Abstract
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. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). 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). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. 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. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. 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. 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. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. 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.Key points• OmpC is the target of a recently identified ECO157-specific mAb 2G12.• Eight peptides were identified from the OmpC by using LC–MS/MS.• The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.
Highlights
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 2016)
Typhimurium were mainly attributed to the sequence, length, and position of loop 2. These results revealed that the “LGVING” part of loop 2 on OmpC was the dominant part that contributed to the specificity of monoclonal antibody (mAb) 2G12 of Escherichia coli O157:H7 (ECO157)
The target protein and the epitope of mAb 2G12 were first purified and isolated for better understanding its specificity at the protein and sequence levels
Summary
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 2016). One of the reasons for these cross-reactions is that the target antigen and the epitope of many previously identified antibodies remained largely unknown. Applied Microbiology and Biotechnology (2021) 105:6819–6833 use of monoclonal antibodies and the development as well as improvement of the specificity of antibody-antigen-based immunoassays. There is an urgent need for additional studies to identify the target(s) of antibodies on the surface of bacteria
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