Applied Microbiology and Biotechnology

, Volume 103, Issue 13, pp 5285–5299 | Cite as

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 YangEmail author
Applied genetics and molecular biotechnology


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.


Outer membrane protein A (OmpA) Escherichia coli strain K1 Phage display technology Bacterial meningitis Antigenic epitopes Single-chain variable fragment (scFv) antibody 


Authors’ contributions

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).

Informed consent

The authors declare no experiment has been performed with human participants.

Supplementary material

253_2019_9761_MOESM1_ESM.pdf (250 kb)
ESM 1 (PDF 249 kb)


  1. Abe Y, Haruta I, Yanagisawa N, Yagi J (2013) Mouse monoclonal antibody specific for outer membrane protein A of Escherichia coli. Monoclon Antib Immunodiagn Immunother 32(1):32–35. Google Scholar
  2. Ali S, Hussain T (2017) Atypical presentation of Streptococcus salivarius meningitis. Int J Res Med Sci 5(9):4164–4166. Google Scholar
  3. Andris-Widhopf J, Rader C, Steinberger P, Fuller R, Barbas III CF (2000) Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242(1-2):159–181.
  4. Atlas RM (2010) Handbook of microbiological media. CRC Press, Boca Raton. Google Scholar
  5. Azzazy HM, Highsmith WE Jr (2002) Phage display technology: clinical applications and recent innovations. Clin Biochem 35(6):425–445. Google Scholar
  6. Bachtiar EW, Soejoedono RD, Bachtiar BM, Henrietta A, Farhana N, Yuniastuti M (2015) Effects of soybean milk, chitosan, and anti-Streptococcus mutans IgY in malnourished rats’ dental biofilm and the IgY persistency in saliva. Interv Med Appl Sci 7(3):118–123. Google Scholar
  7. Barbas CF, Kang AS, Lerner RA, Benkovic SJ (1991) Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc Natl Acad Sci U S A 88(18):7978–7982. Google Scholar
  8. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112(2):195–203. Google Scholar
  9. De Louvois J, Halket S, Harvey D (2005) Neonatal meningitis in England and Wales: sequelae at 5 years of age. Eur J Pediatr 164(12):730–734. Google Scholar
  10. Du X-J, Wu Y-N, Zhang W-W, Dong F, Wang S (2010) Construction and quality examination of murine naive T7 phage display antibody library. Food Agric Immunol 21(1):81–90. Google Scholar
  11. Engvall E, Perlmann P (1971) Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G. Immunochemistry 8(9):871–874. Google Scholar
  12. Frieder D, Larijani M, Tang E, Parsa J-Y, Basit W, Martin A (2006) Antibody diversification: mutational mechanisms and oncogenesis. Immunol Res 35(1–2):75–87. Google Scholar
  13. Griffiths AD, Duncan AR (1998) Strategies for selection of antibodies by phage display. Curr Opin Biotechnol 9(1):102–108. Google Scholar
  14. Guan Q, Wang X, Wang X, Teng D, Mao R, Zhang Y, Wang J (2015) Recombinant outer membrane protein A induces a protective immune response against Escherichia coli infection in mice. Appl Microbiol Biotechnol 99(13):5451–5460. Google Scholar
  15. Hoogenboom HR, de Bruine AP, Hufton SE, Hoet RM, Arends JW, Roovers RC (1998) Antibody phage display technology and its applications. Immunotechnology 4(1):1–20. Google Scholar
  16. Hudson PJ, Souriau C (2003) Engineered antibodies. Nat Med 9(1):129. Google Scholar
  17. Huston JS, Levinson D, Mudgett-Hunter M, Tai MS, NovotnyJ, Margolies MN, … Crea R (1988) Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A, 85(16):5879–5883.
  18. Jara-Acevedo R, Díez P, González-González M, Dégano RM, Ibarrola N, Gongora R, Fuentes M (2016) Methods for selecting phage display antibody libraries. Curr Pharm Des 22(43):6490–6499. Google Scholar
  19. Kim KS (2002) Strategy of Escherichia coli for crossing the blood-brain barrier. J Infect Dis 186(Suppl_2):S220–S224. Google Scholar
