Lu Mingmin, Panebra Alfredo, Kim Woo H, Lillehoj Hyun S
Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, 20705, USA.
Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, MD, 20705, USA.
Dev Comp Immunol. 2021 Jun;119:104023. doi: 10.1016/j.dci.2021.104023. Epub 2021 Jan 23.
CCL5 (formerly RANTES) belongs to the CC (or β) chemokine family and is associated with a plethora of inflammatory disorders and pathologic states. CCL5 is mainly produced and secreted by T cells, macrophages, epithelial cells, and fibroblasts and acts as a chemoattractant to recruit effector cells to the inflammation sites. Chicken CCL5 (chCCL5) protein is closely related to avian CCL5 orthologs but distinct from mammalian orthologs, and its modulatory roles in the immune response are largely unknown. The present work was undertaken to characterize the immunological properties of chCCL5 using the new sets of anti-chCCL5 mouse monoclonal antibodies (mAbs). Eight different mAbs (6E11, 6H1, 8H11, 11G1, 11G11, 12H1, 13D1, and 13G3) were characterized for their specificity and binding ability toward chCCL5. Two (13G3 and 6E11) of them were selected to detect native chCCL5 in chCCL5-specific antigen-capture ELISA. Using 13G3 and 6E11 as capture and detection antibodies, respectively, the ELISA system detected serum chCCL5 secretions in Clostridium perfringens- and Eimeria-infected chickens. The intracellular expressions of chCCL5 in primary cells or cell lines derived from chickens were validated in immunocytochemistry and flow cytometry assays using both 13G3 and 6E11 mAbs. Furthermore, 6E11, but not 13G3, neutralized chCCL5-induced chemotaxis in vitro using chicken PBMCs. These molecular characteristics of chCCL5 demonstrate the potential application of anti-chCCL5 mAbs and CCL5-specific antigen-capture detection ELISA for detecting native chCCL5 in biological samples. The availability of these new immunological tools will be valuable for fundamental and applied studies in avian species.
CCL5（原名RANTES）属于CC（或β）趋化因子家族，与多种炎症性疾病和病理状态相关。CCL5主要由T细胞、巨噬细胞、上皮细胞和成纤维细胞产生和分泌，作为一种趋化因子，将效应细胞招募到炎症部位。鸡CCL5（chCCL5）蛋白与禽类CCL5直系同源物密切相关，但与哺乳动物直系同源物不同，其在免疫反应中的调节作用在很大程度上尚不清楚。本研究旨在利用新的抗chCCL5小鼠单克隆抗体（mAb）来表征chCCL5的免疫学特性。对八种不同的单克隆抗体（6E11、6H1、8H11、11G1、11G11、12H1、13D1和13G3）针对chCCL5的特异性和结合能力进行了表征。其中两种（13G3和6E11）被选用于在chCCL5特异性抗原捕获ELISA中检测天然chCCL5。分别使用13G3和6E11作为捕获抗体和检测抗体，该ELISA系统检测了产气荚膜梭菌和艾美耳球虫感染鸡的血清chCCL5分泌情况。使用13G3和6E11单克隆抗体在免疫细胞化学和流式细胞术分析中验证了chCCL5在鸡原代细胞或细胞系中的细胞内表达。此外，6E11而非13G3在体外使用鸡外周血单核细胞中和了chCCL5诱导的趋化作用。chCCL5的这些分子特性证明了抗chCCL5单克隆抗体和CCL5特异性抗原捕获检测ELISA在检测生物样品中天然chCCL5方面的潜在应用。这些新免疫工具的可用性对于禽类的基础研究和应用研究将具有重要价值。
