# EAACI Molecular Allergology User's Guide 2.0

Stephanie Dramburg, Christiane Hilger, Alexandra Santos, Leticia de las Vecillas, Rob Aalberse, Nathalie Acevedo, Lorenz Aglas, Friedrich Altmann, Karla Arruda, Riccardo Asero, et al.

► To cite this version:

Stephanie Dramburg, Christiane Hilger, Alexandra Santos, Leticia de las Vecillas, Rob Aalberse, et al. EAACI Molecular Allergology User's Guide 2.0. Pediatric Allergy and Immunology, 2023, 34 (S28), pp.e13854. 10.1111/pai.13854. hal-04615555

HAL Id: hal-04615555

https://ut3-toulouseinp.hal.science/hal-04615555

Submitted on 18 Jun 2024

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<figure><img src="image_1.png" /></figure>
# EAACI Molecular Allergology User's Guide 2.0

Stephanie Dramburg, Christiane Hilger, Alexandra Santos, Leticia de las Vecillas, Rob Aalberse, Nathalie Acevedo, Lorenz Aglas, Friedrich Altmann, Karla Arruda, Riccardo Asero, et al.

► To cite this version:

Stephanie Dramburg, Christiane Hilger, Alexandra Santos, Leticia de las Vecillas, Rob Aalberse, et al. EAACI Molecular Allergology User's Guide 2.0. Pediatric Allergy and Immunology, 2023, 34 (S28), pp.e13854. 10.1111/pai.13854. hal-04615555

HAL Id: hal-04615555

https://ut3-toulouseinp.hal.science/hal-04615555v1

Submitted on 18 Jun 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
EAACI Molecular Allergology User's Guide 2.0

Stephanie Dramburg¹ ID | Christiane Hilger² ID | Alexandra F. Santos³,⁴,⁵ ID |

Leticia de las Vecillas⁶ ID | Rob C. Aalberse⁷,⁸ ID | Nathalie Acevedo⁹ ID | Lorenz Aglas¹⁰ ID |

Friedrich Altmann¹¹ ID | Karla L. Arruda¹² ID | Riccardo Asero¹³ ID |

Barbara Ballmer-Weber¹⁴,¹⁵ ID | Domingo Barber¹⁶,¹⁷ ID | Kirsten Beyer¹ ID |

Tilo Biedermann¹⁸ ID | Maria Beatrice Bilo¹⁹,²⁰ ID | Simon Blank²¹ ID |

Philipp P. Bosshard¹⁵ ID | Heimo Breiteneder²² ID | Helen A. Brough³,⁵ ID |

Merima Dublin²² ID | Dianne Campbell²³,²⁴ ID | Luis Caraballo⁹ ID |

Jean Christoph Caubet²⁵ ID | Giorgio Celi²⁶ ID | Martin D. Chapman²⁷ ID |

Maksymilian Chruszcz²⁸ ID | Adnan Custovic²⁹ ID | Rebecca Czolk²,³⁰ ID |

Janet Davies³¹,³² ID | Nikolaos Douladiris³³ ID | Bernadette Eberlein¹⁸ ID |

Motohiro Ebisawa³⁴ ID | Anna Ehlers³⁵,³⁶,⁶⁰ ID | Philippe Eigenmann²⁵ ID |

Gabriele Gadermaier¹⁰ ID | Mattia Giovannini³⁷ ID | Francisca Gomez³⁸,³⁹ ID |

Rebecca Grohman⁴⁰ ID | Carole Guillet¹⁵,⁴¹ ID | Christine Hafner⁴² ID |

Robert G. Hamilton⁴³ ID | Michael Hauser¹⁰ ID | Thomas Hawranek⁴⁴ ID |

Hans Jürgen Hoffmann⁴⁵,⁴⁶ ID | Thomas Holzhauser⁴⁷ ID | Tomona Iizuka⁴⁸ ID |

Alain Jacquet⁴⁹ ID | Thilo Jakob⁵⁰ ID | Bente Janssen-Weets²,⁵¹ ID | Uta Jappe⁵²,⁵³,⁵⁴ ID |

Marek Jutel⁵⁵ ID | Tanja Kalic²²,⁴² ID | Sandip Kamath⁵⁶,⁵⁷ ID | Sabine Kespohl⁵⁸ ID |

Jörg Kleine-Tebbe⁵⁹ ID | Edward Knol⁶⁰ ID | André Knulst⁶⁰ ID | Jon R. Konradsen⁶¹,⁶² ID |

Peter Korošec⁶³,⁶⁴ ID | Annette Kuehn² ID | Gideon Lack³,⁴,⁵ ID | Thuy-My Le³⁶,⁶⁰ ID |

Andreas Lopata⁵⁶,⁵⁷ ID | Olga Luengo¹⁷,⁶⁵ ID | Mika Mäkelä⁶⁶,⁶⁷ ID |

Alessandro Maria Marra⁶⁸ ID | Clare Mills⁶⁹ ID | Martine Morisset⁷⁰ ID |

Antonella Muraro⁷¹ ID | Anna Nowak-Wegrzyn⁷²,⁷³ ID | Roni Nugraha⁵⁷,⁷⁴ ID |

Markus Ollert²,⁵¹ ID | Kati Palosuo⁷⁵ ID | Elide Anna Pastorello⁷⁶ ID |

Sarita Ulhas Patil⁷⁷,⁷⁸ ID | Thomas Platts-Mills⁷⁹ ID | Anna Pomés²⁷ ID |

Pascal Poncet⁸⁰,⁸¹ ID | Ekaterina Potapova¹ ID | Lars K. Poulsen⁸² ID |

Christian Radauer²² ID | Suzana Radulovic³,⁵ ID | Monika Raulf⁵⁸ ID | Pierre Rouge⁸³ ID |

Joaquin Sastre⁸⁴ ID | Sakura Sato³⁴ ID | Enrico Scala⁸⁵ ID | Johannes M. Schmid⁴⁶ ID |

Peter Schmid-Grendelmeier¹⁵,⁸⁶ ID | Denise Schrama⁸⁷ ID | Hélène Sénéchal⁸¹ ID |

Stephanie Dramburg, Christiane Hilger, Alexandra Santos and Leticia de las Vecillas are shared first authors.

Paolo M. Matricardi and Karin Hoffmann-Sommergruber are shared last authors.

This is an open access article under the terms of the [Creative Commons Attribution-NonCommercial License](https://creativecommons.org/licenses/by-nc), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

© 2023 The Authors. *Pediatric Allergy and Immunology* published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.
EAACI Molecular Allergology User's Guide 2.0

Stephanie Dramburg¹ ID | Christiane Hilger² ID | Alexandra F. Santos³,⁴,⁵ ID |
Leticia de las Vecillas⁶ ID | Rob C. Aalberse⁷,⁸ ID | Nathalie Acevedo⁹ ID | Lorenz Aglas¹⁰ ID |
Friedrich Altmann¹¹ ID | Karla L. Arruda¹² ID | Riccardo Asero¹³ ID |
Barbara Ballmer-Weber¹⁴,¹⁵ ID | Domingo Barber¹⁶,¹⁷ ID | Kirsten Beyer¹ ID |
Tilo Biedermann¹⁸ ID | Maria Beatrice Bilo¹⁹,²⁰ ID | Simon Blank²¹ ID |
Philipp P. Bosshard¹⁵ ID | Heimo Breiteneder²² ID | Helen A. Brough³,⁵ ID |
Merima Dublin²² ID | Dianne Campbell²³,²⁴ ID | Luis Caraballo⁹ ID |
Jean Christoph Caubet²⁵ ID | Giorgio Celi²⁶ ID | Martin D. Chapman²⁷ ID |
Maksymilian Chruszcz²⁸ ID | Adnan Custovic²⁹ ID | Rebecca Czolk²,³⁰ ID |
Janet Davies³¹,³² ID | Nikolaos Douladiris³³ ID | Bernadette Eberlein¹⁸ ID |
Motohiro Ebisawa³⁴ ID | Anna Ehlers³⁵,³⁶,⁶⁰ ID | Philippe Eigenmann²⁵ ID |
Gabriele Gadermaier¹⁰ ID | Mattia Giovannini³⁷ ID | Francisca Gomez³⁸,³⁹ ID |
Rebecca Grohman⁴⁰ ID | Carole Guillet¹⁵,⁴¹ ID | Christine Hafner⁴² ID |
Robert G. Hamilton⁴³ ID | Michael Hauser¹⁰ ID | Thomas Hawranek⁴⁴ ID |
Hans Jürgen Hoffmann⁴⁵,⁴⁶ ID | Thomas Holzhauser⁴⁷ ID | Tomona Iizuka⁴⁸ ID |
Alain Jacquet⁴⁹ ID | Thilo Jakob⁵⁰ ID | Bente Janssen-Weets²,⁵¹ ID | Uta Jappe⁵²,⁵³,⁵⁴ ID |
Marek Jutel⁵⁵ ID | Tanja Kalic²²,⁴² ID | Sandip Kamath⁵⁶,⁵⁷ ID | Sabine Kespohl⁵⁸ ID |
Jörg Kleine-Tebbe⁵⁹ ID | Edward Knol⁶⁰ ID | André Knulst⁶⁰ ID | Jon R. Konradsen⁶¹,⁶² ID |
Peter Korošec⁶³,⁶⁴ ID | Annette Kuehn² ID | Gideon Lack³,⁴,⁵ ID | Thuy-My Le³⁶,⁶⁰ ID |
Andreas Lopata⁵⁶,⁵⁷ ID | Olga Luengo¹⁷,⁶⁵ ID | Mika Mäkelä⁶⁶,⁶⁷ ID |
Alessandro Maria Marra⁶⁸ ID | Clare Mills⁶⁹ ID | Martine Morisset⁷⁰ ID |
Antonella Muraro⁷¹ ID | Anna Nowak-Wegrzyn⁷²,⁷³ ID | Roni Nugraha⁵⁷,⁷⁴ ID |
Markus Ollert²,⁵¹ ID | Kati Palosuo⁷⁵ ID | Elide Anna Pastorello⁷⁶ ID |
Sarita Ulhas Patil⁷⁷,⁷⁸ ID | Thomas Platts-Mills⁷⁹ ID | Anna Pomés²⁷ ID |
Pascal Poncet⁸⁰,⁸¹ ID | Ekaterina Potapova¹ ID | Lars K. Poulsen⁸² ID |
Christian Radauer²² ID | Suzana Radulovic³,⁵ ID | Monika Raulf⁵⁸ ID | Pierre Rouge⁸³ ID |
Joaquin Sastre⁸⁴ ID | Sakura Sato³⁴ ID | Enrico Scala⁸⁵ ID | Johannes M. Schmid⁴⁶ ID |
Peter Schmid-Grendelmeier¹⁵,⁸⁶ ID | Denise Schrama⁸⁷ ID | Hélène Sénéchal⁸¹ ID |

