Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies

Autor:	Yu Ching Lee, Tony Eight Lin, Pharaoh Fellow Mwale, Chi Hsin Lee, Yun-Ju Huang, Liang Tzung Lin, Yi Yuan Yang, Hsueh-Hsia Wu, Sy Jye Leu
Rok vydání:	2019
Předmět:	Phage display medicine.drug_class Immunoglobulins Enzyme-Linked Immunosorbent Assay medicine.disease_cause Monoclonal antibody Applied Microbiology and Biotechnology Epitope law.invention Epitopes 03 medical and health sciences Antigen law Escherichia coli medicine Animals Single-chain variable fragment Meningitis Escherichia coli Infections 030304 developmental biology 0303 health sciences biology 030306 microbiology Chemistry Antibodies Monoclonal General Medicine respiratory system bacterial infections and mycoses Molecular biology Recombinant Proteins biology.protein Recombinant DNA bacteria Female Immunization Antibody Cell Surface Display Techniques Chickens Bacterial Outer Membrane Proteins Single-Chain Antibodies Biotechnology
Zdroj:	Applied Microbiology and Biotechnology. 103:5285-5299
ISSN:	1432-0614 0175-7598
DOI:	10.1007/s00253-019-09761-8
Popis:	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.
Databáze:	OpenAIRE
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