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

Pharaoh Fellow Mwale, Chi-Hsin Lee, Sy-Jye Leu, Yu-Ching Lee, Hsueh-Hsia Wu, Liang-Tzung Lin, Tony Eight Lin, Yun-Ju Huang, Yi-Yuan Yang

研究成果: 雜誌貢獻文章

摘要

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.
原文英語
期刊Applied Microbiology and Biotechnology
DOIs
出版狀態打印前電子出版 - 四月 26 2019

指紋

Single-Chain Antibodies
Epitopes
Escherichia coli
Amino Acids
OMPA outer membrane proteins
Proteins
Meningitis
Antibodies
Escherichia coli Meningitis
Monoclonal Antibodies
Bacterial Meningitides
Immunoblotting
Sodium Dodecyl Sulfate
Bacteriophages

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

引用此文

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title = "Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies",
abstract = "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.",
keywords = "Antigenic epitopes, Bacterial meningitis, Escherichia coli strain K1, Outer membrane protein A (OmpA), Phage display technology, Single-chain variable fragment (scFv) antibody",
author = "Mwale, {Pharaoh Fellow} and Chi-Hsin Lee and Sy-Jye Leu and Yu-Ching Lee and Hsueh-Hsia Wu and Liang-Tzung Lin and Lin, {Tony Eight} and Yun-Ju Huang and Yi-Yuan Yang",
year = "2019",
month = "4",
day = "26",
doi = "10.1007/s00253-019-09761-8",
language = "English",
journal = "Applied Microbiology and Biotechnology",
issn = "0175-7598",
publisher = "Springer Verlag",

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TY - JOUR

T1 - Antigenic epitopes on the outer membrane protein A of Escherichia coli identified with single-chain variable fragment (scFv) antibodies

AU - Mwale, Pharaoh Fellow

AU - Lee, Chi-Hsin

AU - Leu, Sy-Jye

AU - Lee, Yu-Ching

AU - Wu, Hsueh-Hsia

AU - Lin, Liang-Tzung

AU - Lin, Tony Eight

AU - Huang, Yun-Ju

AU - Yang, Yi-Yuan

PY - 2019/4/26

Y1 - 2019/4/26

N2 - 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.

AB - 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.

KW - Antigenic epitopes

KW - Bacterial meningitis

KW - Escherichia coli strain K1

KW - Outer membrane protein A (OmpA)

KW - Phage display technology

KW - Single-chain variable fragment (scFv) antibody

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U2 - 10.1007/s00253-019-09761-8

DO - 10.1007/s00253-019-09761-8

M3 - Article

C2 - 31028439

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

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