figure 1 shows the results of the previous stage experiments. It was found that the protein patterns of the rib sound in the acute phase and the recovery period after the disease were significantly different; Proteins with molecular weights of 20kDa (P20) and 25kDa (P25) are particularly significant, and protein bands with 40kDa (P40) regions are also very different (p2O, p25, and P40 have been confirmed to be α_2 binding globulin, lipoprotein AI , And β-binding globulin and α-acid glycoprotein, see below). Because one of the protein bands shown in one-way electrophoresis may be composed of multiple winning units, in order to further this paper standard China National Standards (CNS) Α4 grid (21〇X 297 stitches) Central Sample Coordination, Shellfish Consumer Cooperative, Du printed 406190 a? _____B7 _V. Description of the Invention (4) To analyze its composition, we perform two-dimensional colloidal electrophoresis analysis. Figure 2 shows a comparison of serum protein components between typical KD patients and normal children. Based on the analysis and comparison of equal protein masses, it is clear that several proteins have increased significantly, such as isoforms of P20 and P40; however, there are also reduced species , Such as P25. In order to understand the identity of each job, we transferred it to the transmembrane and performed amine end sequence analysis. The sequencing results showed that both isoforms of P20 were 0: 2_binding globulin (a2-haptoglobin, Hp α2), and 卩 40, which was seen on the unidirectional electrophoretic colloid, was Ling-binding globulin (stone-haptoglobin, Ηρβ) and α-acid glycoprotein (AGP), P25 is lipoprotein octa-Kapolipoprotein AI (A1). The above results were also determined by serum protein mapping (Anderson et al., The Plasma Proteins, Vol. IV P221 '1984) and Western immunoassay analysis. "Two-dimensional protein colloid electrophoresis, amine end sequence analysis, and immunoassay analysis results provided My two important research directions, one is the acute phase] (What are the changes in serum protein in D patients? And whether such changes are related to clinical symptoms? According to the results of unidirectional gel electrophoresis analysis, 16 KD patients and 10 The protein patterns of normal people (healthy control group) are indeed significantly different, and ρρα2 in the patient's serum increased by 5.2 times, while A1 decreased by 34% (Figure 1 and Table 1). In addition, in order to solve the KD serum protein The specificity of the change, we selected patients with acute pneumonia (η = 10) similar to the main symptoms of KD as the disease control group. The results in Figure 1 and Table 1 also show the average Ηρ α 2 in the serum of patients with pneumonia. The performance is 1.6 times that of normal people, and A1 is faster than the normal group and uses the Chinese National Standard Ridge (CNS) A4 specification (210X297 male f) (please read the precautions on the back before filling this page)订 Order. 