  20. Kim KS (2008) Mechanisms of microbial traversal of the blood–brain barrier. Nat Rev Microbiol 6(8):625–634. Google Scholar
  21. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685.
  22. Lee W, Atif AS, Tan SC, Leow CH (2017) Insights into the chicken IgY with emphasis on the generation and applications of chicken recombinant monoclonal antibodies. J Immunol Methods 447:71–85. Google Scholar
  23. Leu S-J, Lee Y-C, Shih N-Y, Huang I-J, Liu K-J, Lu H-F, … Yang Y-Y (2010) Generation and characterization of anti-α-enolase single-chain antibodies in chicken. Vet Immunol Immunopathol, 137(3–4):251–260.
  24. Li Z, Woo CJ, Iglesias-Ussel MD, Ronai D, Scharff MD (2004) The generation of antibody diversity through somatic hypermutation and class switch recombination. Genes Dev 18(1):1–11. Google Scholar
  25. Lin M, Todoric D, Mallory M, Luo BS, Trottier E, Dan H (2006) Monoclonal antibodies binding to the cell surface of Listeria monocytogenes serotype 4b. J Med Microbiol 55(3):291–299. Google Scholar
  26. Mandal P, Biswas A, Choi K, Pal U (2011) Methods for rapid detection of foodborne pathogens: an overview. Am J Food Technol 6(2):87–102. Google Scholar
  27. Maruvada R, Kim KS (2011) Extracellular loops of the Eschericia coli outer membrane protein A contribute to the pathogenesis of meningitis. J Infect Dis 203(1):131–140. Google Scholar
  28. McConnell MJ, Pachón J (2010) Active and passive immunization against Acinetobacter baumannii using an inactivated whole cell vaccine. Vaccine 29(1):1–5. Google Scholar
  29. Mittal R, Prasadarao NV (2011) gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis. Nat Commun 2:552. Google Scholar
  30. Nelson AL, Dhimolea E, Reichert JM (2010) Development trends for human monoclonal antibody therapeutics. Nat Rev Drug Discov 9(10):767–774. Google Scholar
  31. Nizet V, Jo K (2016) Chapter 6 - bacterial Sepsis and meningitis. In: Remington JS, Klein JO, Wilson CB, Baker CJ (eds) Infectious diseases of the fetus and newborn infant (6th edition), 8th edn. W.B. Saunders, Philadelphia, p 217Google Scholar
  32. Prasadarao NV, Wass CA, Weiser JN, Stins MF, Huang S-H, Kim KS (1996) Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells. Infect Immun 64(1):146–153Google Scholar
  33. Ramakrishnan M, Qu J, Pocanschi CL, Kleinschmidt JH, Marsh D (2005) Orientation of β-barrel proteins OmpA and FhuA in lipid membranes. Chain length dependence from infrared dichroism. Biochemistry 44(9):3515–3523. Google Scholar
  34. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual (3-volume set), vol 999. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  35. Sarker SA, Casswall TH, Juneja LR, Hoq E, Hossain I, Fuchs GJ, Hammarström L (2001) Randomized, placebo-controlled, clinical trial of hyperimmunized chicken egg yolk immunoglobulin in children with rotavirus diarrhea. J Pediatr Gastroenterol Nutr 32(1):19–25Google Scholar
  36. Schmitt J, Hess H, Stunnenberg HG (1993) Affinity purification of histidine-tagged proteins. Mol Biol Rep 18(3):223–230. Google Scholar
  37. Scott J (2001) In: Barbas CF III, Burton DR, Scott JK, Silverman GJ (eds) Phage-display vectors (2.1-2.19) in phage display: a laboratory manual. Cold Spring Harbor, Laboratory Press, Cold Spring HarborGoogle Scholar
  38. Shah DK, Daley AJ, Hunt RW, Volpe JJ, Inder TE (2005) Cerebral white matter injury in the newborn following Escherichia coli meningitis. Eur J Paediatr Neurol 9(1):13–17. Google Scholar
  39. Shanmuganathan MV, Krishnan S, Fu X, Prasadarao NV (2014) Escherichia coli K1 induces pterin production for enhanced expression of Fcgamma receptor I to invade RAW 264.7 macrophages. Microbes Infect 16(2):134–141. Google Scholar
  40. Smith SG, Mahon V, Lambert MA, Fagan RP (2007) A molecular Swiss army knife: OmpA structure, function and expression. FEMS Microbiol Lett 273(1):1–11. Google Scholar