Stephanie Dramburg, Christiane Hilger, Alexandra Santos and Leticia de las Vecillas are shared first authors.

Paolo M. Matricardi and Karin Hoffmann-Sommergruber are shared last authors.

This is an open access article under the terms of the [Creative Commons Attribution-NonCommercial License](https://creativecommons.org/licenses/by-nc), which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.

© 2023 The Authors. *Pediatric Allergy and Immunology* published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.
Claudia Traidl-Hoffmann<sup>86,88 ID</sup>

<figure><img src="image_2.png" /></figure>

Marcela Valverde-Monge<sup>84 ID</sup>

<figure><img src="image_3.png" /></figure>

Marianne van Hage<sup>89,90 ID</sup>

<figure><img src="image_4.png" /></figure>

Ronald van Ree<sup>91 ID</sup>

<figure><img src="image_5.png" /></figure>

Kitty Verhoeckx<sup>60 ID</sup>

<figure><img src="image_6.png" /></figure>

Stefan Vieths<sup>47 ID</sup>

<figure><img src="image_7.png" /></figure>

Magnus Wickman<sup>92 ID</sup>

<figure><img src="image_8.png" /></figure>

Josefina Zakzuk<sup>9 ID</sup>

<figure><img src="image_9.png" /></figure>

Paolo M. Matricardi<sup>1 ID</sup>

<figure><img src="image_10.png" /></figure>

Karin Hoffmann-Sommergruber<sup>22 ID</sup>

<figure><img src="image_11.png" /></figure>

<footnote n="1"><sup>1</sup>Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité Universitätsmedizin Berlin, Berlin, Germany</footnote>

<footnote n="2"><sup>2</sup>Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg</footnote>

<footnote n="3"><sup>3</sup>Department of Women and Children's Health (Pediatric Allergy), School of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, London, United Kingdom</footnote>

<footnote n="4"><sup>4</sup>Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King's College London, London, United Kingdom</footnote>

<footnote n="5"><sup>5</sup>Children's Allergy Service, Evelina London, Guy's and St Thomas' Hospital, London, United Kingdom</footnote>

<footnote n="6"><sup>6</sup>Department of Allergy, La Paz University Hospital, IdiPAZ, Madrid, Spain</footnote>

<footnote n="7"><sup>7</sup>Sanquin Research, Dept Immunopathology, University of Amsterdam, Amsterdam, The Netherlands</footnote>

<footnote n="8"><sup>8</sup>Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands</footnote>

<footnote n="9"><sup>9</sup>Institute for Immunological Research, University of Cartagena, Cartagena de Indias, Colombia, Colombia</footnote>

<footnote n="10"><sup>10</sup>Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria</footnote>

<footnote n="11"><sup>11</sup>Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria</footnote>

<footnote n="12"><sup>12</sup>Department of Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Sao Paulo, Brasil, Brazil</footnote>

<footnote n="13"><sup>13</sup>Ambulatorio di Allergologia, Clinica San Carlo, Paderno Dugnano, Italy</footnote>

<footnote n="14"><sup>14</sup>Klinik für Dermatologie und Allergologie, Kantonsspital St. Gallen, St. Gallen, Switzerland</footnote>

<footnote n="15"><sup>15</sup>Department of Dermatology, University Hospital Zurich, Zurich, Switzerland</footnote>

<footnote n="16"><sup>16</sup>Institute of Applied Molecular Medicine Nemesio Diez (IMMAND), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Madrid, Spain</footnote>

<footnote n="17"><sup>17</sup>RETIC ARADYAL and RICORS Enfermedades Inflamatorias (REI), Madrid, Spain</footnote>

<footnote n="18"><sup>18</sup>Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University Munich, Munich, Germany</footnote>

<footnote n="19"><sup>19</sup>Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy</footnote>

<footnote n="20"><sup>20</sup>Allergy Unit Department of Internal Medicine, University Hospital Ospedali Riuniti di Ancona, Torrette, Italy</footnote>

<footnote n="21"><sup>21</sup>Center of Allergy and Environment (ZAUM), Technical University of Munich, School of Medicine and Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany</footnote>

<footnote n="22"><sup>22</sup>Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria</footnote>

<footnote n="23"><sup>23</sup>Department of Allergy and Immunology, Children's Hospital at Westmead, Sydney Children's Hospitals Network, Sydney, New South Wales, Australia</footnote>

<footnote n="24"><sup>24</sup>Child and Adolescent Health, Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia</footnote>

<footnote n="25"><sup>25</sup>Pediatric Allergy Unit, Department of Child and Adolescent, University Hospitals of Geneva, Geneva, Switzerland</footnote>

<footnote n="26"><sup>26</sup>Centro DH Allergologia e Immunologia Clinica ASST- MANTOVA (MN), Mantova, Italy</footnote>

<footnote n="27"><sup>27</sup>InBio, Charlottesville, Virginia, USA</footnote>

<footnote n="28"><sup>28</sup>Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, USA</footnote>

<footnote n="29"><sup>29</sup>National Heart and Lung Institute, Imperial College London, London, UK</footnote>

<footnote n="30"><sup>30</sup>Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg</footnote>

<footnote n="31"><sup>31</sup>Queensland University of Technology, Centre for Immunology and Infection Control, School of Biomedical Sciences, Herston, Queensland, Australia</footnote>

<footnote n="32"><sup>32</sup>Metro North Hospital and Health Service, Emergency Operations Centre, Herston, Queensland, Australia</footnote>

<footnote n="33"><sup>33</sup>Allergy Department, 2nd Paediatric Clinic, National and Kapodistrian University of Athens, Athens, Greece</footnote>

<footnote n="34"><sup>34</sup>Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan</footnote>

<footnote n="35"><sup>35</sup>Chemical Biology and Drug Discovery, Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="36"><sup>36</sup>Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="37"><sup>37</sup>Allergy Unit, Department of Pediatrics, Meyer Children's University Hospital, Florence, Italy</footnote>

<footnote n="38"><sup>38</sup>Allergy Unit IBIMA-Hospital Regional Universitario de Malaga, Malaga, Spain</footnote>

<footnote n="39"><sup>39</sup>Spanish Network for Allergy research RETIC ARADYAL, Malaga, Spain</footnote>

<footnote n="40"><sup>40</sup>NYU Langone Health, Department of Internal Medicine, New York, New York, USA</footnote>

<footnote n="41"><sup>41</sup>Faculty of Medicine, University of Zurich, Zurich, Switzerland</footnote>

<footnote n="42"><sup>42</sup>Department of Dermatology, University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, St. Poelten, Austria</footnote>

<footnote n="43"><sup>43</sup>Johns Hopkins University School of Medicine, Baltimore, Maryland, USA</footnote>

<footnote n="44"><sup>44</sup>Department of Dermatology and Allergology, Paracelsus Private Medical University, Salzburg, Austria</footnote>

<footnote n="45"><sup>45</sup>Institute for Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark</footnote>
Claudia Traidl-Hoffmann<sup>86,88 ID</sup>

<figure><img src="image_2.png" /></figure>

Marcela Valverde-Monge<sup>84 ID</sup>

<figure><img src="image_3.png" /></figure>

Marianne van Hage<sup>89,90 ID</sup>

<figure><img src="image_4.png" /></figure>

Ronald van Ree<sup>91 ID</sup>

<figure><img src="image_5.png" /></figure>

Kitty Verhoeckx<sup>60 ID</sup>

<figure><img src="image_6.png" /></figure>

Stefan Vieths<sup>47 ID</sup>

<figure><img src="image_7.png" /></figure>

Magnus Wickman<sup>92 ID</sup>

<figure><img src="image_8.png" /></figure>

Josefina Zakzuk<sup>9 ID</sup>

<figure><img src="image_9.png" /></figure>

Paolo M. Matricardi<sup>1 ID</sup>

<figure><img src="image_10.png" /></figure>

Karin Hoffmann-Sommergruber<sup>22 ID</sup>

<figure><img src="image_11.png" /></figure>

<footnote n="1"><sup>1</sup>Department of Pediatric Respiratory Medicine, Immunology and Critical Care Medicine, Charité Universitätsmedizin Berlin, Berlin, Germany</footnote>