丨 Ball 406190 V. Description of the invention (J) 5%, but the difference between the two is not statistically significant. On the other hand, 'If comparing the amount of HP0: 2 and A1 in the serum of KD and pneumonia patients', two are found There were significant differences between the patients (Table I, P < 〇01). In terms of the development of serum markers, because Hp α2 belongs to the category of acute phase proteins, there are many patients with Kawasaki disease and their control groups. Clinical characteristics and measured values of various biochemicals * Clinical characteristics and measured values of the control group Healthy pneumonia Kawasaki disease group Number of samples 10 10 16 Gender (male / female) 8/2 8/2 9/7 Month age 31.10 ± 4.98 * 25.70 ± 4.47 lb 16.20 ± 10.86b Fever number — 8.30 ± 9.71 7.27 ± 2.02 Coronary artery (normal / abnormal) 1— 11/5 platelets (xl000 / ram3) * 303.30 ± 43.62b 343.70 ± 27.00b 651.47 * 31.71 * white blood cells (xl〇 〇〇 / im3) Ice 9.65 ± 1.03b 14.48 ± 2.70 Λ 16.89 ± 1.221 Hp〇2 (absorbance value) 33.7645.6911 52.40 ± 11.03s 176.26 ± 18.35a AK absorbance value) 206.10 ± 8.56 * 195.61 ± 7.18 * 135.71 ± 8.48 b Hpcu / Al 0.17 ± 0.03b 0.28 ± 0.06b 1.36 ± 0.15a Difference (meaiuS. E.) Table ^ * indicates that the highest observed value a'b in the course of treatment is the same with the same superscript letters in the same column. The difference is not significant. I policy ----- M i tn 11! I:-II 1 -.. (Please read the precautions on the back before filling out this page) / The stimulus for printed cooperatives of the Consumer Cooperatives of the Central Standards Bureau of the Ministry of Economic Affairs can cause an increase in its quantity, which may be insufficient to represent the specificity of the nature of KD disease, so it is necessary to Add other cofactors (Uen () et al.,
Clin Exp Immunol 76 : 337 ’ 1989) » 本研究調查Kd病 人與對照組血清中蛋白質樣式及其量之改變，發現“的 變化一致性甚高（前述），而且此結果也由最近研究^〇之 主要學者所證實（Kim等人，Acta Paediatr Jpn 37 : 672，1995 ; Chiang 等人，Clin Chim Acta 260 : 7 “ 中_家料（CNS)切!蝽。|〇:< 297公釐). --：~~— 經濟部中央樣隼局W工消費合作社印製 406190 五、發明説明（J) 115， 1997 ; Cabana等人，Pediatr Res 42 : 651， 1997)。又因A1是血清令之主要蛋白而且是高密度膽固醇 粒子95%以上之蛋白質成分’所以吾人選擇A1當為KD血清 標記之輔助因子·> 以A1之量當為分母求得Ηρ α VAl比值，經過統計分 析，結果顯示Ηρα VAl在KD與對照組之間呈現極顯著差 異（表一 ’ P<〇. 001);而KD病人血清中Ηρα2/Α1比值各 為正常人和肺炎病人之8. 0倍和4. 9倍。以上結果明確顯 示，臨床醫師可以HpaVAl比值來評估及分辨患者是否 可能患有KD，其陽性及陰性預測值可達93. 75%及90%(表 表二利用Ηροκ/ΑΙ診斷川崎症之價值評估木 分割值+ 敏感性 (.%) 專一性 (¾) 湯性預測值 (X) 陰性預測值 0.535 93.75 90 93.750 90 * 計算方式根據 0ΙΕ 诊斷棵準手冊(OIE Manual of Standard for Diagnostic Test and Vaccines, Office International des EpizcxDties, 1997, nn 8-15) +以肺炎為疾病對照組，分割值(cut-off value)相當於川崎症組平均值減去 5.5SE. 、 進一步研究〇患者恢復期血清蛋白變化（圖三），可以 發現HpaVAl在IVIG治療約1個月以後降至正常值（此即 表示巳恢復正常），而且其變化與KD各症狀在恢復期消褪 之趨勢平行。其他與發炎反應有關之臨床生化值如企小 板數目，白血球數目，及C反應蛋白質雖皆有變化但卻無 毛或汝又度適用中國國家標準（CNS )厶4坟格（210X291公聲） --1 n ------k I I___—丁 l·__I___ ， V . (請先閱讀背面之注意事項再填寫本頁) A7 B7 40619° 五、發明説明（7 ) 法充分反應疾病之恢復趨勢。 就吾人檢索醫學文獻資料，發現目前並無文獻論及以 Ηρα VAl作為檢驗KD的血清標記。綜合以上所述，利用 極為簡便之單向膠體電泳分析並配合自動儀器之使用， 由血清至得出Ηρα VAl比值之結果可在一小時内達成； 將來製成快速檢驗試劑組更可在1分鐘之内得到結果，不 只地區及教學醫院醫師可以施行此項檢驗，一般中小型 診所也可以使用，此對於KD之早期診斷和治療時機有相 當大之改進作用。 3.發明詳細說明：（實施例說明） (1).血清之採集： 高雄醫學院小兒科臨床醫師根據美國疾病管制中心所 訂的KD診斷標準（如前所述）包括以連續發燒五天，而有 皮庸紅疹、眼結膜炎、口腔黏膜變化（如草莓樣舌，口唇 乾裂等）、四肢之變化（紅腫、脫皮等）等六項之五項以上 症狀時即遇定，並進行靜脈採血工作。以採血管自靜脈 採出約5c. c.，並即時以3000rpm(在室溫）離心5分鐘， 即可取得血清樣本。正常對照組樣本採自門診複診（己無 疾病症狀）病人；另外，疾病對照組樣本來自發燒而無KD 其他症狀之急性肺炎病人。各組血清樣本保持於4艺不超 過24小時g卩行測定各生化值’或保存於_2代於一個月内 再以乾冰保存快遞（2天之内）至臺灣養豬科學研究所比較 醫學系，特血清蛋自成分分析。