| 摘要: |
| 以重氮化法制备盐酸克伦特罗-牛血清白蛋白（CBL-BSA）抗原并免疫家兔制备多克隆抗体,将表面固定有BSA的金磁微粒（金磁微粒-BSA）与多克隆抗体反应,抗BSA抗体通过抗原抗体反应被吸附在金磁微粒表面,磁性分离,利用ELISA法对纯化前后抗CBL及抗BSA抗体的效价进行测定,确定最佳纯化条件,得到高纯度的抗CBL抗体.结果表明,通过一步反应,可实现载体蛋白BSA在金磁微粒表面的固定化,每毫克金磁微粒固定BSA的容量可达236μg;在最佳纯化条件（温度4 C,反应时间2 h,金磁微粒表面BSA质量与抗体质量比为7：1）下,金磁微粒-BSA可有效地去除偶联物多克隆抗体中的抗BSA抗体,而纯化前后抗CBL抗体的效价基本保持不变,分离纯化效果较好。该方法可方便、快速地去除多克隆抗体中的无关抗体,对小分子半抗原多克隆抗体的纯化具有重要意义. |
| 关键词: 盐酸克伦特罗 金磁微粒 亲和吸附 抗体纯化 |
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| 基金项目:国家高技术研究发展计划项目(2003AA2Z2080)；国家自然科学基金项目(20273050) |
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| Purification of anti-clenbuterol hydrochloride polyclonal antibody by GoldMag nanoparticles |
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| Abstract: |
| Using GoldMag (Fe3O4/Au composite) nanoparticles as a separating medium,a new method for removing the anti-BSA antibody from anti-clenbuterol polyclonal antibody was investigated.As an immunogenic complex,CBL was conjugated with BSA via diazotized method first,then the polyclonal antibodies were produced in immunized rabbit with CBL-BSA complex.BSA was successfully immobilized onto the GoldMag particles of 50 nm diameter,then the polyclonal antibodies reacted with the BSA on the surface of GoldMag particles and the anti-BSA antibody could be collected on the particle surface,the purified anti-CBL antibody was left in the liquid phase after magnetic separation,the titer of antibodies against CBL and BSA before and after purification was determined by indirect ELISA.The result showed that the immobilization capacity of BSA reached 236 μg on 1 mg GoldMag particles by one-step.When the ratio of BSA-GoldMag complex and the polyclonal antibody was 7∶1 and reacted more than 2 hours at 4℃,the antibodies against BSA can be removed effectively while the titration of antibodies against CBL remained at the same level.This method may become a simple,rapid and effective method for removing the unrelated antibodies in polycolnal antibodies against small molecules. |
| Key words: clenbuterol hydrochloride GoldMag particles affinity adsorption antibody purification |
