Farias Thais H V, Silva Ketherson R, Mariguela Viviane C, Montassier Hélio J, Pilarski Fabiana
Laboratório de Patologia de Organismos Aquáticos, Centro de Aquicultura, Universidade Estadual Paulista, Via de Acesso Prof. Paulo Donato Castellane, 14884-900 Jaboticabal, SP, Brazil.
Centro Universitário Barão de Mauá, Rua Ramos de Azevedo, 423, 14090-180 Ribeirão Preto, SP, Brazil.
An Acad Bras Cienc. 2018 Oct-Dec;90(4):3327-3335. doi: 10.1590/0001-3765201820170438. Epub 2018 Jun 25.
The pacu is one of the most important species for Brazilian fish farming and is considered emerging in the global aquaculture. Despite its importance, no effective tool for evaluation of the adaptive immune response of this species has been developed. Therefore, this study aimed the development and standardization of indirect ELISA for the measurement of pacu antigen-specific antibodies using polyclonal rabbit anti-pacu IgM used as detector antibody. For this purpose was isolated and purificated of pacu IgM using mannose-binding protein affinity chromatography and produced specific polyclonal antibodies against heavy and light chains pacu IgM, that showed a molecular weight of 72 kDa and 26 kDa, respectively. Polyclonal antibodies obtained demonstrated specificity with heavy and light Ig chains of pacu serum in western blotting. These polyclonal antibodies allowed the development of an indirect ELISA assay of high sensitivity and specificity for the detection and quantification of pacu IgM antibodies immunized with bovine IgG. In conclusion, this approach has great potential to improve the monitoring of vaccine-induced immune responses and help develop immunodiagnostic and epidemiological studies of infectious diseases in pacu systems.
淡水白鲳是巴西鱼类养殖中最重要的物种之一，在全球水产养殖中被视为新兴物种。尽管其具有重要性，但尚未开发出评估该物种适应性免疫反应的有效工具。因此，本研究旨在开发并标准化间接ELISA法，以使用兔抗淡水白鲳IgM多克隆抗体作为检测抗体来测量淡水白鲳抗原特异性抗体。为此，使用甘露糖结合蛋白亲和层析法分离并纯化了淡水白鲳IgM，并制备了针对淡水白鲳IgM重链和轻链的特异性多克隆抗体，其分子量分别为72 kDa和26 kDa。所获得的多克隆抗体在蛋白质印迹法中显示出与淡水白鲳血清的重链和轻链Ig具有特异性。这些多克隆抗体使得能够开发出一种高灵敏度和特异性的间接ELISA检测方法，用于检测和定量用牛IgG免疫的淡水白鲳IgM抗体。总之，这种方法具有极大潜力来改善对疫苗诱导免疫反应的监测，并有助于开展淡水白鲳系统中传染病的免疫诊断和流行病学研究。
