探討不同免疫模式對龜殼花蛇毒抗體效價之影響及建立其體外之中和抗體效價檢測系統

Established in vitro detection system on the antibody neutralization titer and influence of the different immunized strategies on the antibody titer to Trimeresurus mucrosquamatus snake venom

顏旭汶(Hsu-Wen Yen)

指導教授 ： 林春福

中臺科技大學/健康科學院/生命科學研究所/碩士(2010年)

摘要

龜殼花 (Trimeresurus mucrosquamatus,TM) 為台灣的六大毒蛇之一，主要造成出血毒性，為咬傷率最高的蛇種之一。由於以龜殼花蛇毒之粗毒免疫馬匹後抗體效價不易提升，因此抗龜殼花蛇毒血清之製備不易，為探討此問題，本研究將龜殼花蛇毒應用陽離子交換樹脂層析法 (Ion exchange chromatography) 分離，利用溶血試驗及 ICR 小鼠毒力試驗，成功確認分離出龜殼花蛇毒之溶血及非溶血毒素蛋白，進而設計以粗毒、純化毒、先純化毒後粗毒及先粗毒後純化毒等四組不同免疫模式進行 ICR 小鼠之免疫，另馬匹以前三組之免疫模式進行免疫。為探討不同免疫模式所產生對抗龜殼花蛇毒蛋白抗體效價之影響，同時應用捕捉酵素連結免疫吸附法（capture enzyme-linked immunosorbent assay, C-ELISA）及抗龜殼花蛇毒蛋白之體外 (in vitro) 中和抗體試驗進行分析。IgY capture ELISA 分析結果顯示在小鼠及馬匹之試驗均以粗毒免疫及先純化毒後粗毒免疫之組別，其最終抗體效價反應較佳，另經先粗毒後純化毒免疫小鼠之抗體效價反應與粗毒免疫相似。利用兔血液培養基進行商品化抗龜殼花及赤尾鮐蛇毒血清、免疫ICR小鼠及馬匹後之抗龜殼花蛇毒血清中和龜殼花蛇毒溶血之能力試驗，於37℃反應9小時後其溶血圈直徑大小為0.2-0.3公分時，均為≧60田中單位，故本方法在8小時如沒有出現溶血圈，即代表該血清已初步符合產製，因此可作為引起溶血毒性之蛇毒免疫馬匹後之快速初步中和抗體效價篩選平台，以早期偵測不同馬匹之中和抗體效價。

關鍵字

龜殼花 ； 陽離子交換樹脂層析法 ； 捕捉酵素連結免疫吸附分析

並列摘要

Trimeresursus mucrosquamatus (TM) was one of six major vipers in Taiwan and it has hemorrhagic toxicity with highly biting rate among the six vipers. Due to the low neutralization titers of antivenin when immunized the horse with TM crude venom, which caused difficult manufacture of commercial TM antivenin. For investigate this, we firstly were separated the TM crude venom by ion exchange chromatography then used the hemolytic test and LD50 of ICR mice for identify the toxicity portions with hemolytic and non-hemolytic fractions (phospholipase A2, PLA2) . Secondly, we designed four immunized strategies to immunize ICR mice, which including: (A) TM crude venom, (B) toxicity portions of TM crude venom, (C) two vaccinations with toxicity portions of TM crude venom before TM crude venom, and (D) two vaccinations with TM crude venom before toxicity portions of TM crude venom and we also had immunized horse with (A) to (C) strategies. For understand the antibody titers to TM venom among different immunized strategies, we develop capture ELISA and in vitro neutralization antibody titer assay of TM crude venom to assay the antivenin of mice and horses. These results shown that A, C and D strategies have better antibody titers than B and the horses antivenin of in vivo neutralization antibody titers ≧ 60 Tanaka units (TU) neutralized TM crude venom were 0.2-0.3 cm hemolytic zone at 9 hrs, 37°C in rabbit blood agar. We conclude the TM crude venom could cause good antibody titer in mice and horse and the in vitro neutralization antibody titer assay could easy to preliminary screen the neutralization antibody titers of horse antivenin in this study.

並列關鍵字

Ion exchange chromatography ； Trimeresurus mucrosquamatus ； Capture enzyme-linked immunosorbent assay

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