<footnote n="2"><sup>2</sup>Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg</footnote>

<footnote n="3"><sup>3</sup>Department of Women and Children's Health (Pediatric Allergy), School of Life Course Sciences, Faculty of Life Sciences and Medicine, King's College London, London, United Kingdom</footnote>

<footnote n="4"><sup>4</sup>Peter Gorer Department of Immunobiology, School of Immunology and Microbial Sciences, King's College London, London, United Kingdom</footnote>

<footnote n="5"><sup>5</sup>Children's Allergy Service, Evelina London, Guy's and St Thomas' Hospital, London, United Kingdom</footnote>

<footnote n="6"><sup>6</sup>Department of Allergy, La Paz University Hospital, IdiPAZ, Madrid, Spain</footnote>

<footnote n="7"><sup>7</sup>Sanquin Research, Dept Immunopathology, University of Amsterdam, Amsterdam, The Netherlands</footnote>

<footnote n="8"><sup>8</sup>Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands</footnote>

<footnote n="9"><sup>9</sup>Institute for Immunological Research, University of Cartagena, Cartagena de Indias, Colombia, Colombia</footnote>

<footnote n="10"><sup>10</sup>Department of Biosciences and Medical Biology, Paris Lodron University Salzburg, Salzburg, Austria</footnote>

<footnote n="11"><sup>11</sup>Department of Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria</footnote>

<footnote n="12"><sup>12</sup>Department of Medicine, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Sao Paulo, Brasil, Brazil</footnote>

<footnote n="13"><sup>13</sup>Ambulatorio di Allergologia, Clinica San Carlo, Paderno Dugnano, Italy</footnote>

<footnote n="14"><sup>14</sup>Klinik für Dermatologie und Allergologie, Kantonsspital St. Gallen, St. Gallen, Switzerland</footnote>

<footnote n="15"><sup>15</sup>Department of Dermatology, University Hospital Zurich, Zurich, Switzerland</footnote>

<footnote n="16"><sup>16</sup>Institute of Applied Molecular Medicine Nemesio Diez (IMMAND), Department of Basic Medical Sciences, Facultad de Medicina, Universidad San Pablo CEU, CEU Universities, Madrid, Spain</footnote>

<footnote n="17"><sup>17</sup>RETIC ARADYAL and RICORS Enfermedades Inflamatorias (REI), Madrid, Spain</footnote>

<footnote n="18"><sup>18</sup>Department of Dermatology and Allergy Biederstein, School of Medicine, Technical University Munich, Munich, Germany</footnote>

<footnote n="19"><sup>19</sup>Department of Clinical and Molecular Sciences, Università Politecnica delle Marche, Ancona, Italy</footnote>

<footnote n="20"><sup>20</sup>Allergy Unit Department of Internal Medicine, University Hospital Ospedali Riuniti di Ancona, Torrette, Italy</footnote>

<footnote n="21"><sup>21</sup>Center of Allergy and Environment (ZAUM), Technical University of Munich, School of Medicine and Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany</footnote>

<footnote n="22"><sup>22</sup>Department of Pathophysiology and Allergy Research, Medical University of Vienna, Vienna, Austria</footnote>

<footnote n="23"><sup>23</sup>Department of Allergy and Immunology, Children's Hospital at Westmead, Sydney Children's Hospitals Network, Sydney, New South Wales, Australia</footnote>

<footnote n="24"><sup>24</sup>Child and Adolescent Health, Faculty of Medicine and Health, University of Sydney, Sydney, New South Wales, Australia</footnote>

<footnote n="25"><sup>25</sup>Pediatric Allergy Unit, Department of Child and Adolescent, University Hospitals of Geneva, Geneva, Switzerland</footnote>

<footnote n="26"><sup>26</sup>Centro DH Allergologia e Immunologia Clinica ASST- MANTOVA (MN), Mantova, Italy</footnote>

<footnote n="27"><sup>27</sup>InBio, Charlottesville, Virginia, USA</footnote>

<footnote n="28"><sup>28</sup>Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina, USA</footnote>

<footnote n="29"><sup>29</sup>National Heart and Lung Institute, Imperial College London, London, UK</footnote>

<footnote n="30"><sup>30</sup>Faculty of Science, Technology and Medicine, University of Luxembourg, Esch-sur-Alzette, Luxembourg</footnote>

<footnote n="31"><sup>31</sup>Queensland University of Technology, Centre for Immunology and Infection Control, School of Biomedical Sciences, Herston, Queensland, Australia</footnote>

<footnote n="32"><sup>32</sup>Metro North Hospital and Health Service, Emergency Operations Centre, Herston, Queensland, Australia</footnote>

<footnote n="33"><sup>33</sup>Allergy Department, 2nd Paediatric Clinic, National and Kapodistrian University of Athens, Athens, Greece</footnote>

<footnote n="34"><sup>34</sup>Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara National Hospital, Kanagawa, Japan</footnote>

<footnote n="35"><sup>35</sup>Chemical Biology and Drug Discovery, Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="36"><sup>36</sup>Center for Translational Immunology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands</footnote>

<footnote n="37"><sup>37</sup>Allergy Unit, Department of Pediatrics, Meyer Children's University Hospital, Florence, Italy</footnote>

<footnote n="38"><sup>38</sup>Allergy Unit IBIMA-Hospital Regional Universitario de Malaga, Malaga, Spain</footnote>

<footnote n="39"><sup>39</sup>Spanish Network for Allergy research RETIC ARADYAL, Malaga, Spain</footnote>

<footnote n="40"><sup>40</sup>NYU Langone Health, Department of Internal Medicine, New York, New York, USA</footnote>

<footnote n="41"><sup>41</sup>Faculty of Medicine, University of Zurich, Zurich, Switzerland</footnote>

<footnote n="42"><sup>42</sup>Department of Dermatology, University Hospital St. Poelten, Karl Landsteiner University of Health Sciences, St. Poelten, Austria</footnote>

<footnote n="43"><sup>43</sup>Johns Hopkins University School of Medicine, Baltimore, Maryland, USA</footnote>

<footnote n="44"><sup>44</sup>Department of Dermatology and Allergology, Paracelsus Private Medical University, Salzburg, Austria</footnote>

<footnote n="45"><sup>45</sup>Institute for Clinical Medicine, Faculty of Health, Aarhus University, Aarhus, Denmark</footnote>
⁴⁶Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark

⁴⁷Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany

⁴⁸Laboratory of Protein Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan

⁴⁹Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

⁵⁰Department of Dermatology and Allergology, University Medical Center, Justus Liebig University Gießen, Gießen, Germany

⁵¹Odense Research Center for Anaphylaxis, University of Southern Denmark, Odense, Denmark

⁵²Division of Clinical and Molecular Allergology, Priority Research Area Asthma and Allergy, Research Center Borstel, Borstel, Germany

⁵³Leibniz Lung Center, Airway Research Center North (ARCN), Member of the German Center for Lung Research, Germany

⁵⁴Interdisciplinary Allergy Outpatient Clinic, Dept. of Pneumology, University of Lübeck, Lübeck, Germany

⁵⁵Department of Clinical Immunology, Wroclaw Medical University, Wroclaw, Poland

⁵⁶Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, Queensland, Australia

⁵⁷Molecular Allergy Research Laboratory, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Queensland, Australia

⁵⁸Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr- Universität Bochum, Bochum, Germany

⁵⁹Allergy & Asthma Center Westend, Outpatient Clinic and Clinical Research Center, Berlin, Germany

⁶⁰Department of Immunology and Dermatology/ Allergology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

⁶¹Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden

⁶²Pediatric Allergy and Pulmonology Unit at Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden

⁶³University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia

⁶⁴Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

⁶⁵Allergy Section, Internal Medicine Department, Vall d'Hebron University Hospital, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain

⁶⁶Division of Allergy, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

⁶⁷Pediatric Department, Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland

⁶⁸ASST Rhodense, Ospedale di Garbagnate Milanese, Milan, Italy

⁶⁹Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK

⁷⁰Allergy Unit, Angers University Hospital, Angers, France

⁷¹Food Allergy Referral Centre, Department of Woman and Child Health, Padua University Hospital, Padua, Italy

⁷²Division of Pediatric Allergy and Immunology, NYU Grossman School of Medicine, Hassenfeld Children's Hospital, New York, New York, USA

⁷³Department of Pediatrics, Gastroenterology and Nutrition, Collegium Medicum, University of Warmia and Mazury, Olsztyn, Poland

⁷⁴Department of Aquatic Product Technology, Faculty of Fisheries and Marine Science, IPB University, Bogor, Indonesia

⁷⁵Department of Allergology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

⁷⁶Centro di Allergologia, Casa di Cura Ambrosiana Cesano Boscone, Milan, Italy

⁷⁷Division of Rheumatology, Allergy and Immunology, Departments of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

⁷⁸Division of Allergy and Immunology, Department of Pediatrics, Massachusetts General Hospital, Boston, Massachusetts, USA

⁷⁹Division of Allergy and Clinical Immunology, University of Virginia, Charlottesville, Virginia, USA

⁸⁰Institut Pasteur, Immunology Department, Paris, France

⁸¹Allergy & Environment Research Team Armand Trousseau Children Hospital, APHP, Paris, France

⁸²Allergy Clinic, Department of Dermatology and Allergy, Copenhagen University Hospital-Herlev and Gentofte, Copenhagen, Denmark

⁸³UMR 152 PharmaDev, IRD, Université Paul Sabatier, Faculté de Pharmacie, Toulouse, France