以上保存及傳遞過程 (請先閱讀背面之注意事項再填寫本頁) •I於' 訂 經濟部中央標準局貞工消費合作杜印製 經濟部中央標準局員工消費合作社印裝 4061*90 a7 _____B7 五、發明説明（？） 已確定不會造成保存與新鮮樣本間測定值之差異。 (2) 血清蛋白樣本液製備： 冷;東或新鮮樣本經回溫後取1〇μ1加入含240μ1樣本緩 衝液’内含62. 5mM Tris-HCl，ρΗ6. 8，2%十二烷基氫硫 酸鈉鹽（sodium dodecyl sulfate)，5%硫醇基乙醇 (β-mercaptoethanol) ’ 10% 甘油（glycerol)，和 0.002%溴酚藍（bromophenol blue)，並即置入沸水中 反應5分鐘。樣本液經離心（室溫，12000rpm)5分鐘後， 即載入聚丙醯胺膠内，進行單向電泳分離。對欲進行雙 向電泳分離之樣本，取80μ1加入含160μ1分解液，内含 9.5Μ尿素（urea)，2% Nonident Ρ-40，2%雙性分解子 (ampholytes)，和 5%piercaptoethanol，並以urea 調整體積至260μ1即可進行雙向電泳分離血清樣本之蛋 白質濃度以Lowry法測定，並以小牛血清蛋白當為參考標 準。 (3) .蛋白質電泳和呈色： 單向電泳所需聚丙醯胺膠（0. 075x14x16cm)之製備及 進行方法見Laemmli 等人（Nature，227 : 680，1970)。 樣本體積10μ1加入20個槽模之膠片，在上層丙醯胺濃度 為4.75%，下層為12.5%。電泳約進行3小時（以每片膠 30mA固定電流），即可下片染色。膠片在新鮮染色液 (0.125% Coomassie blue R-250 之50% 甲醇及 10% 醋 酸溶液）内浸潤30分鐘，隨即以去染溶液甲（含50%甲醇及 10%醋酸溶液）去染40分鐘，並再度以去染溶液乙（含5%甲 10 本紙悵尺度適用中國國家標準（CNS ) A4况格（210X297公釐） ! -- - I . I I ^^1 I m I-*水 I I I I ——-I ^^1 I I ^^1 I - I »4^ (請先閲讀背面之注意事項再填寫本頁) 406190 A7 ________B7_ 五、發明説明（f ) 醇及7%醋酸溶液）去染至少丨個小時，即可進行蛋白 確定及定量分析。 帶 雙向電泳分析根據〇，Farreii方法（J Biol Che 250:4007 ’ 1975)進行。約6〇〇μβ (3〇μ1樣本液）之蛋白 質載入第一面向之等電聚焦聚丙醯胺膠體（直徑〇.， 長度12cm)上端，即行4〇〇伏特共16小時和之後8〇〇伏特^ 小時之電泳分離。第二面向之聚丙醯胺膠之製作方式、 其電泳過程、和下片後膠體之染色，去染皆與單向電泳 分析相同。 (4).定量分析： 單向電泳膠片上蛋白質帶以光密度掃瞄儀（軟體為Clin Exp Immunol 76: 337 '1989) »This study investigated changes in the protein pattern and amount of serum in Kd patients and control groups, and found that" the consistency of the changes is very high (mentioned above), and this result is also from a recent study ^ 〇 之Confirmed by major scholars (Kim et al., Acta Paediatr Jpn 37: 672, 1995; Chiang et al., Clin Chim Acta 260: 7 "Medium_family material (CNS) cut! 蝽. | 〇: < 297 mm). -: ~~ — Printed by W Industry Consumer Cooperative of the Central Bureau of Samples of the Ministry of Economic Affairs 406190 V. Description of Invention (J) 115, 1997; Cabana et al., Pediatr Res 42: 651, 1997). And because A1 is the main protein of serum, and it is a protein component of more than 95% of high-density cholesterol particles, so I chose A1 as the cofactor for KD serum labeling. ≫ Calculate the Ηρ α VAl ratio using the amount of A1 as the denominator. After statistical analysis, the results showed that Ηρα VAl showed a very significant difference between KD and the control group (Table 1 'P <0.001); and the ratio of Ηρα2 / Α1 in the serum of KD patients was 8% of that of normal people and pneumonia patients. 0 times and 4.9 times. The above results clearly show that clinicians can evaluate and distinguish whether patients may have KD with HpaVAl ratio, and their positive and negative predictive values can reach 93.75% and 90% (Table 2 Evaluation of the value of Ηροκ / ΑΙ in the diagnosis of Kawasaki disease) Wood split value + sensitivity (.%) Specificity (¾) soup predictive value (X) negative predictive value 0.535 93.75 90 93.750 90 * The calculation method is based on the OIE Manual of Standard for Diagnostic Test and Vaccines, Office International des EpizcxDties, 1997, nn 8-15) + Pneumonia was used as the disease control group, and the cut-off value was equivalent to the Kawasaki disease group average minus 5.5SE. Further study. Serum protein changes in patients during recovery (Figure 3), it can be found that HpaVAl drops to normal value after about 1 month of IVIG treatment (this means that the tadpoles return to normal), and the changes are parallel to the tendency of the symptoms of KD to fade during the recovery period. Others are related to the inflammatory response The clinical biochemical values such as the number of small plates, the number of white blood cells, and the C-reactive protein are all changed, but they are hairless or you can apply the Chinese National Standard (CNS) 厶 4 grave (2 10X291 voice) --1 n ------ k I I ___— ding l · __I ___, V. (Please read the notes on the back before filling this page) A7 B7 40619 ° V. Description of Invention (7) Method Fully reflect the recovery trend of the disease. According to my search of medical literature, it is found that there is no literature on the use of Ηρα VAl as a serum marker for testing KD. Based on the above, the extremely simple one-way gel electrophoresis analysis is used in conjunction with automatic instruments. Use, the results from serum to Ηρα VAl ratio can be achieved within one hour; in the future, the rapid test reagent set can be obtained within 1 minute, not only regional and teaching hospital doctors can perform this test, generally It can also be used in small clinics, which greatly improves the early diagnosis and treatment timing of KD. 3. Detailed description of the invention: (exemplary description) (1). Collection of serum: Pediatric clinician of Kaohsiung Medical College based on American disease The KD diagnostic criteria set by the control center (as described above) include five days of continuous fever with skin rash, ocular conjunctivitis, and changes in oral mucosa (such as strawberry-like tongue) Lip cracks, etc.), changes in extremities (redness, swelling, peeling, etc.) when five or more of the six symptoms are met, and venous blood collection work. Take about 5c.c. from the vein with blood collection tube, and immediately at 3000rpm Centrifuge (at room temperature) for 5 minutes to obtain serum samples. The normal control group samples were collected from outpatient follow-up (no disease symptoms); in addition, the disease control group samples were from patients with acute pneumonia who had fever without other symptoms of KD. Serum samples of each group were kept at 4 g for no more than 24 hours, and the biochemical values were measured. Or stored in _2 generations within one month and then stored on dry ice for express delivery (within 2 days) to the Taiwan Pig Science Institute for Comparative Medicine. Line, special serum egg self-component analysis. The above preservation and transfer process (please read the precautions on the back before filling this page) • I order 'Chengdu Consumer Cooperation of the Central Standards Bureau of the Ministry of Economic Affairs and Du Duan printed by the Central Consumers Bureau of the Ministry of Economic Affairs and printed on the consumer cooperatives 4061 * 90 a7 _____B7 5. Description of the invention (?) It has been determined that there will be no difference in measured values between the preserved and fresh samples. (2) Preparation of serum protein sample solution: cold; after the sample is warmed up, take 10μ1 and add 240μ1 sample buffer 'containing 62. 