入侵紅火蟻毒液蛋白及其抗體於老鼠體內的偵測及其應用

Abstract

入侵紅火蟻 (Solenopsis invicta, red imported fire ant, RIFA) 是一種危害相當嚴重的入侵種生物，由於其具有很強的競爭力，且在入侵地缺乏天敵的壓制，因此對野生動物及生物多樣性造成很大的衝擊；此外，也對農牧業、健康安全及公共設施有所威脅，而為了解決這些問題以及進行防治工作，更造成經濟上龐大的負擔。入侵紅火蟻經常會攻擊和牠們具有相似棲息環境的生物，其中居住在地表或地下的野鼠等齧齒動物，便很容易和入侵紅火蟻遭遇而受到叮咬。RIFA的毒液中主要含有4種毒性蛋白分子，可能引起嚴重的過敏性反應。而這些毒蛋白在進入老鼠體內後，可能會滯留一段時間，並誘使老鼠免疫系統製造RIFA毒蛋白的抗體。若對遭受RIFA叮咬的老鼠進行血清的分析，偵測其中是否含有毒蛋白或是其抗體，便可推測老鼠活動範圍內是否有RIFA出沒，而能發展出另一種監測RIFA擴散情形的方法。在進行老鼠血清檢測之前，我們先建立了RIFA毒蛋白之單元抗體Rf-E7 的應用方法，並對RIFA毒液於不同生態因子中加以分析比較。結果顯示以雙抗體三明治酵素連結免疫吸著分析法 (double antibodies sandwich enzyme-linked immunosorbent assay, DAS-ELISA) 對整隻螞蟻粗萃取樣本做分析鑑別時，最佳的抗體稀釋倍率是105X；而對純毒液進行分析時，稀釋倍率在105到106X之間的效果均很相似。對RIFA不同生物因子，包括社會型 (單蟻后型或多蟻后型)、體型及工作任務的工蟻毒液蛋白質，利用DAS-ELISA和SDS-PAGE進行分析比較，結果顯示在不同社會型間並無顯著差異，而不同體型工蟻則略有不同，表示毒蛋白組成比例可能因體型不同而有所差異。在老鼠血清分析方面，將毒液注射入老鼠體內後，以DAS-ELISA對毒液蛋白質的滯留進行檢測，發現毒蛋白量在鼠體內的濃度會先遞增，大約在4 h後達到高峰，至12 h後開始逐漸降低，而在約24 h後，分析所得之ELISA值便降至注射毒液前的程度；而老鼠體內由毒蛋白誘發產生之抗體，則以間接式ELISA (Indirect ELISA) 進行偵測，結果顯示注射過RIFA毒液的小鼠血液，檢測結果呈陽性，而未注射毒液的小鼠，檢測結果則呈陰性，以此方法可檢測出8個月前注射RIFA毒液的小鼠血液中之抗體。若能夠將此檢測方法應用於野外鼠類，便可作為RIFA擴散及偵測的生物指標，對RIFA的監測及防治工作有所助益。

Red imported fire ant (RIFA), Solenopsis invicta Buren, is one of important invasive insect species. It seriously impacts the wildlife, agriculture, public health, and economy. They usually attack creatures which have similar habitats with them. Therefore, RIFA usually brings impacts to the wildlife and biodiversity. Rodents living on or under the ground probably encounter the RIFA and become victims. The venom of RIFA contains four major proteins that might be the allergens causing serious allergy reaction in hosts. These proteins may remain in rodent body for a retention period then induce the production of antibodies against these proteins. This thesis was dedicated to test the serum of the injured rodents to investigate the retention of RIFA venom proteins and production of antibodies, which may be applied to monitor the spreading and distribution of RIFA. For the detection of mouse blood, we applied the immunological assays with the monoclonal antibody Rf-E7 against RIFA to study several ecological factors for RIFA. The results demonstrated that the most efficient assay for a single RIFA (whole body extracts) test could be obtained when 105 fold dilution of Rf-E7 was used from the original stock. For pure venom assay, 105-106 fold dilution of Rf-E7 could achieve best results. The comparative immunological assays were conducted among different social forms, worker sizes, and worker tasks by DAS-ELISA and SDS-PAGE. The results showed no significant differences between two social forms. There were slight differences among different worker sizes and might be resulted from the different composition ratio of venom proteins. In the test of mouse serum after the mouse was injected with RIFA venom, the mouse blood was collected and the venom proteins were detected by DAS-ELISA. The ELISA value was elevated immediately after the venom injection and reached the highest level 4 h later. After 12 h, the ELISA value decreased gradually through time. Finally, the ELISA value decreased to a low level similar to the value without injection about 24 h later. These results indicated that the venom proteins could not remain in mouse body for long time. The induced antibodies were detected by indirect ELISA after venom proteins declined. The results showed positive reaction in venom-injected mouse and negative reaction in non-injected mouse. The antibodies from mouse was still detected 8 months after injected with RIFA venom. The detection of the RIFA venom and its antibodies in wild rats can be applied as a bio-indicator for the monitoring of RIFA spreading.