在2009年世界衛生組織已把蛇傷歸類於被忽視的熱帶疾病，世界各地每年將近540萬起蛇咬傷事件，並造成8萬至14萬的死亡人數，更有死亡數三倍的人們飽受截肢之苦，面臨永久性殘疾，因此具備有效用的抗蛇毒血清是必要的，在台灣每年約有1000筆蛇傷案例，其中眼鏡蛇因數量較多且與人們生活環境重疊，為常見咬傷案例的蛇種。
台灣眼鏡蛇毒主要毒性成分為心臟毒素、神經毒素及磷酸水解酶A2，先前有研究指出，現今市面上利用粗蛇毒做抗原，進行免疫所生產出的抗蛇毒血清，主要認含量少且較無毒性的高分子量蛋白，而相較之下，毒性較強蛋白部分，反而血清辨認能力較弱。同時研究指出，若單純利用心臟毒素、神經毒素及磷酸水解酶A2做抗原，生產出的血清會大幅改善此問題，並有效提升血清的效價。且若抗原中添加Scrambled CTXA3製造出的血清，會增加認心臟毒素的種類，因此我們團隊利用主成分為心臟毒素、神經毒素及磷酸水解酶A2的免疫原，生產出對粗蛇毒有更高中和效價的增效型血清及近一步在免疫成分加了Scrambled CTXA3的廣效型血清，在對各型的血清利用HPLC抗蛇毒血清分析技術、酵素免疫吸附法及小鼠實驗來進一步分析中和活性，並比較這些技術的優點。最後，透過ELISA和NMR滴定實驗研究神經毒素的抗原決定位。

The World Health Organization already classified snake injuries as a neglected tropical disease in 2009. Near 5.4 million snake bites occur every year in the world, causing 80,000 to 140,000 deaths and more than three times of people suffering from amputation and permanent disability. Thus, to prepare an effective antivenom is essential. There are about 1,000 cases of snake injuries each year in Taiwan. Among them, the injuries caused from Taiwan cobra is relatively common because the higher population and living area overlapping with human living environment. The main toxic components of cobra venom are cardiotoxin, neurotoxin and phospholipase A2. Literatures have reported that the current antivenom on the market mainly recognizes the high-molecular proteins in cobra venom, which is less toxic and with very low ratio. In contrast, the toxic part is less recognized. Here, we perform a study to manufacture new-type antivenom by only immunizing cardiotoxin, neurotoxin and phospholipase A2. The strategy improves the yield of antivenom production and meanwhile, effectively increases the serum titer. We also test another procedure by adding scrambled cardiotoxin A3 into the antigen to enhance the ability of serum to recognize all types of cardiotoxins. The different antivenom are produced by immunizing horses. The antigen containing only cardiotoxin, neurotoxin and phosphohydrolase A2 creates synergistic effect in neutralizing crude venom. The antigen further including scrambled CTXA3 therefore demonstrate broad effect. The serum of each preparation is analyzed by HPLC antivenom analysis method, ELISA and animal test to define the neutralization activity. We compare the advantages of the individual methods. In final, the epitope of neurotoxin is explored by ELISA and NMR titration experiments.

目錄
ABSTRACT I
中文摘要 II
中英對照表 III
英文縮寫 VI
第一章、 前言 1
1-1蛇毒 1
1-2台灣眼鏡蛇毒蛋白組成成分 2
1-2-1神經毒素 2
1-2-2心臟毒素 3
1-2-3磷酸水解酶A2 4
1-2-4高分子量蛋白 4
1-3抗蛇毒血清 5
1-4馬匹血清製成 6
1-5眼鏡蛇抗蛇毒血清遭遇困境 6
1-6研究目的 7
第二章、 材料與方法 12
2-1原料製備 12
2-1-1 神經毒素純化 12
2-1-2 馬血清IgG抗體純化 12
2-1-3 神經、心臟毒素胜肽製備 13
2-1-4 蛋白質表現以及純化 13
2-1-5 NMR樣品製備 14
2-2馬匹免疫(疾病管制署協助進行馬匹免疫及採血) 15
2-2-1 對照組 15
2-2-2 增效型組 15
2-2-3 廣效型組 16
2-3抗蛇毒血清和能力分析 (Antivenom分析技術) 17
2-3-1 抗蛇毒血清測試管柱製備 17
2-3-2 分析原料準備過程及Antivenom技術分析 18
2-4酵素免疫吸附法實驗 (ELISA) 19
2-4-1 ELISA實驗過程 19
2-4-2 各週馬血清與粗蛇毒、神經毒素及心臟毒素結合實驗 20
2-4-3 檢測增效型馬血凍晶與三大地區眼鏡粗蛇毒及雨傘節結合實驗 20
2-4-4 神經毒素抗原決定位(epitope)實驗 20
2-5異核氫氮二維核磁共振光譜滴定實驗 21
2-6 小鼠實驗 (委託歐美生技進行) 21
第三章、 結果 33
3-1 Antivenom分析技術 33
3-1-1對照組 33
3-1-2 增效型組 34
3-1-3 廣效型組 35
3-1-4馬血清與NTX及CTX系列蛋白親和能力變化趨勢 36
3-1-5 中途改變施打的免疫原 38
3-2 酵素結合免疫吸附法實驗 (ELISA) 40
3-2-1 各週增效型組及廣效型組馬血清與眼鏡蛇Crude Venom 結合情形 41
3-2-2 各週增效型組及廣效型組馬血清與眼鏡蛇NTX蛋白結合情形 43
3-2-3 各週增效型組及廣效型組馬血清與眼鏡蛇CTXA3蛋白結合情形 45
3-2-4 檢測增效型馬血凍晶與三大地區眼鏡粗蛇毒及雨傘節結合實驗 46
3-2-5 增效型血清進一步純化認神經、心臟毒素胜肽結果 47
3-3異核氫氮二維核磁共振光譜滴定實驗 48
3-4小鼠實驗整理 49
3-4-1對照組 49
3-4-2增效型組 49
3-4-3廣效型組 49
第四章、 討論 72
參考文獻 83

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