⁸⁴Allergy Service, Fundación Jiménez Díaz; CIBER de Enfermedades Respiratorias (CIBERES); Faculty of Medicine, Universidad Autonoma de Madrid, Madrid, Spain

⁸⁵Clinical and Laboratory Molecular Allergy Unit - IDI-IRCCS, Fondazione L M Monti Rome, Rome, Italy

⁸⁶Christine Kühne Center for Allergy Research and Education CK-CARE, Davos, Switzerland

⁸⁷Centre of Marine Sciences (CCMAR), Universidade do Algarve, Faro, Portugal

⁸⁸Department of Environmental Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany

⁸⁹Department of Medicine Solna, Division of Immunology and Allergy, Karolinska Institutet, Stockholm, Sweden

⁹⁰Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden

⁹¹Department of Experimental Immunology and Department of Otorhinolaryngology, Amsterdam University Medical Centers, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

⁹²Department of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
⁴⁶Department of Respiratory Diseases and Allergy, Aarhus University Hospital, Aarhus, Denmark

⁴⁷Division of Allergology, Paul-Ehrlich-Institut, Langen, Germany

⁴⁸Laboratory of Protein Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan

⁴⁹Department of Biochemistry, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand

⁵⁰Department of Dermatology and Allergology, University Medical Center, Justus Liebig University Gießen, Gießen, Germany

⁵¹Odense Research Center for Anaphylaxis, University of Southern Denmark, Odense, Denmark

⁵²Division of Clinical and Molecular Allergology, Priority Research Area Asthma and Allergy, Research Center Borstel, Borstel, Germany

⁵³Leibniz Lung Center, Airway Research Center North (ARCN), Member of the German Center for Lung Research, Germany

⁵⁴Interdisciplinary Allergy Outpatient Clinic, Dept. of Pneumology, University of Lübeck, Lübeck, Germany

⁵⁵Department of Clinical Immunology, Wroclaw Medical University, Wroclaw, Poland

⁵⁶Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, Queensland, Australia

⁵⁷Molecular Allergy Research Laboratory, College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, Queensland, Australia

⁵⁸Institute for Prevention and Occupational Medicine of the German Social Accident Insurance, Institute of the Ruhr- Universität Bochum, Bochum, Germany

⁵⁹Allergy & Asthma Center Westend, Outpatient Clinic and Clinical Research Center, Berlin, Germany

⁶⁰Department of Immunology and Dermatology/ Allergology, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

⁶¹Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden

⁶²Pediatric Allergy and Pulmonology Unit at Astrid Lindgren Children's Hospital, Karolinska University Hospital, Stockholm, Sweden

⁶³University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia

⁶⁴Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

⁶⁵Allergy Section, Internal Medicine Department, Vall d'Hebron University Hospital, Vall d'Hebron Research Institute (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain

⁶⁶Division of Allergy, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

⁶⁷Pediatric Department, Skin and Allergy Hospital, Helsinki University Central Hospital, Helsinki, Finland

⁶⁸ASST Rhodense, Ospedale di Garbagnate Milanese, Milan, Italy

⁶⁹Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK

⁷⁰Allergy Unit, Angers University Hospital, Angers, France

⁷¹Food Allergy Referral Centre, Department of Woman and Child Health, Padua University Hospital, Padua, Italy

⁷²Division of Pediatric Allergy and Immunology, NYU Grossman School of Medicine, Hassenfeld Children's Hospital, New York, New York, USA

⁷³Department of Pediatrics, Gastroenterology and Nutrition, Collegium Medicum, University of Warmia and Mazury, Olsztyn, Poland

⁷⁴Department of Aquatic Product Technology, Faculty of Fisheries and Marine Science, IPB University, Bogor, Indonesia

⁷⁵Department of Allergology, Helsinki University Hospital and University of Helsinki, Helsinki, Finland

⁷⁶Centro di Allergologia, Casa di Cura Ambrosiana Cesano Boscone, Milan, Italy

⁷⁷Division of Rheumatology, Allergy and Immunology, Departments of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA

⁷⁸Division of Allergy and Immunology, Department of Pediatrics, Massachusetts General Hospital, Boston, Massachusetts, USA

⁷⁹Division of Allergy and Clinical Immunology, University of Virginia, Charlottesville, Virginia, USA

⁸⁰Institut Pasteur, Immunology Department, Paris, France

⁸¹Allergy & Environment Research Team Armand Trousseau Children Hospital, APHP, Paris, France

⁸²Allergy Clinic, Department of Dermatology and Allergy, Copenhagen University Hospital-Herlev and Gentofte, Copenhagen, Denmark

⁸³UMR 152 PharmaDev, IRD, Université Paul Sabatier, Faculté de Pharmacie, Toulouse, France

⁸⁴Allergy Service, Fundación Jiménez Díaz; CIBER de Enfermedades Respiratorias (CIBERES); Faculty of Medicine, Universidad Autonoma de Madrid, Madrid, Spain

⁸⁵Clinical and Laboratory Molecular Allergy Unit - IDI-IRCCS, Fondazione L M Monti Rome, Rome, Italy

⁸⁶Christine Kühne Center for Allergy Research and Education CK-CARE, Davos, Switzerland

⁸⁷Centre of Marine Sciences (CCMAR), Universidade do Algarve, Faro, Portugal

⁸⁸Department of Environmental Medicine, Faculty of Medicine, University of Augsburg, Augsburg, Germany

⁸⁹Department of Medicine Solna, Division of Immunology and Allergy, Karolinska Institutet, Stockholm, Sweden

⁹⁰Department of Clinical Immunology and Transfusion Medicine, Karolinska University Hospital, Stockholm, Sweden

⁹¹Department of Experimental Immunology and Department of Otorhinolaryngology, Amsterdam University Medical Centers, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

⁹²Department of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden
**Correspondence**

Karin Hoffmann-Sommergruber,
Department of Pathophysiology and
Allergy Research, Medical University of
Vienna, Vienna, Austria.
Email: karin.hoffmann@meduniwien.ac.at

Editor: Ömer KALAYCI

**Abstract**

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

**KEYWORDS**

allergy, allergy diagnosis, anaphylaxis, asthma, atopic dermatitis, basophil activation test, component-resolved diagnosis, cross-reactive carbohydrates, diagnostic algorithms, food allergy, gibberellin-regulated proteins, pollen allergy, IgE, IgE cross-reactivity, lipocalins, microarray, molecular allergology, non-specific lipid transfer proteins, oleosins, pan-allergens, parvalbumins, pathogenesis-related protein family 10, precision medicine, polcalcins, profilins, seed storage proteins, serum albumins, tropomyosins
**Correspondence**

Karin Hoffmann-Sommergruber,
Department of Pathophysiology and
Allergy Research, Medical University of
Vienna, Vienna, Austria.
Email: karin.hoffmann@meduniwien.ac.at

Editor: Ömer KALAYCI

**Abstract**

Since the discovery of immunoglobulin E (IgE) as a mediator of allergic diseases in 1967, our knowledge about the immunological mechanisms of IgE-mediated allergies has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of the allergic disease for individual patients. The first edition of the "EAACI Molecular Allergology User's Guide" (MAUG) in 2016 rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine. Nevertheless, the field of molecular allergology is moving fast, and after 6 years, a new EAACI Taskforce was established to provide an updated document. The Molecular Allergology User's Guide 2.0 summarizes state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care professionals through the overwhelming list of different allergen molecules available for testing. Further, it provides diagnostic algorithms on the clinical relevance of allergenic molecules and gives an overview of their biology, the basic mechanisms of test formats, and the application of tests to measure allergen exposure.

**KEYWORDS**

allergy, allergy diagnosis, anaphylaxis, asthma, atopic dermatitis, basophil activation test, component-resolved diagnosis, cross-reactive carbohydrates, diagnostic algorithms, food allergy, gibberellin-regulated proteins, pollen allergy, IgE, IgE cross-reactivity, lipocalins, microarray, molecular allergology, non-specific lipid transfer proteins, oleosins, pan-allergens, parvalbumins, pathogenesis-related protein family 10, precision medicine, polcalcins, profilins, seed storage proteins, serum albumins, tropomyosins
## Preface from the EAACI President

Allergies are on the rise with significant burden both on affected individuals and on the societies. Despite representing a major public health issue and constant public awareness campaigns, allergic diseases are still under-recognized.

There is a need to (a) improve the care of allergic patients, (b) provide up-to-date education of healthcare professionals, (c) increase awareness of the public, and (d) raise a voice for patients. All these goals are in line with the Mission of the **European Academy of Allergy and Immunology - EAACI**. The translation of best knowledge into best practice is one of the key aims of EAACI. Atlases, Guidelines, and books published by EAACI represent a reference for physicians, scientific and health care organizations, as well as health policy makers, and are highly cited.

**The Molecular Allergology User's Guide**, published in 2016, was a good example of a well-received overview on new technologies that facilitated **Molecular Allergology** and took allergy diagnostics and patient care to the next level.

However, since 2016, a lot of new evidence has been accumulated, and new allergen sources and allergenic molecules have been identified. Diagnostic methods have subsequently been developed and adopted. Cohort studies were performed documenting polymolecular sensitization patterns and methods were developed to help assessing the risk of allergen exposure. With such methods, detailed studies on allergen-specific immune responses leading to an allergic reaction became feasible. Also, new findings on allergen families and their relevance for immune responses told us to challenge the previously developed diagnostic algorithms.

Therefore, EAACI identified the need for an updated version and a dedicated Task Force on **"The Molecular Allergology User's Guide 2.0"** was set by the EAACI Executive Committee in 2021.

