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小鼠腹水IgM型单抗的纯化 |
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刘继明 邱玉华 张 毅 张学光 孙晓燕
中国图书分类号 R392.11
摘 要 目的：为了建立一种从腹水中获得高纯度IgM抗体的纯化方法。方法：分别以硫酸铵沉淀或优球蛋白沉淀结合凝胶过滤法,从腹水中纯化IgM型单克隆抗体。结果：用优球蛋白沉淀结合凝胶过滤，纯化的IgM抗体纯度＞90%，明显优于硫酸铵沉淀结合凝胶过滤法纯化的IgM抗体纯度(＜60%)。结论：优球蛋白沉淀结合凝胶过滤是一种获得高纯度IgM抗体的方法。
关键词 IgM 单克隆抗体 凝胶过滤 优球蛋白
Purification of IgM monoclonal antibodies from mouse ascites fluid
LIU Ji-Ming, QIU Yu-Hua, ZHANG Yi et al
(Department of Immunology, Suzhou Medical College, Suzhou 215007)
SUN Xiao-Yan
(Suzhou Health School,Suzhou 215002)
Abstract Objective：To obtain high purity and immunoreactive IgM antibody from ascites. Methods：Ascites fluid was subjected to ammonium sulfate precipitation or euglobulin precipitation. The precipitate was redissolved and subsequently subjected to gel filtration on SuperdexTM-200. The purity of the antibody was assessed by the reducing SDS gel electrophoresis. Immuno-activity of the antibody was assessed by an indirect enzyme-linked immunosorbent assay. Results：The purity of the antibody was estimated to be greater than 90% after euglobulin precipitation combined with gel filtration, but less than 60% after ammonium sulfate precipitation combined with gel filtration. There is no obviously difference between two method s recovery of immuno-activity. Conclusion：Euglobulin precipitation combined with gel filtration is a good method for purification IgM Mabs from ascites.
Key words IgM Monoclonal antibody Gel filtration Euglobulin
分离纯化IgM的方法有多种，包括各种沉淀法和层析法［1-5］。腹水含有大量的杂蛋白，采用一步纯化法，不可能得到高纯度的IgM抗体，而多步骤纯化因耗时长，抗体活性损失大。为了寻找一种从腹水中获得高纯度IgM型抗体的最佳纯化方法。本文通过优球蛋白沉淀结合凝胶过滤法纯化腹水IgM型抗体，并与传统的硫酸铵沉淀结合凝胶过滤法相比较。
1 材料与方法
1.1 主要试剂和设备 鼠抗人IL-8单抗WS-4和HPLC纯rhIL-8 (日本金泽大学松岛教授赠送); 碱性磷酸酶(ALP)标记羊抗小鼠免疫球蛋白多抗(Immunotech inc.，France);蛋白质定量用标准蛋白(中国生物制品鉴定所);电泳低分子量标准蛋白(上海东风生化试剂公司); FPLC分离纯化系统和SuperdexTM-200 prep grade(26/600)预装柱(Pharmacia-LKB,Sweden);酶标仪(model 550, Bio-Rad, USA)。
1.2 抗体制备 用本室研制的rhIL-8免疫Balb/[1] [2] [3] 下一页
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