Keggan Alison, Freer Heather, Rollins Alicia, Wagner Bettina
Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA.
Vet Immunol Immunopathol. 2013 Jun 15;153(3-4):187-93. doi: 10.1016/j.vetimm.2013.02.010. Epub 2013 Feb 19.
Horses have 11 immunoglobulin isotypes: IgM, IgD, IgA, IgE, and seven IgG subclasses designated as IgG1-IgG7, each of which are distinguished by separate genes encoding the constant heavy chain regions. Immunoglobulin (Ig) isotypes have different functions during the immune response and pathogen-specific isotypes can be used as indicators for immunity and protection from disease. In addition to existing monoclonal antibodies to various equine Igs, quantification of the individual isotypes requires pure isotype standards. In this report, we describe a fusion between X63-Ag8.653 mouse myeloma cells and horse PBMC to create equine-murine heterohybridomas. Initial screening for Ig production was performed by ELISA. Further testing was performed by a new 5-plex fluorescent bead-based assay able to simultaneously detect equine IgM, IgG1, IgG4/7, IgG5, and IgG6. Production of IgG3 and IgE was tested by separate bead assays. Seven stable heterohybridoma clones producing monoclonal equine IgM, IgG1, IgG3, IgG4/7, IgG5, IgG6 and IgE were created. Purified Ig isotypes were then tested by SDS-PAGE. The pure, monoclonal equine Ig isotypes and the new equine Ig multiplex testing developed here are valuable tools to quantify antibody responses and to accurately determine individual isotypes concentrations in horses.
马有11种免疫球蛋白同种型：IgM、IgD、IgA、IgE，以及7种IgG亚类，分别命名为IgG1 - IgG7，每一种都由编码恒定重链区域的不同基因所区分。免疫球蛋白（Ig）同种型在免疫反应中具有不同功能，病原体特异性同种型可作为免疫和预防疾病的指标。除了现有的针对各种马Ig的单克隆抗体外，对单个同种型进行定量需要纯同种型标准品。在本报告中，我们描述了X63 - Ag8.653小鼠骨髓瘤细胞与马外周血单核细胞之间的融合，以创建马 - 鼠异种杂交瘤。通过酶联免疫吸附测定（ELISA）对Ig产生进行初步筛选。进一步的检测通过一种新的基于5重荧光微珠的检测方法进行，该方法能够同时检测马IgM、IgG1、IgG4/7、IgG5和IgG6。通过单独的微珠检测对IgG3和IgE的产生进行检测。创建了7个稳定的异种杂交瘤克隆，分别产生单克隆马IgM、IgG1、IgG3、IgG4/7、IgG5、IgG6和IgE。然后通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）对纯化的Ig同种型进行检测。这里开发的纯单克隆马Ig同种型和新的马Ig多重检测方法是量化抗体反应以及准确测定马体内单个同种型浓度的有价值工具。
