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Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products

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Abstract

Birch (Betula) pollen is a major cause of allergy in northern and central Europe. The allergenic potency of products for diagnosis and therapy of birch pollen allergy is adjusted nearly exclusively to the major birch pollen allergen Bet v 1. Although every fifth patient is additionally sensitized to Bet v 4, both content and variability of this minor allergen in birch allergen products remain unclear due to a lack of simple and cost-effective quantitative methods. This study aimed to develop and in-house validate the first Bet v 4-specific sandwich enzyme-linked immunosorbent assay (ELISA). Based on a murine monoclonal antibody in combination with a polyclonal rabbit antiserum, the ELISA proved to be highly sensitive, with a lower limit of quantification of 30 pg/ml Bet v 4. After confirmation of satisfactory accuracy, reproducibility, and robustness, the ELISA was utilized to quantify Bet v 4 in 30 authorized birch allergen products. The allergen was detected in all samples tested, ranging from 0.2 to 4.4 μg/ml. No significant correlation of Bet v 4 was found with the respective amount of Bet v 1. In contrast to Bet v 1, also no correlation of Bet v 4 with total protein content or total allergenic activity could be observed. Thus, it seems presently unfeasible to base birch allergen product standardization additionally on Bet v 4. In light of these results, the continuous monitoring of Bet v 4 in birch allergen products with the presented ELISA will provide a basis for the understanding of the clinical relevance of minor allergens.

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Abbreviations

AIT:

Allergen immunotherapy

Bet v:

Birch allergen (Betula verrucosa, nomenclature according to the International Union of Immunological Societies)

ELISA:

Enzyme-linked immunosorbent assay

mAb:

Monoclonal antibody

MS:

Mass spectrometry

nBet v:

Native birch allergen

PTS:

Prick test solution

rBet v:

Recombinant birch allergen

SLIT:

Sublingual immunotherapy

TMB:

3,3′,5,5′-Tetramethylbenzidine

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Acknowledgments

This study was realized thanks to the support of ALK-Abelló Arzneimittel GmbH, Allergopharma GmbH & Co. KG, Bencard Allergie GmbH, HAL Allergy GmbH, Leti Pharma GmbH, Stallergenes GmbH (listed in alphabetical order), and their representatives, giving consent for the use of their birch pollen extracts designated for batch release control at the PEI. Many thanks go to Prof. Dr. Paul Rösch, University of Bayreuth, Germany, for providing the recombinant Bet v 4.

Conflict of interest

In the past 5 years, Stefan Vieths has received a speaker’s honorarium from Phadia (now Thermo Fisher Scientific), Uppsala; an honorarium for organizing and chairing an educational seminar on food allergens from Fresenius Academy, Dortmund; honoraria for acting as expert reviewer for the Allergen Online Database of the Food Allergy Resource and Research Program, Lincoln NE; and consultancy fees by the Institute for Product Quality, Berlin.

Thomas Holzhauser had consultant arrangements with Institut für Produktqualität and Monsanto Company. He received fees from Die Akademie Fresenius for speaking and organizing education. He received reimbursement for lectures from the European Academy of Allergology and Clinical Immunology, World Allergy Organization, Deutscher Allergie- und Asthmabund e.V., International Life Sciences Institute/Health and Environmental Sciences Institute, International Association for Food Protection, AOAC International, Chinesisch-Deutsches Zentrum für Wissenschaftsförderung, Phadia AB, and SAG Süderelbe Projektgesellschaft AG & Co. KG.

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Authors and Affiliations

  1. Division of Allergology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225, Langen, Germany

    Oliver Dehus, Julia Zimmer, Sascha Döring, Frank Führer, Thomas Holzhauser, Florian Neske, Daniel Strecker, Stefan Vieths & Susanne Kaul

  2. Division EU Cooperation/Microbiology, Paul-Ehrlich-Institut, Paul-Ehrlich-Straße 51-59, 63225, Langen, Germany

    Kay-Martin Hanschmann & Jan-Hendrik Trösemeier

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  1. Oliver Dehus
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  2. Julia Zimmer
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  3. Sascha Döring
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  4. Frank Führer
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  5. Kay-Martin Hanschmann
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  6. Thomas Holzhauser
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  7. Florian Neske
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  8. Daniel Strecker
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  9. Jan-Hendrik Trösemeier
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  10. Stefan Vieths
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  11. Susanne Kaul
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Correspondence to Susanne Kaul.

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Oliver Dehus and Julia Zimmer contributed equally to this work.

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Dehus, O., Zimmer, J., Döring, S. et al. Development and in-house validation of an allergen-specific ELISA for quantification of Bet v 4 in diagnostic and therapeutic birch allergen products. Anal Bioanal Chem 407, 1673–1683 (2015). https://doi.org/10.1007/s00216-014-8418-z

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