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http://dx.doi.org/10.4014/jmb.1810.10009

Generation of a Human Monoclonal Antibody to Cross-Reactive Material 197 (CRM197) and Development of a Sandwich ELISA for CRM197 Conjugate Vaccines  

Kim, Dain (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Yoon, Hyeseon (Eubiologics Co., Ltd.)
Kim, Sangkyu (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Wi, Jimin (Scripps Korea Antibody Institute)
Chae, Heesu (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Jo, Gyunghee (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Yoon, Jun-Yeol (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Kim, Heeyoun (Eubiologics Co., Ltd.)
Lee, Chankyu (Eubiologics Co., Ltd.)
Kim, Se-Ho (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Hong, Hyo Jeong (Department of Systems Immunology, College of Biomedical Science, Kangwon National University)
Publication Information
Journal of Microbiology and Biotechnology / v.28, no.12, 2018 , pp. 2113-2120 More about this Journal
Abstract
Cross-reactive material 197 ($CRM_{197}$) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. $CRM_{197}$ has been used as a carrier protein for poorly immunogenic polysaccharide antigens to improve immune responses. In this study, to develop a sandwich ELISA that can detect $CRM_{197}$ and $CRM_{197}$ conjugate vaccines, we generated a human anti-$CRM_{197}$ monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-$CRM_{197}$ polyclonal antibody. The affinity ($K_D$) of 3F9 for $CRM_{197}$ was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of $CRM_{197}$. The sandwich ELISA was carried out using 3F9 as a capture antibody and the mouse polyclonal antibody as a detection antibody. The detection limit of the sandwich ELISA was <1 ng/ml $CRM_{197}$. In addition, the 3F9 antibody bound to the $CRM_{197}$-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of $CRM_{197}$ and $CRM_{197}$ conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against $CRM_{197}$ and to develop a sandwich ELISA for $CRM_{197}$ conjugate vaccines.
Keywords
Cross-reactive material 197; vaccines; human monoclonal antibody; phage display; sandwich ELISA;
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