抗哇巴因鸡蛋黄抗体IgY的制备

Preparation of anti-ouabain IgY in hen egg yolk

目的制备出高特异性的抗哇巴因多克隆抗体,为进一步提高内源性哇巴因的检测的敏感性和特异性提供实验基础。方法采用不同剂量哇巴因-牛血清白蛋白耦联物(0.5 mg/kg;1.0 mg/kg)免疫母鸡,收集鸡蛋;采用聚乙二醇法分离、纯化鸡蛋黄中抗哇巴因抗体;用酶联免疫吸附法检测IgY(yolk immunoglobulin)的抗体效价。SDS-PAGE电泳分析IgY纯度。结果免疫后1周,鸡蛋黄中可检测出IgY;免疫后6周,鸡蛋黄中IgY的效价迅速升高,最高效价达到1∶10 240,持续至少4周。结果还显示,大剂量免疫母鸡获得高效价抗体持续时间较长。结论采用大剂量哇巴因-牛血清白蛋白耦联物免疫母鸡,可以获得较高效价的抗哇巴因IgY。该方法简单,高效价抗体持续时间长,是制备抗体较为有效的方法。

Objective To prepare highly selective anti-ouabain polyclonal antibody to further improve the sensitivity and specificity level of the detection of endogenous ouabain. Methods Different doses of ouabain-BSA conjugate （0. 5 mg/kg; 1.0 mg/kg） was used to immunize hens. Eggs were collected one week after the first immunization. The egg yolks were separated and purified by PEG-6 000 method. The purity was analyzed by 12% SDS-PAGE, and the bioactivity was tested by ELISA. Results IgY could be tested one week after first immunization, but the titer was very low. The titer was increased fast after the second immunization. The highest titer was 1 ： 10 240. And this level lasted for at least 4 weeks. At the same time, the results showed that the more antigen was used, the longer time the high titer IgY lasted. Conclusion High titer IgY can be prepared by multiple immunizations of hens. The method is simple and effective, and the highest titer IgY lasts longer. It is probably an effective way to prepare anti-ouabain IgY.