入侵紅火蟻毒液單元抗體之製備與應用

Abstract

入侵紅火蟻 (Solenopsis invicta Buren) 是台灣當前最嚴重的入侵害蟲。當蟻巢受到騷動時，火蟻會以腹部末端的螫針將毒液注入人體，毒液中的毒蛋白會引發人體的免疫反應，嚴重時甚至會休克、死亡，因此相當受到重視。而台灣目前紀錄有270種螞蟻，其中與火蟻外形相似者不在少數，依形態鑑別不易。同屬火家蟻屬 (Solenopsis) 的熱帶火蟻 (S. geminata)，以及單家蟻屬 (Monomorium)、大頭家蟻屬 (Pheidole) 及擬大頭家蟻屬 (Pheidologeton) 等三個同屬於家蟻亞科 (Myrmicinae) 的螞蟻形態便極為相近，易造成民眾難以分辨而引起恐慌。有基於此，本論文即利用融合瘤技術製備入侵紅火蟻毒蛋白的單元抗體，藉其高專一性及靈敏度的偵測特性，應用於火蟻的快速鑑定上。自野外採集入侵紅火蟻與其他台灣常見螞蟻種類，分別取其毒囊進行SDS-PAGE的蛋白質電泳分析，結果發現入侵紅火蟻毒蛋白條帶明顯與他種螞蟻不同，顯示入侵紅火蟻的毒蛋白確實具其特異性。自毒液中分離出4個蛋白條帶，其中一條帶位於13 kDa左右，另3條帶則位於24 kDa附近，是為毒蛋白所在位置。接著以毒蛋白為抗原注射小白鼠，製備單元抗體。結果發現在抗血清階段即可將火蟻屬的入侵紅火蟻及熱帶火蟻 與他種螞蟻區別，但無法區別入侵紅火蟻與熱帶火蟻。而後經融合瘤技術篩選到30個對入侵紅火蟻有特異性的細胞株，其中編號Rf-E7的細胞株特異性最佳。將該細胞株大量培養並打入小白鼠腹腔產生抗腹水，經純化後得到該抗體IgG，以供入侵紅火蟻之快速偵測。不論純化的抗體或抗腹水稀釋至105倍皆仍可以酵素連結免疫吸著分析法 (ELISA) 明顯區分入侵紅火蟻及熱帶火蟻。另外在偵測的應用上也發現蟲體的乾燥失水會影響抗體偵測效果，殺蟲劑與保存的溶液 (酒精、肥皂水) 也會產生些微干擾，但一般只要在蟲體死亡或處理3日之內便做偵測，仍可達良好的鑑識效果。將蟲體以水或緩衝液保存，或直接冷藏於4℃或-20℃都可以維持毒蛋白的穩定性達1週以上，抗體偵測仍可順利進行。由溫度試驗也得知溫度對於入侵紅火蟻毒液含量的影響有限，所以不管在35、25或15℃環境下，都不會明顯影響抗體的偵測值。由本論文結果得知，利用此單元抗體配合間接酵素連結免疫法 (Indirect-ELISA)，確實可快速鑑別入侵紅火蟻，未來將應用此單元抗體於膠金標誌抗體試劑 (Colloidal gold-labeled antibody strip) 之製備，提供民間或政府機關快速檢測之用。

The red imported fire ant (RIFA, Solenopsis invicta Buren) is one of the most destructive imported insect pests in Taiwan. They are not only invaders that bring serious ecological problems, but also aggressive medical pests that cause various human damages and symptoms such as the formation of characteristic sterile pustules, urticaria, edema, dermal necrosis, and even anaphylactic shock and death in rare cases. Approximately 270 species of ants were discovered in Taiwan, and some are very similar to S. invicta in their morphological characters. For example, the tropical fire ant (Solenopsis geminata) and several members of subfamily Myrmicinae such as Monomorium, Pheidole and Pheidologeton are not easy to be directly and quickly distinguished each other according to their morphology. For rapid and accurate identification of S. invicta to avoid people’s panic, monoclonal antibodies against the specific venom in the venomous gland of S. invicta were developed through the hybridoma technology. S. invicta and several ant species were collected for the venom analysis by SDS-PAGE (SDS-polyacrylamide gel electrophoresis), and the results demonstrated that the protein pattern in SDS-PAGE of S. invicta is different from the others. It indicated that the venom proteins are different among S. invicta as well as the other ant species. Four proteins were isolated and identified from venom. One of them is located in 13 kDa, the others are near around 24 kDa. The venom was used to immunize the mouse to develop monoclonal antibody. In the beginning, the polyclonal antiserum against venom of S. invicta showed positive to S. invicta and S. geminata, and negative to the other ant species. And in later totally 30 hybridoma cell lines have been selected to produce specific antibodies against S. invicta. A cell line named Rf-E7 showing the best specificity and sensitivity was selected for the mass-production of monoclonal antibodies. Furthermore, the monoclonal antibody Rf-E7 was proven to be able to accurately differentiate S. invicta from S. geminata. Both ascetic fluid and purified IgG of Rf-E7 showed the good specificity and sensitivity in the ELISA tests with 105 diluted antibodies. Dried, dead bodies and the samples treated with insecticides as well as preservative solutions obtained the slightly lower ELISA value than fresh samples. However, their ELISA values were still useful for identification of S. invicta. In the temperature experiment, the ELISA tests did not show apparently different results among different S. invicta populations residing in 15, 25, or 35℃. It revealed that alteration of temperature would not affect the quantity of RIFA venom. Based on the results in this thesis, the developed monoclonal antibody Rf-E7 has been proven to be an excellent probe for rapid identification of S. invicta. This antibody will be further dedicated to the preparation of the colloidal gold-labeled antibody strip that provides a more rapid and simple detection of S. invicta without any analytic instrument. People can identify whether their ant samples are S. invicta or not by themselves at home, which can reduce public panic for S. invicta.