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標題: 吳郭魚周邊血液單核球衍生之巨噬細胞單株抗體的建立與應用
Development and Application of Monoclonal Antibodies to Peripheral Blood Monocyte Derived Macrophages of Hybrid Tilapia (Oreochromis niloticus Oreochromis Mossambicus)
作者: 謝育錚
Hsieh, Yu-Jung
關鍵字: tilapia;吳郭魚;macrophages;monoclonal antibodies;Oreochromis niloticus;單株抗體;巨噬細胞
出版社: 獸醫學系暨研究所
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本實驗的目的於製備抗吳郭魚周邊血液來源之巨噬細胞單株抗體。將培養後的貼附性巨噬細胞免疫Balb/c 小鼠,以細胞融合技術將小鼠骨髓瘤細胞 (NS-1)與經過免疫之小鼠脾細胞融合成為融合瘤細胞,再經三次極限稀釋並以間接酵素連結免疫分析法 (indirect ELISA)篩選具有分泌抗體的融合瘤細胞。最後結果得到八株單株抗體,分別命名為mAbM1~mAbM8。經過力價測試後發現mAbM1、mAbM2、mAbM6、mAbM7之力價較高,故選擇這四株單株抗體進行抗體特性研究。先以螢光免疫染色法與流式細胞儀分析這四株單株抗體與週邊血液單核球來源 (PBM)、頭腎來源 (HKM)、及脾臟來源 (SM)之巨噬細胞的結合活性,結果發現mAbM1、mAbM2、mAbM6、mAbM7與PBM和HKM有很高的陽性細胞率 (最高可達80%),與對照組比較皆有顯著差異 (P<0.05),尤其是mAbM6和mAbM7的差異更明顯(P<0.01)。但是對SM的陽性細胞率與陰性對照組皆無極顯著的差異(P>0.05)。因此再選取mAbM6和mAbM7這兩株單株抗體檢測與不同物種 (日本鰻、鯉魚、小鼠、紐西蘭大白兔、雞、綿羊、狗及人類)之週邊血液巨噬細胞之結合能力,結果顯示日本鰻、小鼠、雞和羊的陽性細胞比例與陰性對照組有極大的顯著差異 (P<0.01) (可達40~60%),並發現有螢光標定的陽性細胞幾乎均落在細胞型態較大的象限。此外,再以間接螢光免疫染色法配合雷射共軛焦掃描顯微鏡分析,結果發現mAbM6與mAbM7所辨識的抗原皆分布在細胞上,抗體與抗原接合位置呈現帽狀或環狀。同時發現利用mAbM6與mAbM7間接免疫螢光染色法可將新鮮的吳郭魚周邊血液白血球染色,再配合流式細胞儀分選細胞功能,成功地從周邊血液白血球區別出巨噬細胞。

The aim of this study is to develop and apply the monoclonal antibodies (MAbs) to the macrophages from hybrid tilapia (Oreochromis niloticus × Oreochromis Mossambicus) because there are a limited number of MAbs to fish leukocytes have been described until today. Our study results show that we are able to get eight MAbs, mAbM1~mAbM8, produced from Balb/c mice immunized with peripheral blood monocyte derived macrophages (PBM) of hybrid tilapia. Those mAbs have been screened from a lot of hybridoma clones by indirect ELISA and analyzed with macrophages from peripheral blood leucocytes (PBL). The result reveals that the antibody titers of mAbM1, mAbM2, mAbM6 and mAbM7 are higher than the others. Those four MAbs reacted with PBM (peripheral blood macrophages), HKM (head kidney macrophages) and SM (spleen mcrophages) are used to be analyzed their characterizations by the indirect immunofluorescence assay test (IIFAT) and flow cytometry (FCM). The results suggest these four MAbs have very high activities with PBM and HKM, especially mAbM6 and mAbM7. Furthermore, mAbM6 and mAbM7 are chosen for testing the cross-reactivity with peripheral blood monocyte derived macrophages from different species including eel, carp, mice, rabbit, chicken, sheep, dog, and human. The results indicate that both mAbM6 and mAbM7 have significantly higher reactivity with PBM from eel, mice, sheep and chicken. Interestingly , the capping or ring forms of the mAbM6+ and mAbM7+ cells are recognized and found by the confocal microscope. Therefore, we presume that both MAbs binding epitopes are on the cell surface of the macrophages. Finally, we find that our developed mAbM6 and mAbM7 MAbs could be sorted the macrophages from peripheral blood leukocytes successfully. Indeed, our MAbs could serve as a useful tool to identify fish macrophages for further studies.
其他識別: U0005-0707200818475800
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