Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

趋化因子（CC基序）配体（CCL）4是一个保守的半胱氨酸残基的顺序定位所定义的CC趋化因子亚家族成员。在细胞表面结合G蛋白偶联受体后，CCL4介导了一系列生物过程，包括趋化性，肿瘤发生，体内平衡和胸腺生成。尽管20年前就已经阐明了哺乳动物CCL4的生理作用，但是关于鸡CCL4（chCCL4）的生物学活性的信息却很少。在本研究中，我们开发并鉴定了针对chCCL4的小鼠单克隆抗体（mAb），以更好地表征chCCL4的免疫学特性。在最初筛选的400多个克隆中，有两个检测chCCL4、1A12和15D9的单克隆抗体，使用蛋白质印迹法和chCCL4特异性抗原捕获酶联免疫吸附测定法进行鉴定和表征，并通过chCCL4诱导的外周血单核细胞趋化测定法验证其中和活性。此外，使用1A12和15D9 mAb通过免疫细胞化学和流式细胞术证实了chCCL4在各种鸡细胞中的细胞内表达。这些结果共同表明1A12和15D9 mAb可以特异性检测鸡CCL4，它们将成为禽免疫学基础研究和应用研究的有价值的免疫试剂。免疫细胞化学和流式细胞仪检测chCCL4在各种鸡细胞中的细胞内表达，方法是使用1A12和15D9 mAb。这些结果共同表明1A12和15D9 mAb可以特异性检测鸡CCL4，它们将成为禽免疫学基础研究和应用研究的有价值的免疫试剂。免疫细胞化学和流式细胞仪检测chCCL4在各种鸡细胞中的细胞内表达，方法是使用1A12和15D9 mAb。这些结果共同表明1A12和15D9 mAb可以特异性检测鸡CCL4，它们将成为禽免疫学基础研究和应用研究的有价值的免疫试剂。