Deciphering antigen-responding antibody repertoires by using next-generation sequencing and confirming them through antibody-gene synthesis
| Autor: | Hiroyuki Toh, Manabu Ato, Osamu Numata, Shigeyuki Itamura, Lin Sun, Naoko Kono, Hanbing Xue, Takeyuki Shimizu, Kazuo Ohnishi |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Biophysics Biology Protein Engineering Biochemistry DNA sequencing Antigen-Antibody Reactions 03 medical and health sciences Mice 0302 clinical medicine Antibody Repertoire Antigen Sequence Analysis Protein Drug Discovery Animals Molecular Biology Genetics Cell Biology Antigen recognition Acquired immune system High-Throughput Screening Assays Mice Inbred C57BL 030104 developmental biology biology.protein Female Antigen response Antibody Gene synthesis Epitope Mapping 030215 immunology |
| Zdroj: | Biochemical and biophysical research communications. 487(2):300-306 |
| ISSN: | 0006-291X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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