B.abortus 544A和B.melitensis 16M多克隆抗体的制备及鉴定

目的制备高效价、高纯度的布鲁菌多克隆抗体,作为检测布鲁菌的诊断试剂。方法采用弗氏佐剂乳化抗原家兔背部皮下多点免疫,制备抗体,纯化后用SDS-PAGE进行鉴定,ELISA法检测抗体水平,紫外分光光度仪检测蛋白质含量,AlphaScreenRabbitIgGDetectionKit检测IgG含量,并计算纯度。结果SDS-PAGE结果显示多抗片段大小与预期相符,B.melitensis16M和B.abortus544A抗体效价分别为1∶25600和1∶51200,纯化后抗体蛋白含量分别为11·32和14·03mg/ml,IgG含量分别为10·45和13·12mg/ml,且纯度分别达到92·3%和93·5%。结论已获得高效价、高纯度的多克隆抗体IgG,为建立布鲁菌免疫检测方法奠定了基础。

Preparation and Characterization of Polyclonal Antibody against B. abortus 544Aand B. melitensis 16M

Objective To prepare polyclonal antibody against Brucella,with high titer and purity,as the diagnostic kit for brucelliasis.Methods Prepare B.melitensis 16M and B.abortus 544A antigens containing Freund adjuvant by routine method.Immunize rabbits with the prepared antigens by subcutaneous injection in various sites on back.Collect the sera of immunize rabbits for preparation of polyclonal antibody by extraction with caprylic acid-ammonium sulfate and purification with HiTrap Protein G HP affinity chromatography.Identify the purified antibody by SDS-PAGE,then determine its titer,protein content and IgG content by ELISA,ultraviolet spectroscopy and AlphaScreen Rabbit IgG Detection Kit respectively,and calculate its purity.Results SDS-PAGE showed that the relative molecular weight of prepared antibody was identical to that expected.The titer,protein content,IgG content and purity of antibody against B.melitensis 16M were 1:25 600,11.32 mg/ml,10.45 mg/ml and 92.3%,and those of antibody against B.abortus 544A were 1:51 200,14.03 mg/ml,13.12 mg/ml and 93.5% respectively.Conclusion The polyclonal antibodies against Brucella,with high titer and purity,were successfully prepared.It laid a foundation of development of method for detection of Brucella.