氯霉素高特异抗体的制备与纯化

Preparation and Purfication of High Specific IgG Antibody for the Affinity Chromatograpy of Chloramphenicol

目的自制免疫亲和层析柱,采用亲和层析法获取高纯度的抗氯霉素抗体并对其进行活性分析。方法采用硫酸铵分级沉淀法对抗体进行初步提取。然后选用3,3′-二氨丙基亚胺(DADPA)作为亲和凝胶与配基氯霉素之间的"间隔臂",制备分离纯化抗氯霉素特异性IgG的亲和层析柱,对IgG粗品进行亲和层析纯化,并用ELISA法检测其活性。结果经饱和硫酸铵分级沉淀,可除去大部分的杂蛋白,较好实现对目标蛋白的初步分离;采用自制的免疫亲和层析柱,可获得高纯度的兔抗氯霉素抗体,检测其活性比纯化前提高了4倍,交叉反应结果显示抗体对CAP有高的特异性,对其他类似药物无反应活性。结论应用本试验所建立的方法可得到高纯度的抗氯霉素抗体,为氯霉素的免疫检测奠定了基础。

Objective To prepare a self-made immunoalilnity chromatography column for the high purified anti-Chloramphenicol polyclonal antibody and investigate its biological activity. Method Purification of antibodies with ammonium sulphate fractional precipitations was conducted initially. Bis （3-aminoprepg） amine was used as a ＂spacer＂ between gel and Chloramphenicol （CAP） ligand of affinity chromatography column to purify and differentiate the specific IgG for anti-CAP antibody from other crude IgG. The activity of antibody purified by affinity chromatography was determined by ELISA. Results The target proteins were precipitated by saturated ammonium sulfate, and most other proteins can be removed. Highly purified rabbit anti-chloramphenicol antibody can be obtained by the self-prepared immunoaffinity chromatography column. The biological activity of the target protein was 4 times higher than that before purification. Cross reaction showed that the IgG was highly specific to CAP, and litter response to other selected antimicrebial agents. Conclusion The established method can be used to obtain purified specific IgG from Chloramphenicol antibody, which may lay a foundation for the immunological examination of Chloramphenicol.