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  • Open Access

Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut

  • 1,
  • 2,
  • 3,
  • 4,
  • 4,
  • 5,
  • 2 and
  • 2
Clinical and Translational Allergy20133 (Suppl 3) :P168

https://doi.org/10.1186/2045-7022-3-S3-P168

  • Published:

Keywords

  • Tandem Mass Spectrometry
  • Size Exclusion Chromatography
  • Allergic Patient
  • Peanut Allergy
  • Antibody Assay

Background

Ara h 6 has a reported seroprevalence similar to Ara h 2, a major peanut allergen. Both allergens are of similar molecular size and share 50% identity in their amino acid sequences. Their similarity has made it difficult to obtain natural Ara h 6 free of Ara h 2 for immunoreactivity studies. The objective of this study was to obtain purified natural Ara h 6 that is essentially free of Ara h 2 and to compare its IgE reactivity and potency in histamine release assays to Ara h 2.

Methods

Natural Ara h 6 was purified from peanut flour extract by affinity chromatography and size exclusion chromatography. Purified Arah 6 was analyzed by silver-stained SDS-PAGE and tandem mass spectrometry (LC/MS-MS). The immunological and biological reactivity of Ara h 6 and Ara h 2 were compared by ELISA and histamine release assays.

Results

SDS-PAGE of the highly purified allergen revealed a single 14.5kD band and the identity of Ara h 6 was confirmed by tandem mass spectrometry (LC/MS-MS). The purified nAra h 6 contained <0.01% traces of Ara h 2 as assessed by ELISA. Natural Ara h 6 had a lower biological activity in basophil histamine release assays than natural Ara h 2. Chimeric ELISA showed that 70 and 75% of peanut allergic patients (n=57) had specific-IgE to natural Ara h 2 and natural Ara h 6 respectively.

Conclusion

Ara h 6 is a major peanut allergen, with comparable immunoreactivity to Ara h 2. The highly purified Ara h 6, free of Ara h 2, will be useful for diagnostic IgE antibody assays, and for molecular and cellular studies to further investigate the immunological mechanisms of peanut allergy.

Disclosure of interest

J Hindley: Employee of INDOOR Biotechnologies Ltd, A Koid: Employee of INDOOR Biotechnologies Inc, R Hamilton: None declared, R van Ree: None declared, S Versteeg: None declared, S Dreskin: None declared, M Chapman: Shareholder of INDOOR Biotechnologies Inc, S Wunschmann: Employee of INDOOR Biotechnologies Inc.

Authors’ Affiliations

(1)
INDOOR Biotechnologies Ltd, Cardiff, UK
(2)
INDOOR Biotechnologies Inc, Charlottesville, VA, USA
(3)
Johns Hopkins University School of Medicine, Baltimore, MD, USA
(4)
Academic Medical Centre, Amsterdam, the Netherlands
(5)
University of Colorado, Denver, CO, USA

Copyright

© Hindley et al; licensee BioMed Central Ltd. 2013

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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