Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies
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Bacterial meningitis is a severe disease that is fatal to one-third of patients. The major cause of meningitis in neonates is Escherichia coli (E. coli) K1. This bacterium synthesizes an outer membrane protein A (OmpA) that is responsible for the adhesion to (and invasion of) endothelial cells. Thus, the OmpA protein represents a potential target for developing diagnostic and therapeutic agents for meningitis. In this study, we expressed recombinant OmpA proteins with various molecular weights in E. coli. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to check the molecular size of OmpA’s full length (FL) and truncated proteins. OmpA-FL protein was purified for immunizing chickens to produce immunoglobulin yolk (IgY) antibodies. We applied phage display technology to construct antibody libraries (OmpA-FL scFv-S 1.1 × 107 and OmpA-FL scFv-L 5.01 × 106) to select specific anti-OmpA-FL scFv antibodies; these were characterized by their binding ability to recombinant or endogenous OmpA using ELISA, immunofluorescent staining, and confirmed with immunoblotting. We found 12 monoclonal antibodies that react to OmpA fragments; seven scFvs recognize fragments spanning amino acid (aa) residues 1–346, aa 1–287, aa 1–167, and aa 60–192, while five scFvs recognize fragments spanning aa 1–346 and aa 1–287 only. Two fragments (aa 246-346 and aa 287-346) were not recognized with any of the 12 scFvs. Together, the data suggest three antigenic epitopes (60 aa–160 aa, 161 aa–167 aa, 193 aa–245 aa) recognized by monoclonal antibodies. These scFv antibodies show strong reactivity against OmpA proteins. We believe that antibodies show promising diagnostic agents for E. coli K1 meningitis.
KeywordsOuter membrane protein A (OmpA) Escherichia coli strain K1 Phage display technology Bacterial meningitis Antigenic epitopes Single-chain variable fragment (scFv) antibody
CHL, SJL, YCL, HHW, YJH, TEL, and LTL offered technical advice. YYY is an advisor and corresponding. PFM conducted the experiments and wrote the manuscript. The final manuscript was read and accepted by all authors.
The authors would like to thank the support by the Ministry of Science and Technology through Professor Yi-Yuan Yang, grant number (MOST105-2622-8-038-001-TB1) and by the Ministry of Health and Welfare surcharge of tobacco products through Professor Yi-Yuan Yang, grant number (MOHW106-TDU-B-212-144001, MOHW107-TDU-B-212-114014, and MOHW108-TDU-B-212-124014).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval statement for the animal experiments
The 24-week-old white leghorn female chickens were kept under approved conditions in the animal center room at Taipei Medical University. Experimental procedures on female chickens (Gallus domesticus) follows standards set by the Animal Care and Use Committee of Taipei Medical University (ethical approval number: LAC-2015-0302, valid on 1 February 2016 to 31 January 2019).
The authors declare no experiment has been performed with human participants.
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