Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha
Yi Yang,
Yining Meng,
Lina Chen,
Maoli Dong,
Huining Zhang,
Ji Wu,
Xiaoli Hao,
Shuangjiang He,
Yunfei Tian,
Zaicheng Gong,
Shaobin Shang
Affiliations
Yi Yang
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China
Yining Meng
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Lina Chen
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Maoli Dong
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Huining Zhang
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Ji Wu
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Xiaoli Hao
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Shuangjiang He
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Yunfei Tian
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Zaicheng Gong
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
Shaobin Shang
Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China; International Corporation Laboratory of Agriculture and Agricultural Products Safety, Yangzhou University, Yangzhou 225009, China; Corresponding author.
Tumor necrosis factor alpha (TNF-α) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-α (chTNF-α) gene has not been identified until very recently due to its high GC content (∼70%) and long GC fragments. The biological functions of this newly-identified cytokine and its detection methods remain to be further investigated. In this study, the extracellular domain of chTNF-α was cloned into prokaryotic vector after codon optimization and recombinant chTNF-α protein was expressed. Subsequently, using recombinant chTNF-ɑ as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-ɑ monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-ɑ expressed in E.coli and transfected COS-7 cells. Further mapping the antigenic region showed that all the mAbs recognized a region of amino acid residues 195–285 of chTNF-ɑ. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-ɑ was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-ɑ expressed by mitogen-activated chicken splenocytes in a dose-dependent manner, with a detection limit of 1 ng/mL. Collectively, our results indicated that the mAbs and pAb against chTNF-α are specific and could be used for the study of the biological functions of chTNF-ɑ and the detection of chTNF-ɑ.
