臺灣博碩士論文加值系統

新尼古丁類殺蟲劑的使用越見廣泛與頻繁，但現有殘留檢測方法耗時傷財，免疫分析法將能提供一種更快速簡易且低成本的殺蟲劑殘留檢測方式。本論文透過小鼠單株抗體的製備，發展酵素免疫分析方法 (ELISA) 及免疫快篩試紙，小鼠在免疫注射益達胺 (imidacloprid)、可尼丁 (clothianidin)、亞滅培 (acetamiprid) 及賽速安 (thiamethoxam) 等四種新尼古丁類殺蟲劑抗原後，血清中皆帶有得以辨識殺蟲劑的抗體，血清稀釋16,000倍仍可辨識到殺蟲劑抗原，並與控制組有顯著差異。取小鼠脾臟細胞與癌細胞融合製備融合瘤細胞，融合瘤細胞的抗體產生率介於1.04-2.94%，並成功篩選出 18 株能辨識新尼古丁類殺蟲劑的單株抗體細胞株。挑選四株分別辨識益達胺、可尼丁、亞滅培及賽速安的單株細胞生產腹水抗體，再運用 ELISA 進行檢測。抗體 1D10 對益達胺的偵測極限為 72.13 ng/mL；抗體 3E9 對賽速安的偵測極限為 37.89 ng/mL，抗體 3E5 與 4C11 對可尼丁及亞滅培的偵測極限更低，分別為1.543 ng/mL及1.308 ng/mL。於特定濃度範圍內製作檢量線，其迴歸線的決定係數 (R2) 皆大於0.967。以菠菜樣本測試基質對抗體檢測時的干擾，顯示菠菜樣本對益達胺抗體的干擾較低，但對其他三株抗體產生輕微的影響。將靈敏度較高的可尼丁、亞滅培及賽速安抗體與膠體金結合製備免疫快篩試紙，三株抗體與膠體金的最適抗體結合量皆低於 80 mg/L。利用免疫快篩試紙檢測殺蟲劑，檢測時間可縮短至15分鐘，可尼丁的肉眼可辨極限 (visible detection limit) 為 10 ng/mL，亞滅培及賽速安則分別為 100 及 1000 ng/mL。運用試紙進行殺蟲劑檢測，相較於ELISA速度更快卻較不靈敏，若能改善試紙的偵測極限，或是製備更高靈敏度的殺蟲劑抗體，將有助於開發免疫殘留檢測方法。

Neonicotinoid pesticides have been used extensively in field. But now, most pesticides detection methods are time-consuming and expensive. Immunoassays may probably provide a fast, easy and economic detection way for pesticides. In this study, monoclonal antibodies were produced to develop enzyme-linked immunosorbent assay (ELISA) and strip tests. Six mice’s serums contained antibodies recognized pesticides after immunization with pesticide antigen. The titers of the serums were all higher than 16,000 times, and showed significant difference with control. Hybridomas were produced by fusing between spleen cells and tumor cells. Eighteen monoclonal hybridomas that secreted neonicotinoid pesticide antibodies were selected, and the antibody production ratio were from 1.04% to 2.94%. Four of them were found to recognize imidacloprid, clothianidin, acetamiprid and thiamethoxam respectively. The limit of detection (LOD) of the imidacloprid antibody 1D10 was 72.13 ng/mL; and was 37.89 ng/mL of the thiamethoxam antibody 3E9. The Detection limits were even lower for the clothianidin antibody 3E5 and the acetamiprid antibody 4Cll (1.543 ng/mL and 1.308 ng/mL, respectively). R2 values for pesticide concentration-inhibitory calibration curves within certain concentration ranges were all higher than 0.967. To test matrix interference to antibodies, spinach matrix spiked samples were used. We found that spinach matrices had a slight effect on all antibodies except for the imidacloprid antibody 1D10. The 3E5, 4C11, and 3E9 antibodies were conjugated with colloidal golds to develop strip tests, with the optimized conjugating ratios being all below 80 mg/L. This way reduced the detection time to less than 15 minutes. The visible detection limits of clothianidin, acetamiprid and thiamethoxam strips were 10 ng/mL, 100 and 1000 ng/mL. Using strip for pesticide detection is fast but insensitive than ELISA technique. To develop immunoassays for pesticide detection, we must have to improve the sensitivities of strips and monoclonal antibodies.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
ABSTRACT v
目錄 vii
表次 x
圖次 xi
壹、前言 1
貳、往昔研究 3
2.1 新尼古丁類殺蟲劑 3
2.2 殘留農藥檢驗方法 5
2.3 單株及多株抗體 6
2.4 酵素免疫分析方法 8
2.5 快速檢驗試紙 9
參、材料與方法 11
3.1 藥品 11
3.1.1 配製殺蟲劑 11
3.1.2 試劑 11
3.2 農藥抗原合成 12
3.3 蛋白質定量 12
3.4 農藥抗體製備 13
3.4.1 實驗動物飼養 13
3.4.2 免疫小鼠 13
3.4.3 血清測試效價 13
3.4.4 血清測試競爭反應 14
3.4.5 小鼠脾臟細胞與癌細胞融合 14
3.4.6 單株抗體細胞株篩選 14
3.4.7 測試單株抗體專一性 14
3.4.8 細胞冷凍與解凍 15
3.5 蛋白質電泳 15
3.6 單株抗體特性測試 15
3.6.1 交互反應 15
3.6.2 偵測極限與線性範圍 15
3.6.3 蔬菜基質添加標準品分析 16
3.7 免疫快篩試紙製作 16
3.7.1 膠體金與單株抗體之最適結合量 16
3.7.2 抗體-膠體金複合物 (colloidal gold-Ab, Cg-Ab) 最適用量 16
3.7.3 運用免疫快篩試紙檢測殺蟲劑 17
肆、結果 18
4.1 農藥抗原的合成 18
4.2農藥抗原免疫小鼠 18
4.3 小鼠脾臟細胞與癌細胞融合 19
4.4 單株抗體細胞株篩選 19
4.5 單株抗體細胞株專一性測試 19
4.6 抗體純化 20
4.7 單株抗體對不同種類農藥之交互反應測試 20
4.8 四株單株抗體製作檢量線 20
4.9 蔬菜基質添加標準品分析 21
4.10 膠體金與單株抗體之最適結合量及pH值 21
4.11 抗體-膠體金複合物最適用量 22
4.12 免疫快篩試紙偵測極限 23
伍、討論 46
5.1 抗體製備 46
5.2 交互反應測試 46
5.3 運用ELISA技術檢測殺蟲劑 47
5.4 菠菜基質測試添加回收率 48
5.5運用免疫快篩試紙檢測殺蟲劑 48
陸、結論 50
柒、參考文獻 52
捌、附錄 60

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