Generation of a human monoclonal antibody to cross-reactive material 197 (CRM197) and development of a sandwich ELISA for CRM197 conjugate vaccines

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.

Generation and Characterization of Monoclonal Antibodies to the Ogawa Lipopolysaccharide of Vibrio cholerae O1 from Phage-Displayed Human Synthetic Fab Library

“…The phage-Fabs were analyzed by ELISAs as described previously [ 35 ]. For the indirect ELISA, the phage-Fabs were incubated with Ogawa, Inaba, O139, or BSA (2 μg/ml) at 37°C for 1 h. After washing, the bound phage-Fabs were incubated with anti-M13 antibody-HRP (1:5000 v/v, GE Healthcare).…”

“…To evaluate whether the mAbs can be used for quantitation of Ogawa LPS in V. cholerae vaccine, each mAb (200 ng/ml) was incubated with serially diluted Euvichol as a coating antigen and an indirect ELISA was conducted. The 96-well microplates (Nunc) were coated with each antigen in coating buffer (Na 2 CO 3 = 15 mM, NaHCO 3 = 35 mM, pH 9.6) at 4°C overnight, while all incubations were performed at 37°C for 1 h, as described previously [ 35 ]. Finally, the TMB reagent was added and incubated for 6 min.…”

“…ScFv fragments often have a high tendency to form multimers as well as aggregates and have been found to lose affinity during conversion to Ig (immunoglobulin)G, whereas Fab fragments have been found to possess comparably higher structural stability by the presence of the CH1 and light-chain constant domain (CL), and thus their binding activities were retained after conversion to IgG [ 32 ]. We previously generated a human synthetic Fab library based on the VH3-23 and VK1-39 framework pairing and have successfully generated mAbs against recombinant proteins and peptide [ 33 - 35 ]…”

Epitope Mapping of the Diphtheria Toxin and Development of an ELISA-Specific Diagnostic Assay

“…DTxs produced by the different corynebacteria are structurally very similar [29][30][31][32] and the identification and characterization of corynebacteria are conducted by phenotypic methods [11,33,34], polymerase chain reaction (PCR) [35][36][37], and mass spectrometry (MALDI-TOFF) [38]. The immunological tests developed for the disease diagnostic were the microcell culture [39], fluorescence immunoassay [40], and duple antigen [41] or sandwich-ELISA [42].…”

Cross-reaction between mouse and rat immunoglobulin G: does it matter in sandwich ELISA?

“…In addition, it is also used in biomarker profiling [ 10 , 11 ] and biomarker-based diagnostic assays for cancer diagnosis [ 12 ], diagnosis of autoimmune diseases [ 13 ], and Alzheimer’s [ 14 ]. Moreover, it has applications in vaccine development [ 15 ] and conjugate vaccine assessment [ 16 ]. Also, it is of use in discrimination of allergic cultivates for human [ 17 ] and even as a basic bio-approach in engineering as in early-diagnostic biosensors [ 18 ].…”