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KMID : 0545120180280122113
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Journal of Microbiology and Biotechnology
2018 Volume.28 No. 12 p.2113 ~ p.2120
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Generation of a human monoclonal antibody to cross-reactive material 197 (CRM197) and development of a sandwich ELISA for CRM197 conjugate vaccines
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Kim Da-In
Yoon Hye-Seon
Kim Sang-Kyu
Wi Ji-Min
Chae Hee-Su
Jo Gyung-Hee
Yoon Jun-Yeol
Kim Hee-Youn
Lee Chan-Kyu
Kim Se-Ho
Hong Hyo-Jeong
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Abstract
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Cross-reactive material 197 (CRM197) is a non-toxic mutant of diphtheria toxin containing a single amino acid substitution of glycine 52 with glutamic acid. CRM197 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 CRM197 and CRM197 conjugate vaccines, we generated a human anti-CRM197 monoclonal antibody (mAb) 3F9 using a phage-displayed human synthetic Fab library and produced mouse anti-CRM197 polyclonal antibody. The affinity (KD) of 3F9 for CRM197 was 3.55 nM, based on Bio-Layer interferometry, and it bound specifically to the B fragment of CRM197. 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 CRM197. In addition, the 3F9 antibody bound to the CRM197-polysaccharide conjugates tested in a dose-dependent manner. This ELISA system will be useful for the quantification and characterization of CRM197 and CRM197 conjugate vaccines. To our knowledge, this study is the first to generate a human monoclonal antibody against CRM197 and to develop a sandwich ELISA for CRM197 conjugate vaccines.
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KEYWORD
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Cross-reactive material, vaccines, human monoclonal antibody, phage display, sandwich ELISA
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