This undertaking could not have been accomplished without dedication, commitment, and the spirit of team work in close interaction between clinicians and scientists with the support of the EAACI leadership and the EAACI Family. Within this Task Force, 100 contributors provided their expertise and data to get the work finalized.

I would like to express special thanks go to the **Editorial team**, Karin Hoffmann-Sommergruber, Christiane Hilger, Stephanie Dramburg, Alexandra Santos, Leticia de las Villas, and all the **Authors**, both the ones that were part of the first edition and the "newcomers" for the current edition. Among all contributors the support from Paolo Matricardi, who was the driving force behind the first edition and also actively engaged in the second edition, needs to be specifically mentioned.

**This Molecular Allergology User's Guide 2.0** provides state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care

professionals through the overwhelming list of different allergen molecules identified available for testing. Further, it provides diagnostic algorithms based on current knowledge about the clinical relevance of allergen molecules and gives an overview on the basic mechanisms of test formats, the biology of allergen molecules and the application of tests to measure allergen exposure.

It is with pride that I endorse this EAACI Publication, hoping that it will serve as the very useful resource to the whole Allergy community.

### Marek Jutel

President of the European Academy of Allergy and Clinical Immunology-EAACI

## Preface from Dr. Paolo Matricardi, initiator of the first EAACI Task Force leading to the production of the first edition of the "Molecular Allergology User's Guide"

In its six years (since 2016), the first edition of this book has received enormous attention from researchers, allergists, doctors, and other readers. Over 400 citations in ISI and over 700 in Google-Scholar demonstrate how urgent the need of systematic information on allergen molecules is as a basis to develop clinical and biological research and improve the diagnostics and therapy of the allergic patient in routine clinical practice. Thanks to the EAACI Molecular Allergology User's Guide, the concepts, technologies, and algorithms published years ago have been more and more integrated into clinical practice of allergists all over the world.

Nevertheless, the field of Molecular Allergology is moving fast and the first edition of the book was quickly becoming "old." New molecules have been discovered, characterized, cloned and their clinical relevance demonstrated. Under the pressure of clinical questions and readers' comments, new chapters have been added, such as the one on Cross-reactive Carbohydrate Determinants (CCDs), edible insects, and new types of cross-reactive molecules (e.g., gibberellin-regulated proteins, oleosins). In addition, the growth of the team and inclusion of new and younger authors, testifies that Molecular Allergology is "young" and future-oriented.

I welcome therefore the great effort of the new Editors of the book, who prepared this second Edition in a short "record" time. I am especially grateful to Dr. Stephanie Dramburg and Dr. Christiane Hilger, who played a central role already in the preparation of the first edition and represent the continuity of the original spirit of the editorial team for a coherent update and progression of the text.

I am also very thankful to EAACI for the continuous support and particularly to the Vice President Communications and Membership, Prof. Dr. Karin Hoffmann-Sommergruber, who not
## Preface from the EAACI President

Allergies are on the rise with significant burden both on affected individuals and on the societies. Despite representing a major public health issue and constant public awareness campaigns, allergic diseases are still under-recognized.

There is a need to (a) improve the care of allergic patients, (b) provide up-to-date education of healthcare professionals, (c) increase awareness of the public, and (d) raise a voice for patients. All these goals are in line with the Mission of the **European Academy of Allergy and Immunology – EAACI**. The translation of best knowledge into best practice is one of the key aims of EAACI. Atlases, Guidelines, and books published by EAACI represent a reference for physicians, scientific and health care organizations, as well as health policy makers, and are highly cited.

**The Molecular Allergology User's Guide**, published in 2016, was a good example of a well-received overview on new technologies that facilitated **Molecular Allergology** and took allergy diagnostics and patient care to the next level.

However, since 2016, a lot of new evidence has been accumulated, and new allergen sources and allergenic molecules have been identified. Diagnostic methods have subsequently been developed and adopted. Cohort studies were performed documenting polymolecular sensitization patterns and methods were developed to help assessing the risk of allergen exposure. With such methods, detailed studies on allergen-specific immune responses leading to an allergic reaction became feasible. Also, new findings on allergen families and their relevance for immune responses told us to challenge the previously developed diagnostic algorithms.

Therefore, EAACI identified the need for an updated version and a dedicated Task Force on **"The Molecular Allergology User's Guide 2.0"** was set by the EAACI Executive Committee in 2021.

This undertaking could not have been accomplished without dedication, commitment, and the spirit of team work in close interaction between clinicians and scientists with the support of the EAACI leadership and the EAACI Family. Within this Task Force, 100 contributors provided their expertise and data to get the work finalized.

I would like to express special thanks go to the **Editorial team**, Karin Hoffmann-Sommergruber, Christiane Hilger, Stephanie Dramburg, Alexandra Santos, Leticia de las Villas, and all the **Authors**, both the ones that were part of the first edition and the "newcomers" for the current edition. Among all contributors the support from Paolo Matricardi, who was the driving force behind the first edition and also actively engaged in the second edition, needs to be specifically mentioned.

**This Molecular Allergology User's Guide 2.0** provides state-of-the-art information on allergen molecules, their clinical relevance, and their application in diagnostic algorithms for clinical practice. It is designed for both, clinicians and scientists, guiding health care

professionals through the overwhelming list of different allergen molecules identified available for testing. Further, it provides diagnostic algorithms based on current knowledge about the clinical relevance of allergen molecules and gives an overview on the basic mechanisms of test formats, the biology of allergen molecules and the application of tests to measure allergen exposure.

It is with pride that I endorse this EAACI Publication, hoping that it will serve as the very useful resource to the whole Allergy community.

Marek Jutel

President of the European Academy of Allergy and Clinical Immunology-EAACI

## Preface from Dr. Paolo Matricardi, initiator of the first EAACI Task Force leading to the production of the first edition of the "Molecular Allergology User's Guide"

In its six years (since 2016), the first edition of this book has received enormous attention from researchers, allergists, doctors, and other readers. Over 400 citations in ISI and over 700 in Google-Scholar demonstrate how urgent the need of systematic information on allergen molecules is as a basis to develop clinical and biological research and improve the diagnostics and therapy of the allergic patient in routine clinical practice. Thanks to the EAACI Molecular Allergology User's Guide, the concepts, technologies, and algorithms published years ago have been more and more integrated into clinical practice of allergists all over the world.

Nevertheless, the field of Molecular Allergology is moving fast and the first edition of the book was quickly becoming "old." New molecules have been discovered, characterized, cloned and their clinical relevance demonstrated. Under the pressure of clinical questions and readers' comments, new chapters have been added, such as the one on Cross-reactive Carbohydrate Determinants (CCDs), edible insects, and new types of cross-reactive molecules (e.g., gibberellin-regulated proteins, oleosins). In addition, the growth of the team and inclusion of new and younger authors, testifies that Molecular Allergology is "young" and future-oriented.

I welcome therefore the great effort of the new Editors of the book, who prepared this second Edition in a short "record" time. I am especially grateful to Dr. Stephanie Dramburg and Dr. Christiane Hilger, who played a central role already in the preparation of the first edition and represent the continuity of the original spirit of the editorial team for a coherent update and progression of the text.

I am also very thankful to EAACI for the continuous support and particularly to the Vice President Communications and Membership, Prof. Dr. Karin Hoffmann-Sommergruber, who not
only directed the Editorial Team of this Second Edition but also guaranteed a continuous link between the authors and the EAACI Leadership.

The seeds that over 60 experts planted, together with my co-editors Markus Ollert, Jörg Kleine-Tebbe, Hans Jürgen Hoffmann and Rudolf Valenta, in 2016, flourished and produced a beautifully

growing plant. Given the trend towards precision medicine, including “precision allergology,” it is easy to predict the success of this second edition and to foresee a third edition before 2030!

Paolo Maria Matricardi - Berlin, 25 May 2022
only directed the Editorial Team of this Second Edition but also guaranteed a continuous link between the authors and the EAACI Leadership.

The seeds that over 60 experts planted, together with my co-editors Markus Ollert, Jörg Kleine-Tebbe, Hans Jürgen Hoffmann and Rudolf Valenta, in 2016, flourished and produced a beautifully

growing plant. Given the trend towards precision medicine, including “precision allergology,” it is easy to predict the success of this second edition and to foresee a third edition before 2030!

Paolo Maria Matricardi - Berlin, 25 May 2022
# A01 - Molecular allergology coming of age—an introduction to the second edition of the EAACI User's Guide (MAUG 2.0)

Since the discovery of immunoglobulin E (IgE) as mediator of allergic diseases in 1967,¹ our knowledge about the immunological mechanisms of IgE-mediated allergic diseases has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. Currently, 1080 molecules from different animals, fungi, and plants have been identified as allergens and are listed in the IUIS/WHO database.²,³

In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of allergic diseases for individual patients. In addition to individual molecules, chip-based test systems have evolved, enabling the simultaneous detection of specific IgE antibodies directed towards more than 100 allergenic molecules within one test run.

The identification of IgE antibodies to specific molecules can, not only improve diagnosis but also have additional applications, such as the risk assessment of sensitized individuals, monitoring of environmental exposure, and detection of specific allergens in foods. Novel allergens are constantly being described and new allergen sources, such as edible insects, for example. The role of immunoactive substances, such as ligands, can contribute to an accurate diagnosis. This ever-changing multitude of new developments and research requires updated literature and shared perspectives from experts in the Molecular Allergology field.

