The immobilization of antibodies on a solid support for separation (e.g., Sepharose agarose, magnetic beads) and the resistance nature of antibodies to enzymatical proteolysis lay the foundation for the two most frequently used approaches: epitope excision and epitope extraction (Opuni et al. In the last three decades, mass spectrometry and enzyme digestion-based methods have been reported to be alternative approaches for rapid and robust epitope mapping (Casina et al. However, they are not cost-effective and are more suitable for linear epitope mapping rather than conformational epitopes (Forsström et al. These methods rely on reactions between synthetic peptides and antibodies and are user-friendly. Peptide microarrays have also been used for studying epitopes. In addition, another limitation associated with these methods is the technical complexity of library construction and expression, which requires considerable expertise in molecular cloning (Najar et al. While these mutagenesis methodologies can be accurate and powerful, local folding defects caused by mutation may affect the results. Another common biological strategy is to construct an amino acid mutant library of the target protein(s) and translate and express them on yeast or phage (Kowalsky et al. 2009), and 2, the X-ray crystallography relies on high degrees of sophistication and training (Opuni et al. However, there is no guarantee of success with these methods, because 1, only a small fraction of Ab–Ag complexes can be crystallized for epitope analysis (Lu et al. 2014) have been reported to be the most accurate approaches as they can determine the interacting atoms between the antigen’s and antibody’s surfaces. Classic structural biology techniques like X-ray crystallography (Malito et al. Several strategies have been reported to study epitopes. Thus, there is an urgent need for additional studies to identify the target(s) of antibodies on the surface of bacteria. Such missing information has limited the use of monoclonal antibodies and the development as well as improvement of the specificity of antibody-antigen-based immunoassays. One of the reasons for these cross-reactions is that the target antigen and the epitope of many previously identified antibodies remained largely unknown. Taking Escherichia coli O157:H7 (ECO157) as an example, cross-reactions of ECO157 monoclonal antibodies with bacterial species such as Escherichia hermannii, Brucella melitensis, and Citrobacter freundii have been reported (Law et al. However, the specificity of such assays has been continuously challenged. Immunoassays based on antibody-antigen reactions have been widely used to recover and detect foodborne pathogens, given their high sensitivity, automation, and simplicity (Valderrama et al. The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.Eight peptides were identified from the OmpC by using LC–MS/MS.OmpC is the target of a recently identified ECO157-specific mAb 2G12.
In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. The topology of OmpC showed that three peptides had extracellular loops. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). mAb 2G12 has shown high specificity for the recovery and detection of ECO157. The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12.
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