  41. Strachan G, McElhiney J, Drever MR, McIntosh F, Lawton L, Porter AJR (2002) Rapid selection of anti-hapten antibodies isolated from synthetic and semi-synthetic antibody phage display libraries expressed in Escherichia coli. FEMS Microbiol Lett 210(2):257–261. Google Scholar
  42. Sukumaran SK, Shimada H, Prasadarao NV (2003) Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A. Infect Immun 71(10):5951–5961. Google Scholar
  43. Suzuki K, Akahori Y, Asano Y, Kurosawa Y, Shiraki K (2007) Isolation of therapeutic human monoclonal antibodies for varicella-zoster virus and the effect of light chains on the neutralizing activity. J Med Virol 79(6):852–862. Google Scholar
  44. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72(2):211–222Google Scholar
  45. Thigpen MC, Whitney CG, Messonnier NE, Zell ER, Lynfield R, Hadler JL, Bennett NM (2011) Bacterial meningitis in the United States, 1998–2007. N Engl J Med 364(21):2016–2025. Google Scholar
  46. Torres AG, Jeter C, Langley W, Matthysse AG (2005) Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures. Appl Environ Microbiol 71(12):8008–8015. Google Scholar
  47. Wang L-F, Yu M (2004) Epitope identification and discovery using phage display libraries: applications in vaccine development and diagnostics. Curr Drug Targets 5(1):1–15. Google Scholar
  48. Wark KL, Hudson PJ (2006) Latest technologies for the enhancement of antibody affinity. Adv Drug Deliv Rev 58(5–6):657–670. Google Scholar
  49. Winter G, Griffiths AD, Hawkins RE, Hoogenboom HR (1994) Making antibodies by phage display technology. Annu Rev Immunol 12(1):433–455. Google Scholar
  50. Wu HH, Yang YY, Hsieh WS, Lee CH, Leu SJ, Chen MR (2009) OmpA is the critical component for Escherichia coli invasion-induced astrocyte activation. J Neuropathol Exp Neurol 68(6):677–690. Google Scholar
  51. Xu JL, Davis MM (2000) Diversity in the CDR3 region of VH is sufficient for most antibody specificities. Immunity 13(1):37–45. Google Scholar
  52. Yuan Q, Jordan R, Brlansky RH, Istomina O, Hartung J (2015) Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display. J Microbiol Methods 117:148–154. Google Scholar
  53. Zhang Y, Stewart S, Joseph T, Taylor H, Caldwell H (1987) Protective monoclonal antibodies recognize epitopes located on the major outer membrane protein of Chlamydia trachomatis. J Immunol 138(2):575–581Google Scholar
  54. Zhang X, Yang T, Cao J, Sun J, Dai W, Zhang L (2016) Mucosal immunization with purified OmpA elicited protective immunity against infections caused by multidrug-resistant Acinetobacter baumannii. Microb Pathog 96:20–25. Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Pharaoh Fellow Mwale
    • 1
    • 2
  • Chi-Hsin Lee
    • 1
    • 2
  • Sy-Jye Leu
    • 3
    • 4
  • Yu-Ching Lee
    • 5
  • Hsueh-Hsia Wu
    • 1
    • 2
  • Liang-Tzung Lin
    • 3
    • 4
  • Tony Eight Lin
    • 6
  • Yun-Ju Huang
    • 2
  • Yi-Yuan Yang
    • 1
    • 2
    • 7
    Email author
  1. 1.Ph.D. Program in Medical Biotechnology, College of Medical Science and TechnologyTaipei Medical UniversityTaipeiTaiwan
  2. 2.School of Medical Laboratory Science and Biotechnology, College of Medical Science and TechnologyTaipei Medical UniversityTaipeiTaiwan
  3. 3.Graduate Institute of Medical Sciences, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  4. 4.Department of Microbiology and Immunology, School of Medicine, College of MedicineTaipei Medical UniversityTaipeiTaiwan
  5. 5.The Center of Translational MedicineTaipei Medical UniversityTaipeiTaiwan
  6. 6.Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and TechnologyTaipei Medical UniversityTaipeiTaiwan
  7. 7.Core Laboratory of Antibody Generation and ResearchTaipei Medical UniversityTaipeiTaiwan

Personalised recommendations