兔抗凋亡诱导因子多克隆抗体的制备、纯化与鉴定

背景：凋亡诱导因子是存在于线粒体膜间隙中的黄素蛋白，它不仅具有氧化还原和电子传递功能，还具有促细胞凋亡功能，从而在维持细胞正常生理活动中具有重要作用。目的：制备抗凋亡诱导因子多克隆抗体并进行特性鉴定。 设计、时间及地点：单一样本观察，于2006-09/2008-08在新乡医学院免疫学研究中心完成。材料：新西兰大白兔雌性2只，体质量1.9~2.1 kg；Jurkat E6-1细胞系购自美国ATCC公司。食管癌组织取自新乡医学院普通外科手术患者。抗原肽-KLH由上海华大天源生物科技公司合成。CNBr活化的Sepharose 4B购自美国Pharmacia公司。方法：利用美国过敏和感染疾病研究所(NIAID)主办的抗原表位分析网站(http://beta.immuneepitope.org/home.php)的在线软件对凋亡诱导因子蛋白的氨基酸序列进行分析设计凋亡诱导因子抗原肽，制备多克隆抗体并纯化。主要观察指标：采用间接ELISA、Western blot、免疫组织化学3种方法对获得的多克隆抗体进行特性鉴定。结果：间接ELISA法检测显示，血清抗凋亡诱导因子抗体的效价达到1∶128 000。Western blot结果显示，存在Mr67 000条带。免疫组织化学检测结果显示，在食管癌细胞的胞质中有棕黄色的阳性颗粒存在，说明此抗体也可以用于免疫组织化学染色。结论：实验获得了高效价的特异性的抗凋亡诱导因子抗体。

Preparation, purification and identification of polyclonal antibody of rabbit anti apoptosis-inducing factor

BACKGROUND: Apoptosis-inducing factor (AIF) is a phylogenetically conserved mitochondrial intermembrane flavoprotein which has the ability to induce apoptosis via a caspase-independent pathway. AIF plays an important role in maintaining normal physiologycal activities.OBJECTIVE: To prepare and identify the polyclonal antibody of rabbit anti AIF.DESIGN, TIME AND SETTING: A single sample observation was performed at the Xinxiang Medical University from September 2006 to August 2008.MATERIALS: Two female New Zealand rabbits, weighting 1.9-3.1 kg. Jurkat E6-1 cell line was purchased from American ATCC Coperation. Human esophageal carcinoma was obtained from patients of Xinxiang Medical University. Antigen peptide-KLH was synthesized by Shanghai HD Biosicences Co., Ltd. CNBr Activated Sepharose 4B was provided by American Pharmacia Company.METHODS: The online software of Immune Epitope Database and Analysis Reaource (http://beta.immuneepitope.org/home.php), which hosted by NIAID, was used to analyze amino acid sequence of AIF and design antigenic peptide of AIF. Then the polyclonal antibody was prepared and purified. MAIN OUTCOME MEASURES: The indirect ELISA, Western blot and immunohistochemistry method was used to characterize polyclonal antibody.RESULTS: The detection of indirect ELISA method showed that titer of serum AIF antibody reaches 1:128 000. The result of Western blot demonstrated that there was a Mr 67 000 band. Brownish-yellow positive particle exits in esophageal carcinoma cytoplasm, which showed that the antibody could be used to immunohistochemistry.CONCLUSION: The high titer specific AIF antibody is obtained in the study.