Fish & Shellfish Immunology ( IF 4.7 ) Pub Date : 2020-12-18 , DOI: 10.1016/j.fsi.2020.12.007 Janet Velázquez , Alianet Rodríguez , Hasel Aragón , Arlette Haidar , Marcos González , Rodolfo Valdés , Hilda Elsa Garay , David Diago Abreu , Yassel Ramos , Ania Cabrales , Antonio Morales , Osmany González , Fidel Herrera , Mario Pablo Estrada , Yamila Carpio
Nile tilapia (Oreochromis niloticus) is a freshwater fish, which is extensively cultivated worldwide and constitutes one of the model species for the study of fish immunology. Monoclonal antibodies are very advantageous molecular tools for studying teleost immune system. Specifically, monoclonal antibodies that react with immunoglobulins are used successfully in the study of the humoral immune response of several fish species. In the present study, we produced and characterized a monoclonal antibody against tilapia IgM heavy chain using a peptide-based strategy. The peptide sequence was selected from the surface-exposed region between CH3–CH4 domains. The specificity of the polyclonal serum and the hybridoma culture supernatant obtained by immunization with the peptide conjugated to keyhole limpet hemocyanin were evaluated by western blotting, both showing reactivity against tilapia serum IgM. The purified mAb was able to recognize secreted IgM by western blotting and ELISA and membrane IgM by flow cytometry. We also demonstrated that the antibody doesn't cross-react with a recombinant IgT fragment. This tool allowed us to study for the first time the stimulation of mucosal immunity after Pituitary Adenylate Cyclase Activating Polypeptide administration. Overall, the results demonstrated the utility of this mAb to characterize humoral immune response in O. niloticus.
中文翻译:
抗尼罗罗非鱼(IeroM)的单克隆抗体IgM重链:检测和定量IgM和IgM +细胞的有价值的工具
尼罗罗非鱼(Oreochromis niloticus)是一种淡水鱼,已在世界范围内广泛种植,是鱼类免疫学研究的典范物种之一。单克隆抗体是研究硬骨免疫系统的非常有利的分子工具。具体而言,与免疫球蛋白反应的单克隆抗体已成功用于几种鱼类体液免疫反应的研究中。在本研究中,我们使用基于肽的策略生产并鉴定了针对罗非鱼IgM重链的单克隆抗体。肽序列选自CH3-CH4结构域之间的表面暴露区域。通过蛋白质印迹法评估了多克隆血清和杂交瘤细胞培养上清液的特异性,其中所述杂交瘤细胞培养上清液是通过与钥孔血蓝蛋白缀合的肽免疫而获得的,两者均显示出对罗非鱼血清IgM的反应性。纯化的mAb能够通过蛋白质印迹和ELISA识别分泌的IgM,并通过流式细胞术识别膜IgM。我们还证明了该抗体不会与重组IgT片段发生交叉反应。该工具使我们首次研究了垂体腺苷酸环化酶激活多肽给药后对粘膜免疫的刺激作用。总体而言,结果表明该单克隆抗体可用于表征小鼠体内的体液免疫应答 该工具使我们首次研究了垂体腺苷酸环化酶激活多肽给药后对粘膜免疫的刺激作用。总体而言,结果表明该单克隆抗体可用于表征小鼠体内的体液免疫反应 该工具使我们首次研究了垂体腺苷酸环化酶激活多肽给药后对粘膜免疫的刺激作用。总体而言,结果表明该单克隆抗体可用于表征小鼠体内的体液免疫应答尼罗罗非鱼。