Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha

Tumor necrosis factor alpha (TNF-alpha) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-alpha (chTNF-alpha) gene has not been identified until very recently due to its high GC content (similar to 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-alpha was cloned into prokaryotic vector after codon optimization and recombinant chTNF-a protein was expressed. Subsequently, using recombinant chTNF-alpha as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-. monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-alpha 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-alpha. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-alpha was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-alpha 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-alpha are specific and could be used for the study of the biological functions of chTNF-alpha and the detection of chTNF-alpha.