The First Edition of the “EAACI Molecular Allergology User’s Guide” (MAUG) was launched in 2016 and rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine, more broadly. From the beginning, the content has been freely available to the community as an e-book at www.eaaci.org and as a supplement of the journal Pediatric Allergy and Immunology, where it quickly became one of the most cited articles.⁴ As science and knowledge keep evolving rapidly, the editors and authors of the first book agreed on the need of an updated edition. A team of five colleagues (Karin Hoffmann-Sommergruber, Christiane Hilger, Stephanie Dramburg (formerly Hofmaier), Alexandra Santos, and Leticia de las Vecillas) came together to coordinate the process and applied for an EAACI Task Force in order to secure the indispensable support of the EAACI family. This new editorial team, led by Karin Hoffmann-Sommergruber, benefited from the support from Paolo Matricardi, chair of the first task force on CRD in Allergology, both as an advisor and author of several chapters of MAUG 2.0. All authors of the first edition were invited to actively

contribute to the update and expert junior members were also included as authors for most chapters. The new edition of the “EAACI Molecular Allergology User’s Guide” (MAUG 2.0) kept the basic structure of the first book with three sections focussing on: general aspects (Section A: General aspects), specific allergens/ allergies (Section B: Molecular Allergology In clinical practice), and cross-reactive allergens (Section C: Cross-reactive molecules and their clinical relevance).

## A: Molecular allergology: General concepts

Section A combines chapters on basic and general aspects of molecular allergology. This includes important information on allergens, their sources and superordinate families (Chapters A02, A07, A08), the role of molecular IgE testing in clinical practice (Chapter A03), methodological aspects of singleplex vs. multiplex testing (Chapter A04), and the role of allergenic molecules within *in vivo* diagnostics (Chapter A06) and basophil activation tests (Chapter 5-A05). New chapters discuss the role of molecular allergology for allergen immunotherapy (AIT) (Chapter A09) and explain the importance of cross-reactive carbohydrate determinants (Chapter A10) for the correct interpretation of test results. A chapter sets a focus on small molecules and introduces “harmless” molecules, such as lipids, glycosylated flavonoids (and derivatives), steroids, fatty acids, or cytokinins as potential modifiers of the innate and adaptive immune response towards allergens A11. Finally, section A concludes with a comprehensive overview on molecular allergen exposure, sampling, and testing devices, and how our knowledge contributes to improved allergenic risk assessment A12.

## B: Molecular allergology in clinical practice

Section B offers updated information regarding specific allergies with a clinical focus. This includes not only new information in the previously established chapters but also completely new contents, such as Chapter B09 on the role of edible insects. Within existing chapters, new insights are reported, for example, a larger section on the alpha-gal syndrome and a diagnostic algorithm for different types of meat allergy in Chapter B14, acknowledging the novelty that carbohydrate epitopes recognized by IgE antibodies are now included in the WHO/IUIS allergen nomenclature database. Another innovative chapter provides guidance on the diagnosis and management of allergies to moulds (Chapter B07), including comprehensive information on a large set of allergenic molecules. Further examples of enriched chapters are the integration of *Anisakis* simplex in Chapter B12 on fish allergy and new content on buckwheat allergy within Chapter B16 (Wheat and Buckwheat Allergy). These innovations are only a small foretaste of the new content in 22 specific chapters.
# A01 - Molecular allergology coming of age—an introduction to the second edition of the EAACI User's Guide (MAUG 2.0)

Since the discovery of immunoglobulin E (IgE) as mediator of allergic diseases in 1967,¹ our knowledge about the immunological mechanisms of IgE-mediated allergic diseases has remarkably increased. In addition to understanding the immune response and clinical symptoms, allergy diagnosis and management depend strongly on the precise identification of the elicitors of the IgE-mediated allergic reaction. Currently, 1080 molecules from different animals, fungi, and plants have been identified as allergens and are listed in the IUIS/WHO database.²,³

In the past four decades, innovations in bioscience and technology have facilitated the identification and production of well-defined, highly pure molecules for component-resolved diagnosis (CRD), allowing a personalized diagnosis and management of allergic diseases for individual patients. In addition to individual molecules, chip-based test systems have evolved, enabling the simultaneous detection of specific IgE antibodies directed towards more than 100 allergenic molecules within one test run.

The identification of IgE antibodies to specific molecules can, not only improve diagnosis but also have additional applications, such as the risk assessment of sensitized individuals, monitoring of environmental exposure, and detection of specific allergens in foods. Novel allergens are constantly being described and new allergen sources, such as edible insects, for example. The role of immunoactive substances, such as ligands, can contribute to an accurate diagnosis. This ever-changing multitude of new developments and research requires updated literature and shared perspectives from experts in the Molecular Allergology field.

The First Edition of the “EAACI Molecular Allergology User’s Guide” (MAUG) was launched in 2016 and rapidly became a key reference for clinicians, scientists, and interested readers with a background in allergology, immunology, biology, and medicine, more broadly. From the beginning, the content has been freely available to the community as an e-book at www.eaaci.org and as a supplement of the journal Pediatric Allergy and Immunology, where it quickly became one of the most cited articles.⁴ As science and knowledge keep evolving rapidly, the editors and authors of the first book agreed on the need of an updated edition. A team of five colleagues (Karin Hoffmann-Sommergruber, Christiane Hilger, Stephanie Dramburg (formerly Hofmaier), Alexandra Santos, and Leticia de las Vecillas) came together to coordinate the process and applied for an EAACI Task Force in order to secure the indispensable support of the EAACI family. This new editorial team, led by Karin Hoffmann-Sommergruber, benefited from the support from Paolo Matricardi, chair of the first task force on CRD in Allergology, both as an advisor and author of several chapters of MAUG 2.0. All authors of the first edition were invited to actively

contribute to the update and expert junior members were also included as authors for most chapters. The new edition of the “EAACI Molecular Allergology User’s Guide” (MAUG 2.0) kept the basic structure of the first book with three sections focussing on: general aspects (Section A: General aspects), specific allergens/ allergies (Section B: Molecular Allergology In clinical practice), and cross-reactive allergens (Section C: Cross-reactive molecules and their clinical relevance).

## A: Molecular allergology: General concepts

Section A combines chapters on basic and general aspects of molecular allergology. This includes important information on allergens, their sources and superordinate families (Chapters A02, A07, A08), the role of molecular IgE testing in clinical practice (Chapter A03), methodological aspects of singleplex vs. multiplex testing (Chapter A04), and the role of allergenic molecules within *in vivo* diagnostics (Chapter A06) and basophil activation tests (Chapter 5-A05). New chapters discuss the role of molecular allergology for allergen immunotherapy (AIT) (Chapter A09) and explain the importance of cross-reactive carbohydrate determinants (Chapter A10) for the correct interpretation of test results. A chapter sets a focus on small molecules and introduces “harmless” molecules, such as lipids, glycosylated flavonoids (and derivatives), steroids, fatty acids, or cytokinins as potential modifiers of the innate and adaptive immune response towards allergens A11. Finally, section A concludes with a comprehensive overview on molecular allergen exposure, sampling, and testing devices, and how our knowledge contributes to improved allergenic risk assessment A12.

## B: Molecular allergology in clinical practice

Section B offers updated information regarding specific allergies with a clinical focus. This includes not only new information in the previously established chapters but also completely new contents, such as Chapter B09 on the role of edible insects. Within existing chapters, new insights are reported, for example, a larger section on the alpha-gal syndrome and a diagnostic algorithm for different types of meat allergy in Chapter B14, acknowledging the novelty that carbohydrate epitopes recognized by IgE antibodies are now included in the WHO/IUIS allergen nomenclature database. Another innovative chapter provides guidance on the diagnosis and management of allergies to moulds (Chapter B07), including comprehensive information on a large set of allergenic molecules. Further examples of enriched chapters are the integration of *Anisakis* simplex in Chapter B12 on fish allergy and new content on buckwheat allergy within Chapter B16 (Wheat and Buckwheat Allergy). These innovations are only a small foretaste of the new content in 22 specific chapters.
C: Cross-reactive molecules

Section C summarizes significant knowledge and latest findings on cross-reactive allergens. While updates are available for profiles (Chapter C01), PR-10 proteins (Chapter C02), non-specific lipid transfer proteins (Chapter C03) (nsLTP), serum albumins (Chapter C04), tropomyosins (Chapter C05), polcalcins (Chapter C06), lipocalins (Chapter C07), and parvalbumins (Chapter C11), new chapters have been integrated on the role of seed storage proteins (Chapter C08), gibberellin-regulated proteins (GRPs) (Chapter C09), and oleosins (Chapter C10). All in all, 45 chapters have been updated or newly written and carefully reviewed by a 100 authors from four contaminants—a truly international team effort. What started with a first online kick-off meeting in April 2021, evolved over several consensus and production stages including regular meetings of the editors and authors up to weekly meetings with the design team during the final phase, resulting in the launch of a comprehensive update on Molecular Allergology: MAUG 2.0 at the EAACI Annual Congress 2022 in Prague.

The editors would like to thank all the authors and contributors to MAUG 2.0 for their continuous efforts, professional contributions,

and team spirit. The editors would like to thank Marcela Ataíde and Olivia Matni for their outstanding artwork in illustrating and type-setting the book, as well as Hoang Yen Do for her excellent work on the references. Finally, the editors are grateful to the EAACI for supporting the initiative and large collaboration of almost 100 experts that allowed updating this valuable resource, the 2nd edition of the Molecular Allergology User's Guide.

We hope the readers enjoy this updated edition and that it proves useful in both clinical practice and continued research on Molecular Allergology!