5mM Tris-HCl, ρΗ6.8, 2% dodecyl hydrogen Sodium dodecyl sulfate, 5% β-mercaptoethanol '10% glycerol, and 0.002% bromophenol blue, and placed in boiling water to react for 5 minutes. After the sample solution was centrifuged (room temperature, 12000 rpm) for 5 minutes, it was loaded into the polyamidamine gel and separated by unidirectional electrophoresis. For samples to be separated by two-dimensional electrophoresis, take 80μ1 and add 160μ1 decomposing solution, which contains 9.5M urea, 2% Nonident P-40, 2% ampholytes, and 5% piercaptoethanol. Adjust the volume of urea to 260μ1 to perform two-dimensional electrophoresis to separate the protein concentration of serum samples by Lowry method, and use calf serum protein as the reference standard. (3). Protein electrophoresis and color development: Laemmli et al. (Nature, 227: 680, 1970) for the preparation and the method of polypropylene gel (0.075x14x16cm) required for unidirectional electrophoresis. A sample volume of 10 μ1 was added to the film of 20 slot molds. The concentration of propylamine in the upper layer was 4.75% and the lower layer was 12.5%. The electrophoresis is performed for about 3 hours (at a fixed current of 30 mA per gel), and the next slide can be stained. The film was soaked in fresh staining solution (0.125% Coomassie blue R-250 in 50% methanol and 10% acetic acid solution) for 30 minutes, and then decolorized with decoloring solution A (containing 50% methanol and 10% acetic acid solution) for 40 minutes. And again with a de-staining solution B (containing 5% A10 paper size) applicable Chinese National Standard (CNS) A4 condition (210X297 mm)!--I. II ^^ 1 I m I- * 水 IIII —— -I ^^ 1 II ^^ 1 I-I »4 ^ (Please read the notes on the back before filling out this page) 406190 A7 ________B7_ V. Description of the invention (f) Alcohol and 7% acetic acid solution) Destain at least one Within hours, protein identification and quantitative analysis can be performed. Band Two-dimensional electrophoresis analysis was performed according to the Farreii method (J Biol Che 250: 4007 '1975). Approximately 600 μβ (30 μ1 sample solution) of the protein was loaded on the top of the first-oriented isoelectrically focused polyimide colloid (diameter 0.1, length 12 cm), that is, a line of 400 volts for a total of 16 hours and 800 volts thereafter. Volt ^ h separation by electrophoresis. The manufacturing method, the electrophoresis process, and the gel staining and de-staining of the second-side polyimide gel are the same as those of unidirectional electrophoresis analysis. (4). Quantitative analysis: The protein bands on the unidirectional electrophoresis film are scanned with optical density (software is
ImageQuant NT，Molecular Dynamics，美國）择入軟 體内，並進行定量分析。吾人感興趣之JJp Q；2和A1帶以相 同大小之四方形（rectangle)選定定量區，並取其體積量 (volume)值即得Hp 〇：2和A1之吸光值。膠片内 (intragel)和膠片間（intergel)所測定吸光值經過模擬 校正’對同一樣本之變異（variati〇n)皆位於之内。 經濟部中央標準局貝工消費合作社印裝 n-l·— I --II--ί -良 I--- τ Γ CT - 、τ (請先聞請背面之注意事項再填寫本頁) 4.本發明之重要性及特點： 本發明專為診斷KD而設計。因為KD之臨床診斷需要出 現六種主要症狀之五種才為確診，所以時常會有失去治 療先機之虞，在保護病患權益與節省醫療資源考量之間 甚難取得平衡點。相當有經驗之醫師可在典型病症出現 五天前即予以確診並予適時之IVIG治療，此時產生冠狀 本紙佚尺度遘用中國國家橾準（CNS ) Μ规格（2I0X297公釐） 經濟部中央標準局員工消费合作社印製 406190 at B7 _ _丨丨 "*' _ 五、發明説明（，〇 ) 動脈瘤的機率可降低至10-15%。然而KD普遍發生於各 地，而目前除病症診斷之外，炎無有效使用之其他診斷 方法，常使得治療失去時機，造成高達50-60%病患產生 冠狀動脈瘤，此可能是造成病患成年之後心臟血管疾病 之重要原因。