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研究生:林卉宜
研究生(外文):Hui-Yi Lin
論文名稱:利用乳膠凝集試驗快速篩檢及應用LipL32重組蛋白質以酵素結合免疫吸附法進行鉤端螺旋體感染症之血清學診斷
論文名稱(外文):Latex Agglutination as a Rapid Screening Test and the Use of Recombinant LipL32 antigen- based Enzyme- linked Immunosorbent Assay for the Serodiagnosis of Human Leptospirosis
指導教授:潘銘正潘銘正引用關係
指導教授(外文):Ming-Jeng Pan
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:獸醫學研究所
學門:獸醫學門
學類:獸醫學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:98
中文關鍵詞:LipL32酵素結合免疫吸附法乳膠凝集試驗鉤端螺旋體感染症
外文關鍵詞:latex agglutination testleptospirosisLipL32ELIS
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鉤端螺旋體感染症是一種世界性分佈的人畜共通傳染疾病,此病無法單獨以臨床症狀作為診斷,必須以實驗室檢查加以輔助確認,目前採用的標準檢驗方法是顯微凝集試驗 (microscopic agglutination test,簡稱MAT),其操作特點是以各種血清型的活菌作為抗原,除了耗時外,對實驗室操作人員而言,具有高度危險性的。本研究製備乳膠凝集試劑 (latex agglutination test,簡稱LAT),其效力與MAT及兩種商品化套組LeptoTek Lateral Flow和LeptoTek Dri-Dot做比較。檢體來源為472個送檢之疑似鉤端螺旋體感染病患的血清,經由合約實驗室進行MAT及Lateral Flow和Dri-Dot的檢驗。以MAT為黃金標準分別評估Lateral- Flow、Dri-Dot 及LAT之敏感性及特異性,敏感性分別為44.4%、52.8%、86.1%,而特異性分別為95.2%、82.5%、78.8%。以Kappa value (κ)來評定這三種試驗與MAT的一致性,其中以LAT有較佳的一致性κ = 0.62,而Lateral-Flow與Dri-Dot的一致性較差,分別為0.44及0.37,故LAT應可用於臨床快速篩檢鉤端螺旋體感染症。由於多種鉤端螺旋體的外膜蛋白質已被選殖且大量表現,故提供具免疫原性的重組蛋白質抗原作為鉤端螺旋體感染症的抗體診斷有其必要性,本研究的另一部分是利用大腸桿菌株表現LipL32重組蛋白抗原,應用酵素結合免疫吸附法 (enzyme-linked immunosorbent assay,簡稱ELISA)來偵測鉤端螺旋體感染症之IgG及IgM抗體。採用上述送檢之疑似鉤端螺旋體感染病患的血清及40個正常人血清樣本進行試驗,利用Two-graph Receiver-Operator Curve (TG-ROC)訂定Cutoff,結果在IgG-ELISA試驗之敏感性為97.1%,特異性為71.9%。在IgM-ELISA試驗之敏感性為80.3 %,特異性為63.0%。此次實驗再度證明LipL32蛋白質在鉤端螺旋體感染症的血清學診斷或閉O一個有用的抗原。

Leptospirosis is a widespread zoonotic disease that affects all mammals indifferent parts of the world. Clinical recognition of leptospirosis is challenging, and the definitive serologic diagnosis assay, the microscopic agglutination test (MAT) is the reference test for diagnosis, but it is time-consuming and requires the maintenances of several leptospiral strains to be used as antigens, live organisms creating a risk of laboratory-acquired infections. We prepared a rapid latex agglutination test (LAT) reagent for the detection of Leptospira- specific antibodies in human serum samples. The efficacy of the LAT was compared with two commercial rapid serologic tests, the LeptoTek Lateral Flow and the LeptoTek Dri-Dot with MAT as gold standard. A total of 419 human serum samples were analyzed by MAT, LAT, Lateral Flow, and Dri-Dot. By Using MAT as gold standard, the sensitivities for detection of leptospiral antibodies were 44.4% by Lateral Flow, 52.8% by Dri-Dot and 86.1% by LAT; specificities were95.2% by Lateral Flow, 82.5% by Dri-Dot, and 78.8% by LAT, respectively. On our study, LAT showed the best agreement with MAT (κ=0.62) and the agreement of the Lateral Flow and Dri-Dot with MAT was low (κ=0.44 and 0.37, respectively). These characteristics make LAT ideal for rapid screening for leptospirosis suspicious patients. Because more leptospiral outer membrane protein genes has been cloned and expressed. It is essential to provide a antigen for antibodies detection. The recombinant LipL32 protein expressed in Escherichia coli BL21 is used as antigen for enzyme-linked immunosorbent assay (ELISA) to screen leptospirosis suspected patients and 40 normal control sera. Sensitivity and specificity are 97.1% and 71.9% in IgG-ELISA, 80.3 % and 63.0% in IgM-ELISA. These findings indicate that LipL32 may be an useful antigen for the serodiagnosis of leptospirosis.

中文摘要 II
英文摘要 IV
目錄 V
表次 VIII
圖次 IX
第一章 緒言 1
第二章 文獻探討 3
第一節 鉤端螺旋體感染症歷史 4
第二節 鉤端螺旋體感染症之分類 6
第三節 鉤端螺旋體感染症之病原與抗原種類 7
第四節 鉤端螺旋體的致病與免疫機制 12
第五節 鉤端螺旋體感染症的傳播途徑與自然宿主 14
第六節 鉤端螺旋體感染症之臨床症狀 16
第七節 鉤端螺旋體感染症之實驗室診斷 18
第八節 鉤端螺旋體感染症之治療、預防與控制 27
第三章 利用乳膠凝集試驗快速篩檢鉤端螺旋體感染症 29
第一節 摘要 30
第二節 前言 31
第三節 材料與方法 33
3.1 血清樣本來源及相關測試 33
3.2 抗原與乳膠粒子的製備 37
3.3 乳膠凝集試驗理想試驗條件測試 39
3.4 乳膠凝集試驗檢測 40
3.5 統計分析 40
第四節 結果 41
第五節 討論 47
第四章 應用鉤端螺旋體LipL32重組蛋白質抗原於酵素結合免
疫吸附法偵測鈎端螺旋體感染症之IgG及IgM抗體 50
第一節 摘要 51
第二節 前言 52
第三節 材料與方法 54
3.1 鉤端螺旋體LipL32重組蛋白質抗原之表現與純化 54
3.2 LipL32重組蛋白質之電泳分析 56
3.3 LipL32重組蛋白質之定量分析 58
3.4 血清樣本來源 59
3.5 酵素結合免疫吸附法理想試驗條件測試 60
3.6 酵素結合免疫吸附法抗體檢測 61
3.7 統計分析 63
第四節 結果 64
第五節 討論 73
參考文獻 75
附錄 84
試藥之製備方法 84
血清樣本原始資料 87


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