We thank from the bottom of our hearts our esteemed colleague and co-author Prof. Dr. med. Jörg Kleine-Tebbe, who passed away on 5.1.2023. He contributed significantly to the creation of both editions of the EAACI Molecular Allergology User's Guide and made an extraordinary contribution to the advancement of molecular allergology.

Stephanie Dramburg, Karin Hoffmann-Sommergruber, Christiane Hilger, Alexandra F. Santos, Leticia de las Vecillas.
C: Cross-reactive molecules

Section C summarizes significant knowledge and latest findings on cross-reactive allergens. While updates are available for profiles (Chapter C01), PR-10 proteins (Chapter C02), non-specific lipid transfer proteins (Chapter C03) (nsLTP), serum albumins (Chapter C04), tropomyosins (Chapter C05), polcalcins (Chapter C06), lipocalins (Chapter C07), and parvalbumins (Chapter C11), new chapters have been integrated on the role of seed storage proteins (Chapter C08), gibberellin-regulated proteins (GRPs) (Chapter C09), and oleosins (Chapter C10). All in all, 45 chapters have been updated or newly written and carefully reviewed by a 100 authors from four contaminants—a truly international team effort. What started with a first online kick-off meeting in April 2021, evolved over several consensus and production stages including regular meetings of the editors and authors up to weekly meetings with the design team during the final phase, resulting in the launch of a comprehensive update on Molecular Allergology: MAUG 2.0 at the EAACI Annual Congress 2022 in Prague.

The editors would like to thank all the authors and contributors to MAUG 2.0 for their continuous efforts, professional contributions,

and team spirit. The editors would like to thank Marcela Ataíde and Olivia Matni for their outstanding artwork in illustrating and type-setting the book, as well as Hoang Yen Do for her excellent work on the references. Finally, the editors are grateful to the EAACI for supporting the initiative and large collaboration of almost 100 experts that allowed updating this valuable resource, the 2nd edition of the Molecular Allergology User's Guide.

We hope the readers enjoy this updated edition and that it proves useful in both clinical practice and continued research on Molecular Allergology!

We thank from the bottom of our hearts our esteemed colleague and co-author Prof. Dr. med. Jörg Kleine-Tebbe, who passed away on 5.1.2023. He contributed significantly to the creation of both editions of the EAACI Molecular Allergology User's Guide and made an extraordinary contribution to the advancement of molecular allergology.

Stephanie Dramburg, Karin Hoffmann-Sommergruber, Christiane Hilger, Alexandra F. Santos, Leticia de las Vecillas.
# A02 - Allergens and the allergenic composition of source materials

Ronald van Ree, Rob C. Aalberse

## Highlights

* Many different types of proteins are allergenic.

* The context of a protein may be a major determinant for its allergenicity.

* Some IgE inducers are not really allergens at all because they do not induce symptoms. This negatively impacts specificity of diagnostic tests, certainly of extract-based tests but also still of molecular tests.

* Allergen extracts are imperfect but not yet obsolete.

* Molecular sensitization profiles are potential biomarkers for disease phenotypes and progression.

* The initial response to an allergen source is possibly characterized by IgE antibodies to one or two "initiator" allergens.

Allergenic sources can vary from biologic sources with very complex composition such as pollen, house dust mites (inhalant allergy), or foods (food allergy), to single molecules such as chemicals (occupational allergy) or drugs (drug allergy). In this chapter, we will focus on the molecular composition of more complex biologic allergen sources that are implicated in causing hay fever, allergic asthma, and food allergy. Around the late sixties and early seventies of last century, first reports were published in which individual molecules were identified that were responsible for binding IgE within different allergen sources such as grass pollen,⁵ ragweed, pollen,⁶ cod fish allergen,⁷ and house dust mite.⁸ By now probably the most important allergens of the most relevant allergen sources have been identified (www.allergen.org; www.comparedatabase.org; www.allergenonline.org; www.allergome.org). Before we can discuss the allergens, we have to introduce the nomenclature of molecularly defined allergens (see Textbox 1).

## 1 - What is an allergen?

The broadest definition of an allergen is that it is any molecule-binding IgE antibodies.¹³ Allergens can differ in several ways (see also Textboxes 2 and 3). In this introduction, the practical consequences of four of these differences will be discussed.

## 2 - Sensitizing versus nonsensitizing allergens

Most, but not all, allergens are sensitizing, which is defined as the ability to induce allergen-specific IgE antibodies. Nonsensitizing allergens can only cause allergic symptoms if previous contact with a related (cross-reactive) allergen has caused sensitization. A prototypic example of a sensitizer is birch allergen Bet v 1 and

### Textbox 1 Allergen nomenclature

Allergen names are based on the scientific (Latin) name of the plant or animal species from which the allergen originates.⁹,¹⁰ For example, the major allergen from birch pollen Bet v 1 is named after the scientific name of the tree Betula verrucosa, in which Betula is the genus and verrucosa the species. The first three letters of the genus (Bet) and the first letter of the species (v) together form the basis of the allergen name, followed by a number. In principle the number is given in order of discovery, so Bet v 1 was the first allergen from birch pollen that was discovered. Related (often cross-reactive) allergens from different species, genus, family or even order, get the same number, if still available. So, the homologue of Bet v 1 in hazel is Cor a 1 and in apple is Mal d 1 but in peanut is Ara h 8 because numbers 1–7 were already occupied by peanut allergens described earlier. Many allergens have molecular variants (isoforms). One example is Cor a 1. One isoform is mainly found in hazel pollen (Cor a 1.01), the other mainly in hazelnut (Cor a 1.04). Some isoforms are so closely related (>90% sequence identity) that they can usually be considered identical. If they need to be distinguished, two more digits are added to the name, for example, Cor a 1.0101 and Cor a 1.0102.

a cross-reactive nonsensitizer is the homologous apple allergen Mal d 1.¹⁴

## 3 - Airborne versus food allergens: crossing different barriers

The two most common sites of entry into the body are the mucosal surfaces of the airways in which the allergen is delivered as part of an airborne particle or aerosol droplet and the digestive tract, which includes the oral cavity where it is introduced as part of a food or drink (see Textbox 4). Also, the skin has been proposed as a route for sensitization, a hypothesis that gained significance since the discovery of filaggrin SNPs associated with the development of allergy. A prototypic example of true food allergens (primary sensitizers) is the shrimp muscle protein tropomyosin that varies in nomenclature depending on the type of shrimp (Pen a 1, Crac 1, Met e 1, Lit v 1, etc.), or other crustaceans and molluscs, such as lobster (Hom a 1) and crayfish (Pro c 1). All these are highly cross-reactive allergens (see the official IUIS website www.allergen.org) or the Allergome website (www.allergome.org); other invertebrate allergens, such as hemocyanin and hemoglobin, are more likely to sensitize via the airways or via skin contact in an occupational setting (seafood preparation, fish food production).¹⁵ Examples of true food allergens in plants are the
# A02 - Allergens and the allergenic composition of source materials

Ronald van Ree, Rob C. Aalberse

## Highlights

* Many different types of proteins are allergenic.

* The context of a protein may be a major determinant for its allergenicity.

* Some IgE inducers are not really allergens at all because they do not induce symptoms. This negatively impacts specificity of diagnostic tests, certainly of extract-based tests but also still of molecular tests.

* Allergen extracts are imperfect but not yet obsolete.

* Molecular sensitization profiles are potential biomarkers for disease phenotypes and progression.

* The initial response to an allergen source is possibly characterized by IgE antibodies to one or two "initiator" allergens.

Allergenic sources can vary from biologic sources with very complex composition such as pollen, house dust mites (inhalant allergy), or foods (food allergy), to single molecules such as chemicals (occupational allergy) or drugs (drug allergy). In this chapter, we will focus on the molecular composition of more complex biologic allergen sources that are implicated in causing hay fever, allergic asthma, and food allergy. Around the late sixties and early seventies of last century, first reports were published in which individual molecules were identified that were responsible for binding IgE within different allergen sources such as grass pollen,⁵ ragweed, pollen,⁶ cod fish allergen,⁷ and house dust mite.⁸ By now probably the most important allergens of the most relevant allergen sources have been identified (www.allergen.org; www.comparedatabase.org; www.allergenonline.org; www.allergome.org). Before we can discuss the allergens, we have to introduce the nomenclature of molecularly defined allergens (see Textbox 1).

## 1 - What is an allergen?

The broadest definition of an allergen is that it is any molecule-binding IgE antibodies.¹³ Allergens can differ in several ways (see also Textboxes 2 and 3). In this introduction, the practical consequences of four of these differences will be discussed.