另一方面，因目前無有效之KD血清標記， 所以無法進行大規模跨中心之研究計畫及建立動物模 式，如能早期監測’即可節省大量醫療資源，並有效預 防冠狀動脈瘤之發生，將可提高國民醫療水準及保護生 命之最終目標。 本發明利用膠體電泳方法測定Ηρ α 2/Α1之比值，更具有 如下特點： (1) 簡單：本法在大部份醫院或檢驗院所可以輕易施行， 不需增添昂貴設備。 (2) 快速.本法若使用自動儀器（如phastSySteffl， Pharmacia ’瑞典）可在1小時内獲得結果。 (3) 經濟：本法只需〇,4μ1血清即可進行檢驗，而且對照 樣本可在同一膠片上作大量（可到2〇個樣本）比對。 (4) 敏感度向：本法直接檢測，並有效地除去 背景值和空白值，為其他方法所不能及。 基於上述特點並結合KD在急性期和恢復期Ηρα 2和Α1 的反應本質’本法具有相當高之專一性，除可辅助臨 床醫師確定診斷之外’並且將成為薛選疑&KD病例及 建立完善動物模式相當重要的工具。 12 用中國闺家標准ί CNS ) 丨0X 297公斧-：-： f ---- I - -'- I - ID 1- - i -- - - - n -- I I - II- - I I il. .f5JJ - I I - -1 - 11 -- - I (請先閱讀背面之注意事項再填寫本頁) 406190_^_ 五、發明説明（I I ) 5.圖式說明 (請先閱讀背面之注意事項再填寫本頁) 圖一川崎症患者和其對照樣本血清蛋白質樣式。第一 階段分析川崎症患者樣本（KDI)，發現(X2-結合球 蛋白（Ηρα2)和脂蛋白原A-I(Al)是變化最明顯而 且與患者病程有關之蛋白質。其他樣本，如第二 階段川崎症（KDII)、健康（HC)、及肺炎（PC)樣 本為各組之抽樣實施例。Μ，為分子量標準，以 千道耳吞（kDa)表示；Β，靜脈注射免疫球蛋白 (IVIG)之前的樣本；A，IVIG完成之後的樣本； C，IVIG之後二個月的樣本；Ηρ+Α1，α2-結合球 蛋白和脂蛋白原A-Ι之蛋白質參照；IG，免疫球 蛋白參照；Ηρβ/AGP，β-結合球蛋白和a-酸性醣 蛋白；Upon，on-結合球蛋白。 圖二川崎症患者和正常兒童血清蛋白雙向電泳圖。代 號如圖一；IEF，等電聚焦電泳；SDS-PAGE，聚 丙醯胺膠電泳；N，正常血清樣本；KD，川崎症 血清樣本。圖下方數字表示IEF所形成之pH 值。 經濟部中央橾準局員工消費合作社印裝 圖三川崎症患者血清中a2-結合球蛋白對脂蛋白原-AI比 值(Hpot2/Al)之變化。恢復期第0天為靜脈注射免疫 球蛋白之時間。負數表示治療前之觀察天數。插圖 為患者KD3a2_結合球蛋白（Hpa2)和脂蛋白原-A I (A1) 變化之實例。 13 夂 罔闺家標准（CN:S 1 (]丨〇:<297 公犛）ImageQuant NT, Molecular Dynamics, USA) was selected into the soft body and subjected to quantitative analysis. We are interested in the JJp Q; 2 and A1 bands. The quantitative region is selected with a rectangle of the same size, and the volume value is taken to obtain the absorbance of Hp 0: 2 and A1. The absorbance measured within the film (intragel) and between the films (intergel) is simulated and corrected, and the variation (variation) of the same sample is included. Printed by the Shellfish Consumer Cooperative of the Central Standards Bureau of the Ministry of Economic Affairs nl · —I--II--ί-良 I --- τ Γ CT-、 τ (Please read the notes on the back before filling out this page) 4. This Importance and characteristics of the invention: The invention is designed for the diagnosis of KD. Because the clinical diagnosis of KD requires five of the six main symptoms to be confirmed, there is often a risk of losing the opportunity to treat, and it is difficult to strike a balance between protecting patient rights and saving medical resources. A fairly experienced physician can be diagnosed five days before the onset of typical symptoms and treated with IVIG in a timely manner. At this time, a coronal paper is produced, using the Chinese National Standard (CNS) M standard (2I0X297 mm). Central standard by the Ministry of Economic Affairs Printed by the Bureau ’s Consumer Cooperatives 406190 at B7 _ _ 丨 丨 " * '_ V. Description of the Invention (, 〇) The probability of aneurysms can be reduced to 10-15%. However, KD generally occurs in various places. At present, in addition to the diagnosis of the disease, other diagnostic methods that are not effectively used, often make the treatment missed, causing up to 50-60% of patients with coronary aneurysms, which may cause adult patients. Important cause of cardiovascular disease afterwards. On the other hand, because there is no effective KD serum marker, it is not possible to conduct large-scale cross-center research projects and establish animal models. If early monitoring can