生物素标记的布鲁菌多克隆抗体的制备  

Preparation of biotin-labeled polyclonal antibody against Brucella abortus

作　　者：李丽[1] 鞠文[1] 殷德辉 孟日增[1,2] 李娟[1] 宋秀玲[1] 徐坤[1] 

机构地区：[1]吉林大学公共卫生学院卫生检验教研室,吉林长春130021 [2]吉林出入境检验检疫局检验检疫技术中心,吉林长春130062

出　　处：《中国生物制品学杂志》2015年第2期147-150,154,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(81072337;81473018);国家自然科学基金青年基金项目(81401721);吉林大学"大学生创新创业训练计划"项目(2014A72317)

摘　　要：目的制备生物素标记的兔抗牛种布鲁菌544A多克隆抗体,并进行鉴定。方法复苏并扩大培养牛种布鲁菌544A,灭活后制备灭活疫苗,经背部皮下多点注射免疫新西兰大白兔,5次免疫后,经心脏采血,分离血清,采用硫酸铵盐析法对血清中的多克隆抗体进行粗提,再通过蛋白亲和柱层析法对抗体进一步纯化,利用SDS-PAGE分析抗体的纯度,间接ELISA法检测抗体的效价和特异性,Bradford法检测抗体的蛋白含量,并利用生物素对抗体进行标记。结果制备的牛种布鲁菌544A的兔多克隆抗体纯度较高,效价为1:256 000,特异性较好,蛋白含量为2.83 mg/ml,每摩尔蛋白中标记的生物素摩尔数为3.09。结论成功制备了生物素标记的牛种布鲁菌多克隆抗体,为下一步研制牛种布鲁菌免疫检测试剂盒奠定了物质基础。Objective To prepare and identify the polyclonal antibody against Brucella abortus 544 A. Methods B. abortus544 A strain was resuscitated, cultured and enriched, then inactivated. New Zealand white rabbits were immunized s. c.with the prepared inactivated vaccine on back in several sites for 5 times, of which the heart blood was collected. Sera were separated, from which polyclonal antibody was crudely extracted by salting out with ammonium sulphate and further purified by protein affinity chromatography, then determined for purity by SDS-PAGE, for titer and specificity by indirect ELISA, and for protein content by Bradford method, and labeled with biotin. Results The prepared rabbit polyclonal antibody against B. abortus 544 A showed a high purity and a good specificity, of which the titer was 1 ∶ 256 000, the protein content was 2. 83 mg / ml, and the labeling efficiency was 3. 09 of biotin to antibody. Conclusion Biotinla beled polyclonal antibody against B. abortus 544 A was prepared successfully, which laid a foundation of development of immunological detection kit for B. abortus.

关 键 词：布鲁菌 多克隆抗体 免疫检测 

分 类 号：R378.5[医药卫生—病原生物学] R392-33[医药卫生—基础医学]