Skip to main content

Measuring Antibody Orientation at the Bacterial Surface

Part of the Methods in Molecular Biology book series (MIMB,volume 1535)


Many bacteria have the ability to interact with antibodies as a means to circumvent the immune response. This includes binding to the Fc portion of antibodies, effectively reversing the antibody orientation and thus decreasing the Fc-mediated immune signaling. Since antibody orientation at the bacterial surface has been shown to be important in human disease, it is valuable to be able to assess how antibodies are interacting with bacterial pathogens. Here, we describe a method to measure the proportion of human IgG that are bound via their Fc or Fabs to a bacterial surface. This is achieved by treating antibody-coated bacteria with the bacterial enzyme IdeS – which will cleave IgG into Fc and Fab fragments – and subsequently detect remaining fragments with fluorescent Fabs. The method is easy and fast, and the principle is most likely also applicable to other systems where distinguishing between antibody Fc and Fab binding is important.

Key words

  • IgG
  • Bacteria
  • Flow cytometry
  • IgG-binding proteins
  • IdeS

This is a preview of subscription content, access via your institution.

Buying options

USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD   119.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more


  1. Frick IM, Wikström M, Forsén S, Drakenberg T, Gomi H, Sjöbring U, Björck L (1992) Convergent evolution among immunoglobulin G-binding bacterial proteins. Proc Natl Acad Sci U S A 89(18):8532–8536

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  2. Björck L, Kronvall G (1984) Purification and some properties of streptococcal protein G, a novel IgG-binding reagent. J Immunol 133(2):969–974

    PubMed  Google Scholar 

  3. Forsgren A, Sjöquist J (1966) Protein A from S. aureus. I. Pseudo-immune reaction with human gamma-globulin. J Immunol 97(6):822–827

    CAS  PubMed  Google Scholar 

  4. Akesson P, Cooney J, Kishimoto F, Björck L (1990) Protein H--;a novel IgG binding bacterial protein. Mol Immunol 27(6):523–531

    CrossRef  CAS  PubMed  Google Scholar 

  5. Nordenfelt P, Waldemarson S, Linder A, Mörgelin M, Karlsson C, Malmström J, Björck L (2012) Antibody orientation at bacterial surfaces is related to invasive infection. J Exp Med 209(13):2367–2381. doi:10.1084/jem.20120325

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  6. Lood R, Waldetoft KW, Nordenfelt P (2015) Localization-triggered bacterial pathogenesis. Future Microbiol 10(10):1659–1668. doi:10.2217/fmb.15.89

    CrossRef  CAS  PubMed  Google Scholar 

  7. von Pawel-Rammingen U, Johansson BP, Björck L (2002) IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 21(7):1607–1615. doi:10.1093/emboj/21.7.1607

    CrossRef  Google Scholar 

  8. Björck L (1988) Protein L. A novel bacterial cell wall protein with affinity for Ig L chains. J Immunol 140(4):1194–1197

    PubMed  Google Scholar 

Download references


This work was supported by the Crafoord Foundation, the Swedish Research Council, and the Swedish Society of Medicine.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Pontus Nordenfelt .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2017 Springer Science+Business Media New York

About this protocol

Cite this protocol

Shannon, O., Nordenfelt, P. (2017). Measuring Antibody Orientation at the Bacterial Surface. In: Nordenfelt, P., Collin, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology, vol 1535. Humana Press, New York, NY.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6671-4

  • Online ISBN: 978-1-4939-6673-8

  • eBook Packages: Springer Protocols