save a lot of medical resources and effectively prevent the occurrence of coronary aneurysms, The ultimate goal is to raise the standard of national medical care and protect lives. The present invention uses the colloidal electrophoresis method to measure the ratio of Ηρ α 2 / Α1, and has the following characteristics: (1) Simple: This method can be easily implemented in most hospitals or testing institutes without adding expensive equipment. (2) Fast. This method can obtain results within 1 hour if using automatic equipment (such as phastSySteffl, Pharmacia 'Sweden). (3) Economic: This method requires only 0.4 μl serum for testing, and the control sample can be compared on a large number of films (up to 20 samples). (4) Sensitivity direction: This method directly detects and effectively removes background and blank values, which is beyond the reach of other methods. Based on the above characteristics and combined with the nature of KD's response to 急性 ρα 2 and A1 in the acute and recovery phases, 'this method has a high degree of specificity, in addition to assisting clinicians in determining the diagnosis', and will become a case of Xue Xuan Su & KD It is a very important tool to establish and perfect animal models. 12 Use Chinese boudoir standard ί CNS) 0X 297 male axe-:-: f ---- I--'- I-ID 1--i----n-II-II--II il . .f5JJ-II--1-11--I (Please read the precautions on the back before filling this page) 406190 _ ^ _ V. Description of the invention (II) 5. Schematic description (Please read the precautions on the back first) (Fill on this page again) Figure 1. Serum protein patterns of Kawasaki patients and their control samples. The first stage analysis of patients with Kawasaki disease (KDI), found that (X2-binding globulin (Ηρα2) and prolipoprotein AI (Al) are the most obvious changes and related to the patient's disease. Other samples, such as the second stage Kawasaki (KDII), health (HC), and pneumonia (PC) samples are sampling examples of each group. M, the molecular weight standard, expressed in thousand channel ear swelling (kDa); B, intravenous immunoglobulin (IVIG) Previous samples; A, samples after IVIG completion; C, samples two months after IVIG; Ηρ + A1, α2-protein reference for binding globulin and lipoprotein A-1; IG, immunoglobulin reference; Ηρβ / AGP, β-binding globulin and a-acid glycoprotein; Onon, on-binding globulin. Figure 2. Two-dimensional electrophoresis pictures of serum proteins of patients with Kawasaki disease and normal children. Codes are shown in Figure 1. IEF, isoelectric focusing electrophoresis; SDS -PAGE, polyacrylamide gel electrophoresis; N, normal serum samples; KD, Kawasaki disease serum samples. The numbers below the figure represent the pH value formed by the IEF. Printed by the Consumer Cooperatives of the Central Government Bureau of the Ministry of Economic Affairs. A2-bond ball Changes in white to lipoprotein-AI ratio (Hpot2 / Al). Day 0 of the recovery period is the time of intravenous immunoglobulin injection. Negative numbers indicate the number of days before treatment. The illustration shows patients KD3a2_binding globulin (Hpa2) and An example of changes in pro-lipoprotein-AI (A1). 13 Standards for Chinese Family (CN: S 1 (] 丨 :: 297 cm)