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Isolation of Natural Anti-FcεRIα Autoantibodies from Healthy Donors

  • Monique VogelEmail author
  • Michael P. Horn
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1643)

Abstract

Natural antibodies are defined as antibodies detected in a healthy individual without active immunization. These antibodies are specific for exoantigens, as well as for autoantigens, mostly without any pathogenic role. Most of the studies conducted with natural (auto-) antibodies have been performed using affinity purified antibodies from individual sera or polyclonal Ig-preparations such as Intravenous Ig (IVIg). For in-depth analysis of such autoantibodies affinity-purified Ig-preparations from healthy individuals are of no use, as they are oligoclonal or polyclonal. Thus, there is a need of human monoclonal autoantibodies. Human monoclonal autoantibodies can be produced from B cells isolated from humans; however, this requires the screening of a large number of antibodies to identify one among them specific to an antigen. Using the phage display technology we generated such autoantibodies against the alpha subunit of the high-affinity IgE receptor (FcεRIα). Here we describe the step-by-step protocol for the generation of such libraries and the isolation of autoantibodies by affinity panning.

Key words

Natural antibodies Anti-FcεRIα autoantibodies IVIg Phage display technology Isolation 

References

  1. 1.
    Hide M, Francis DM, Grattan CE, Hakimi J, Kochan JP, Greaves MW (1993) Autoantibodies against the high-affinity IgE receptor as a cause of histamine release in chronic urticaria. N Engl J Med 328(22):1599–1604. doi: 10.1056/NEJM199306033282204 CrossRefPubMedGoogle Scholar
  2. 2.
    Fiebiger E, Hammerschmid F, Stingl G, Maurer D (1998) Anti-FcepsilonRIalpha autoantibodies in autoimmune-mediated disorders. Identification of a structure-function relationship. J Clin Invest 101(1):243–251. doi: 10.1172/JCI511 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Horn MP, Gerster T, Ochensberger B, Derer T, Kricek F, Jouvin MH et al (1999) Human anti-FcepsilonRIalpha autoantibodies isolated from healthy donors cross-react with tetanus toxoid. Eur J Immunol 29(4):1139–1148CrossRefPubMedGoogle Scholar
  4. 4.
    Lacroix-Desmazes S, Kaveri SV, Mouthon L, Ayouba A, Malanchere E, Coutinho A et al (1998) Self-reactive antibodies (natural autoantibodies) in healthy individuals. J Immunol Methods 216(1–2):117–137CrossRefPubMedGoogle Scholar
  5. 5.
    Mouthon L, Lacroix-Desmazes S, Nobrega A, Barreau C, Coutinho A, Kazatchkine MD (1996) The self-reactive antibody repertoire of normal human serum IgM is acquired in early childhood and remains conserved throughout life. Scand J Immunol 44(3):243–251CrossRefPubMedGoogle Scholar
  6. 6.
    Horn MP, Pachlopnik JM, Vogel M, Dahinden M, Wurm F, Stadler BM et al (2001) Conditional autoimmunity mediated by human natural anti-Fc(epsilon)RIalpha autoantibodies? FASEB J 15(12):2268–2274CrossRefPubMedGoogle Scholar
  7. 7.
    Barbas CF 3rd, 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):7978–7982CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Horn MP, Zuercher AW, Imboden MA, Rudolf MP, Lazar H, Wu H et al (2010) Preclinical in vitro and in vivo characterization of the fully human monoclonal IgM antibody KBPA101 specific for Pseudomonas aeruginosa serotype IATS-O11. Antimicrob Agents Chemother. doi:10.1128/AAC.01142–09. AAC.01142–09 [pii]PubMedPubMedCentralGoogle Scholar
  9. 9.
    Vogel M, Lai L, Rudolf MP, Curcio-Vonlanthen V, Miescher S, Stadler BM (1996) Cross reactive anti-tetanus and anti-melittin Fab fragments by phage display after tetanus toxoid immunisation. Hum Antibodies Hybridomas 7(1):11–20PubMedGoogle Scholar
  10. 10.
    Miescher S, Zahn-Zabal M, De Jesus M, Moudry R, Fisch I, Vogel M et al (2000) CHO expression of a novel human recombinant IgG1 anti-RhD antibody isolated by phage display. Br J Haematol 111(1):157–166CrossRefPubMedGoogle Scholar
  11. 11.
    Stadler BM, Rudolf MP, Zurcher AW, Miescher S, Vogel M (1996) Anti-IgE in allergic sensitization. Immunol Cell Biol 74(2):195–200. doi: 10.1038/icb.1996.27 CrossRefPubMedGoogle Scholar
  12. 12.
    Vogel M, Miescher S, Biaggi C, Stadler BM (1994) Human anti-IgE antibodies by repertoire cloning. Eur J Immunol 24(5):1200–1207CrossRefPubMedGoogle Scholar
  13. 13.
    Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M (2012) scFv antibody: principles and clinical application. Clin Dev Immunol 2012:980250. doi: 10.1155/2012/980250 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lennard S (2002) Standard protocols for the construction of scFv libraries. Methods Mol Biol 178:59–71PubMedGoogle Scholar
  15. 15.
    Giles IP, Haley J, Nagl S, Latchman DS, Chen PP, Chukwuocha RU et al (2003) Relative importance of different human aPL derived heavy and light chains in the binding of aPL to cardiolipin. Mol Immunol 40(1):49–60CrossRefPubMedGoogle Scholar
  16. 16.
    DeKosky BJ, Ippolito GC, Deschner RP, Lavinder JJ, Wine Y, Rawlings BM et al (2013) High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire. Nat Biotechnol 31(2):166–169. doi: 10.1038/nbt.2492 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.University Clinic of Rheumatology, Immunology and Allergology, Inselspital, University Hospital of BernBernSwitzerland
  2. 2.University Institute of Clinical Chemistry and Center of Laboratory Medicine, Inselspital, University Hospital of BernBernSwitzerland

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