Centre de Recherche du CHU de Québec-Université Laval, Quebec City, QC G1V 4G2, Canada.
Département de Microbiologie-Infectiologie et d'Immunologie, Faculté de Médecine, Université Laval, Québec City, QC G1V 0A6, Canada.
Int J Mol Sci. 2020 Sep 25;21(19):7074. doi: 10.3390/ijms21197074.
We have generated polyclonal and monoclonal antibodies by genetic immunization over the last two decades. In this paper, we present our most successful methodology acquired over these years and present the animals in which we obtained the highest rates of success. The technique presented is convenient, easy, affordable, and generates antibodies against mammalian proteins in their native form. This protocol requires neither expensive equipment, such as a gene gun, nor sophisticated techniques such as the conjugation of gold microspheres, electroporation, or surgery to inject in lymph nodes. The protocol presented uses simply the purified plasmid expressing the protein of interest under a strong promoter, which is injected at intramuscular and intradermal sites. This technique was tested in five species. Guinea pigs were the animals of choice for the production of polyclonal antibodies. Monoclonal antibodies could be generated in mice by giving, as a last injection, a suspension of transfected cells. The antibodies detected their antigens in their native forms. They were highly specific with very low non-specific background levels, as assessed by immune-blots, immunocytochemistry, immunohistochemistry and flow cytometry. We present herein a detailed and simple procedure to successfully raise specific antibodies against native proteins.
在过去的二十年中，我们通过遗传免疫产生了多克隆和单克隆抗体。在本文中，我们介绍了多年来获得的最成功的方法，并介绍了获得最高成功率的动物。所提出的技术方便、简单、经济实惠，并能以天然形式针对哺乳动物蛋白产生抗体。该方案既不需要昂贵的设备（如基因枪），也不需要复杂的技术（如金微球缀合、电穿孔或淋巴结注射手术）。所提出的方案仅使用在强启动子下表达感兴趣蛋白的纯化质粒，将其注射到肌肉和皮内部位。该技术在五个物种中进行了测试。豚鼠是生产多克隆抗体的首选动物。通过最后一次注射转染细胞的悬浮液，可以在小鼠中产生单克隆抗体。所检测到的抗体以天然形式检测其抗原。通过免疫印迹、免疫细胞化学、免疫组织化学和流式细胞术评估，它们具有非常高的特异性和非常低的非特异性背景水平。本文详细介绍了一种成功针对天然蛋白产生特异性抗体的简单程序。
