Tan Yue, Xu Zhengzhong, Zhu Zhaocheng, Xia Aihong, Sun Lin, Chen Xiang, Jiao Xinan
Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture and Rural Affairs, Yangzhou University, Yangzhou 225009, China.
Jiangsu Key Laboratory of Zoonosis/Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2019 Mar;35(3):271-276.
Objective To prepare monoclonal antibodies (mAbs) against bovine tumor necrosis factor-alpha (BoTNF-α). Methods Recombinant BoTNF-α with His tag (rHis-BoTNF-α) was expressed in a prokaryotic system as immunogen, and recombinant BoTNF-α with GST tag (rGST-BoTNF-α) was expressed as detection antigen. The mAbs were developed by hybridoma cell technology. The reactivity of mAbs with rHis-BoTNF-α and rGST-BoTNF-α was detected by Western blot analysis. Indirect ELISA was used to detect the titer and specificity of mAbs, and the reactivity to commercialized recombinant BoTNF-α (rBoTNF-α) was also evaluated. The reactivity of mAbs against natural antigens was identified by flow cytometry. Results Seven hybridomas stably secreting anti-rBoTNF-α mAbs were successfully obtained. The results showed that all seven mAbs had good reactivity and specificity. Flow cytometry analysis also showed that mAb 4G4 had good reactivity with natural BoTNF-α antigens. Conclusion The anti-rBoTNF-α mAbs are successfully achieved.
目的 制备抗牛肿瘤坏死因子-α（BoTNF-α）单克隆抗体（mAbs）。方法 以带有His标签的重组牛肿瘤坏死因子-α（rHis-BoTNF-α）作为免疫原在原核系统中表达，以带有GST标签的重组牛肿瘤坏死因子-α（rGST-BoTNF-α）作为检测抗原。采用杂交瘤细胞技术制备单克隆抗体。通过蛋白质印迹分析检测单克隆抗体与rHis-BoTNF-α和rGST-BoTNF-α的反应性。采用间接酶联免疫吸附测定法检测单克隆抗体的效价和特异性，并评估其与商业化重组牛肿瘤坏死因子-α（rBoTNF-α）的反应性。通过流式细胞术鉴定单克隆抗体对天然抗原的反应性。结果 成功获得7株稳定分泌抗rBoTNF-α单克隆抗体的杂交瘤细胞株。结果显示，所有7株单克隆抗体均具有良好的反应性和特异性。流式细胞术分析还表明，单克隆抗体4G4与天然BoTNF-α抗原具有良好的反应性。结论 成功制备了抗rBoTNF-α单克隆抗体。
