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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 characterize the biotinylated rabbit anti-Brucella abortus 544A polyclonal antibody. Methods The Brucella abortus 544A was recovered and expanded. Inactivated vaccine was prepared after inactivation. New Zealand white rabbits were injected subcutaneously in the back. After five immunizations, blood was collected from the heart and the serum was separated. The polyclonal antibody in serum was crudely extracted, and the antibody was further purified by protein affinity column chromatography. The purity of the antibody was analyzed by SDS-PAGE. The titer and specificity of the antibody were detected by indirect ELISA. Protein content, and the use of biotin antibody labeling. Results The prepared rabbit polyclonal antibody against Brucella abortus 544A had a high titer of 1: 256 000 with a good specificity and a protein content of 2.83 mg / ml. The number of moles of labeled biotin per mole of protein was 3.09. Conclusion The biotin-labeled polyclonal antibody against Brucella abortus was successfully prepared, which laid the material foundation for the further development of Brucella immunogen detection kit.