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 x Oreochromis mossambicus)

本實驗的目的在於製備抗吳郭魚周邊血液來源巨噬細胞的單株抗體。將培養後的貼附性吳郭魚周邊血液來源巨噬細胞免疫BALB/c小鼠，以融合瘤技術將小鼠骨髓瘤細胞（NS-1cells）與經過免疫之小鼠脾細胞融合成融合瘤細胞，再經三次極限稀釋，並以間接酵素連結免疫分析法（indirect ELISA）篩選出具有分泌抗體的融合瘤細胞，共得到八株單株抗體，分別命名為mAbM1-mAbM8。之後經抗體力價測試後發現mAbM1、mAbM2、mAbM6及mAbM7呈現高抗體力價。這四株單株抗體（mAbM1、mAbM2、mAbM6及mAbM7）之後則進行抗體特性研究。先以免疫螢光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、頭腎來源及脾臟來源之巨噬細胞的結合能力，發現mAbM1、mAbM2、mAbM6及mAbM7對週邊血液單核球來源巨噬細胞和頭腎來源巨噬細胞有很高的陽性細胞率（最高可達80%），與陰性對照組比較皆有顯著性差異（p＜0.05），尤其以mAbM6和mAbM7具有極顯著性差異（p＜0.01），但是對脾臟來源巨噬細胞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、雞及羊的陽性細胞率與陰性對照組有極顯著差異（p＜0.01）（可達40-60%）。此外，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膜上，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將新鮮的吳郭魚周邊血液白血球進行染色，再配合流式細胞儀細胞分選功能，成功地從周邊血液白血球區別出巨噬細胞。本實驗成功開發吳郭魚巨噬細胞之單株抗體，期望此抗體能作為分類不同魚類血球族群及未來魚類免疫學研究上之工具。

The aim of this study is to develop and apply the monoclonal antibodies (mAbs) against the hybrid tilapia macrophage (Oreochromis niloticus×Oreochromis mossambicus). There are a limited number of mAbs against fish leukocytes have been described until today. Our study results showed that eight mAbs, mAbM1-mAbM8, produced from BALB/c mice immunized with hybrid tilapia peripheral blood monocyte derived macrophages (PBM) with adjuvant. Those mAbs have been screened from a lot of hybridoma clones by indirect ELISA. Reveal of that the antibody titers of mAbM1, mAbM2, mAbM6, and mAbM7 are higher than the others. These four mAbs reacted with PBM, HKM (head kidney macrophages), and SM (spleen mcrophages) were used to be analyzed their characterizations by the indirect immunofluorescence assay test and flow cytometry. The results suggest these four mAbs have very high binding abilities 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 binding abilities 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.