Kono Naoko, Sun Lin, Toh Hiroyuki, Shimizu Takeyuki, Xue Hanbing, Numata Osamu, Ato Manabu, Ohnishi Kazuo, Itamura Shigeyuki
Center for Influenza Virus Research, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.
Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan.
Biochem Biophys Res Commun. 2017 May 27;487(2):300-306. doi: 10.1016/j.bbrc.2017.04.054. Epub 2017 Apr 12.
Vast diversity and high specificity of antigen recognition by antibodies are hallmarks of the acquired immune system. Although the molecular mechanisms that yield the extremely large antibody repertoires are precisely understood, comprehensive description of the global antibody repertoire generated in individual bodies has been hindered by the lack of powerful measures. To obtain holistic understanding of the antibody-repertoire space, we used next-generation sequencing (NGS) to analyze the deep profiles of naive and antigen-responding repertoires of the IgM, IgG1, and IgG2c classes formed in individual mice. The overall landscapes of naive IgM repertoires were almost the same for each mouse, whereas those of IgG1 and IgG2c differed considerably among naive individuals. Next, we immunized mice with a model antigen, nitrophenol (NP)-hapten linked to chicken γ-globulin (CGG) carrier, and compared the antigen-responding repertoires in individual mice. To extract the complete antigen response, we developed an intelligible method for detecting common components of antigen-responding repertoires. The major responding antibodies were IGHV1-72/IGHD1-1/IGHJ2 for NP-hapten and IGHV9-3/IGHD3-1/IGHJ2 for CGG-carrier protein. The antigen-binding specificities of the identified antibodies were confirmed through ELISA after antibody-gene synthesis and expression of the corresponding NGS reads. Thus, we deciphered antigen-responding antibody repertoires by inclusively analyzing the antibody-repertoire space generated in individual bodies by using NGS, which avoided inadvertent omission of key antibody repertoires.
抗体对抗原识别的广泛多样性和高度特异性是获得性免疫系统的标志。尽管精确理解了产生极其庞大抗体库的分子机制，但由于缺乏有力的方法，对个体体内产生的整体抗体库的全面描述受到了阻碍。为了全面了解抗体库空间，我们使用下一代测序（NGS）来分析个体小鼠中形成的IgM、IgG1和IgG2c类别的天然和抗原反应性库的深度概况。每只小鼠的天然IgM库的总体格局几乎相同，而天然个体之间的IgG1和IgG2c格局则有很大差异。接下来，我们用与鸡γ球蛋白（CGG）载体连接的模型抗原硝基苯酚（NP）-半抗原免疫小鼠，并比较个体小鼠中的抗原反应性库。为了提取完整的抗原反应，我们开发了一种可理解的方法来检测抗原反应性库的共同成分。主要的反应抗体对于NP-半抗原是IGHV1-72/IGHD1-1/IGHJ2，对于CGG载体蛋白是IGHV9-3/IGHD3-1/IGHJ2。在抗体基因合成和相应NGS读数表达后，通过ELISA确认了所鉴定抗体的抗原结合特异性。因此，我们通过使用NGS全面分析个体体内产生的抗体库空间来破译抗原反应性抗体库，从而避免了关键抗体库的意外遗漏。
