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.
Vibrio cholerae
, cause of the life-threatening diarrheal disease cholera, can be divided into different serogroups based on the structure of its lipopolysaccharide (LPS), which consists of lipid-A, corepolysaccharide and O-antigen polysaccharide (O-PS). The O1 serogroup, the predominant cause of cholera, includes two major serotypes, Inaba and Ogawa. These serotypes are differentiated by the presence of a single 2-
O
-methyl group in the upstream terminal perosamine of the Ogawa O-PS, which is absent in the Inaba O-PS. To ensure the consistent quality and efficacy of the current cholera vaccines, accurate measurement and characterization of each of these two serotypes is highly important. In this study, we efficiently screened a phage-displayed human synthetic Fab library by bio-panning against Ogawa LPS and finally selected three unique mAbs (D9, E11, and F7) that specifically react with Ogawa LPS. The mAbs bound to
Vibrio cholerae
vaccine in a dose-dependent fashion. Sequence and structure analyses of antibody paratopes suggest that IgG D9 might have the same fine specificity as that of the murine mAbs, which were shown to bind to the upstream terminal perosamine of Ogawa O-PS, whereas IgGs F7 and E11 showed some different characteristics in the paratopes. To our knowledge, this study is the first to demonstrate the generation of Ogawa-specific mAbs using phage display technology. The mAbs will be useful for identification and quantification of Ogawa LPS in multivalent
V. cholerae
vaccines.
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