## 2 - Sensitizing versus nonsensitizing allergens

Most, but not all, allergens are sensitizing, which is defined as the ability to induce allergen-specific IgE antibodies. Nonsensitizing allergens can only cause allergic symptoms if previous contact with a related (cross-reactive) allergen has caused sensitization. A prototypic example of a sensitizer is birch allergen Bet v 1 and

### Textbox 1 Allergen nomenclature

Allergen names are based on the scientific (Latin) name of the plant or animal species from which the allergen originates.⁹,¹⁰ For example, the major allergen from birch pollen Bet v 1 is named after the scientific name of the tree Betula verrucosa, in which Betula is the genus and verrucosa the species. The first three letters of the genus (Bet) and the first letter of the species (v) together form the basis of the allergen name, followed by a number. In principle the number is given in order of discovery, so Bet v 1 was the first allergen from birch pollen that was discovered. Related (often cross-reactive) allergens from different species, genus, family or even order, get the same number, if still available. So, the homologue of Bet v 1 in hazel is Cor a 1 and in apple is Mal d 1 but in peanut is Ara h 8 because numbers 1–7 were already occupied by peanut allergens described earlier. Many allergens have molecular variants (isoforms). One example is Cor a 1. One isoform is mainly found in hazel pollen (Cor a 1.01), the other mainly in hazelnut (Cor a 1.04). Some isoforms are so closely related (>90% sequence identity) that they can usually be considered identical. If they need to be distinguished, two more digits are added to the name, for example, Cor a 1.0101 and Cor a 1.0102.

a cross-reactive nonsensitizer is the homologous apple allergen Mal d 1.¹⁴

## 3 - Airborne versus food allergens: crossing different barriers

The two most common sites of entry into the body are the mucosal surfaces of the airways in which the allergen is delivered as part of an airborne particle or aerosol droplet and the digestive tract, which includes the oral cavity where it is introduced as part of a food or drink (see Textbox 4). Also, the skin has been proposed as a route for sensitization, a hypothesis that gained significance since the discovery of filaggrin SNPs associated with the development of allergy. A prototypic example of true food allergens (primary sensitizers) is the shrimp muscle protein tropomyosin that varies in nomenclature depending on the type of shrimp (Pen a 1, Crac 1, Met e 1, Lit v 1, etc.), or other crustaceans and molluscs, such as lobster (Hom a 1) and crayfish (Pro c 1). All these are highly cross-reactive allergens (see the official IUIS website www.allergen.org) or the Allergome website (www.allergome.org); other invertebrate allergens, such as hemocyanin and hemoglobin, are more likely to sensitize via the airways or via skin contact in an occupational setting (seafood preparation, fish food production).¹⁵ Examples of true food allergens in plants are the
### Textbox 2 The long road from allergen-coding DNA to a molecule interacting with IgE on the mast cell surface

Much of our information on allergen structure comes from proteins that are produced with recombinant DNA technologies (conveniently, but scientifically incorrectly, referred to as "recombinant allergens"). Most currently available recombinant allergens are prepared based on a direct translation of their genomic information. In the real world, the allergen that is knocking at the mast cell's door is often modified. Some of the modifications are well-characterized intracellular biochemical processes known as co-translational or post-translational modifications, such as homo- and hetero-oligomerization, glycosylation, cleavage of a leader peptide, pro-peptide, and other proteolytic events, binding of metallic cofactors or organic ligands, and oxidation of proline to hydroxyproline. Others are due to more random extracellular processes often influenced by environmental conditions (humidity, UV, ozone). Examples are nitration, methionine oxidation, deamidation, and cross-linking by transglutaminases and glycation (a nonenzymatic process also known as the Maillard reaction). Upon water loss, excreted proteins attach to various substrates, both on a nano-scale (homo- and heteroaggregation) and on a microscale (attachment to fibers and dusty particles). The effect of these modifications of the structure of the allergen on allergenicity has only just started to be investigated.<sup>11,12</sup>

2S albumins from legumes (e.g., Ara h 2), tree nuts (e.g., Cor a 14, Jug r 1, and Ana o 3), and seeds (e.g., Ses i 1). Although these allergens share clear structural features based on a common disulphide-bond pattern, their primary sequences are quite diverse resulting in limited cross-reactivity.

## 4 – Initiator allergens versus secondary responding allergens

The initial response to an allergen source is possibly characterized by IgE antibodies to one or two "initiator" allergens, which tend to dominate the subsequent more complex IgE response to the allergen source in question. It is therefore an attractive hypothesis that within an allergen source some allergens are more important than others. It might be tempting to call these "major" allergens, but traditionally, an allergen is referred to as "major" if it is recognized by >50% of the patients that are sensitized to the source (see Textbox 5). Not all these "major" allergens seem to act as "initiator" allergens.

## 5 – Innocent versus dangerous allergens

As expected, a close association is often found between allergen exposure and allergic symptoms. However, for some sensitizers this association is very weak. Several factors may explain the position of an allergen in the allergenicity risk spectrum. In addition to all the three above-mentioned features, it is presumably relevant that some relatively innocuous sensitizers are associated with relatively high IgG/IgE ratios. A prototypic example is tetanus toxoid.<sup>23</sup> The allergenic risk spectrum is further discussed below.

## 6 – What makes an antigen an allergen?

Some, but not all, antigens that pass through our epithelial barrier trigger an IgE response (see Textbox 6). There is an ongoing debate on the features of allergenic proteins (if any) that distinguish these from the more mundane, only IgG-inducing, antigens. It has been proposed that there are few restrictions on the properties of antigens that can induce IgE antibodies<sup>9,19</sup>. On the other hand, it has been argued that only a very restricted set of antigens has been found to induce IgE antibodies. This view has been promoted among others by Breiteneder et al.<sup>25</sup> Many different types of proteins are allergenic. Some features are not intrinsic to the protein itself but rather a consequence of an extrinsic feature: the context of the protein. One such extrinsic feature is the introduction of the protein in the presence of bacterial cell wall components. If a protein enters our body in the context of a bacterial infection, the innate immune response-induced cytokines (such as IL-12) usually skew the adaptive immune response towards the production of Th1-type cytokines (such as gamma-interferon) that are needed to efficiently fight the infection. Gamma-interferon prevents the production of Th2-type cytokines (particularly IL-4) that are needed for the switch to IgE. Regarding intrinsic features, the situation is not so clear-cut. While there is good evidence to support the claim that proteolytic activity may enhance the allergenicity of a protein (example: the mite allergen Der p 1), most allergens are not proteases. Some allergens have enzymatic activities that are unlikely to have an effect on human pathophysiology (example: pectinase activity of pollen allergens). Similarly, many allergens can bind small ligands, but the type of ligand varies considerably. It is not uncommon to find more than 10 different allergenic proteins in a single allergen source material (mite, pollen, peanut, shrimp, etc.). Although it seems contradictory to a basic function of the immune system, i.e., to react to non-self-proteins, it has been suggested that proteins having close homologues to human proteins are intrinsically more likely to be allergenic. Proteins of the lipocalin family are taken as an example.<sup>26</sup> It is clear that some proteins are more allergenic than others. Many factors are known to contribute to these differences, but prediction of the allergenicity (i.e., sensitization risk of a protein not cross-reactive with a known allergen) of a novel protein has thus far proven to be an
### Textbox 2 The long road from allergen-coding DNA to a molecule interacting with IgE on the mast cell surface

Much of our information on allergen structure comes from proteins that are produced with recombinant DNA technologies (conveniently, but scientifically incorrectly, referred to as "recombinant allergens"). Most currently available recombinant allergens are prepared based on a direct translation of their genomic information. In the real world, the allergen that is knocking at the mast cell's door is often modified. Some of the modifications are well-characterized intracellular biochemical processes known as co-translational or post-translational modifications, such as homo- and hetero-oligomerization, glycosylation, cleavage of a leader peptide, pro-peptide, and other proteolytic events, binding of metallic cofactors or organic ligands, and oxidation of proline to hydroxyproline. Others are due to more random extracellular processes often influenced by environmental conditions (humidity, UV, ozone). Examples are nitration, methionine oxidation, deamidation, and cross-linking by transglutaminases and glycation (a nonenzymatic process also known as the Maillard reaction). Upon water loss, excreted proteins attach to various substrates, both on a nano-scale (homo- and heteroaggregation) and on a microscale (attachment to fibers and dusty particles). The effect of these modifications of the structure of the allergen on allergenicity has only just started to be investigated.<sup>11,12</sup>

2S albumins from legumes (e.g., Ara h 2), tree nuts (e.g., Cor a 14, Jug r 1, and Ana o 3), and seeds (e.g., Ses i 1). Although these allergens share clear structural features based on a common disulphide-bond pattern, their primary sequences are quite diverse resulting in limited cross-reactivity.

## 4 – Initiator allergens versus secondary responding allergens

The initial response to an allergen source is possibly characterized by IgE antibodies to one or two "initiator" allergens, which tend to dominate the subsequent more complex IgE response to the allergen source in question. It is therefore an attractive hypothesis that within an allergen source some allergens are more important than others. It might be tempting to call these "major" allergens, but traditionally, an allergen is referred to as "major" if it is recognized by >50% of the patients that are sensitized to the source (see Textbox 5). Not all these "major" allergens seem to act as "initiator" allergens.

## 5 – Innocent versus dangerous allergens

As expected, a close association is often found between allergen exposure and allergic symptoms. However, for some sensitizers this association is very weak. Several factors may explain the position of an allergen in the allergenicity risk spectrum. In addition to all the three above-mentioned features, it is presumably relevant that some relatively innocuous sensitizers are associated with relatively high IgG/IgE ratios. A prototypic example is tetanus toxoid.<sup>23</sup> The allergenic risk spectrum is further discussed below.

## 6 – What makes an antigen an allergen?

Some, but not all, antigens that pass through our epithelial barrier trigger an IgE response (see Textbox 6). There is an ongoing debate on the features of allergenic proteins (if any) that distinguish these from the more mundane, only IgG-inducing, antigens. It has been proposed that there are few restrictions on the properties of antigens that can induce IgE antibodies<sup>9,19</sup>. On the other hand, it has been argued that only a very restricted set of antigens has been found to induce IgE antibodies. This view has been promoted among others by Breiteneder et al.<sup>25</sup> Many different types of proteins are allergenic. Some features are not intrinsic to the protein itself but rather a consequence of an extrinsic feature: the context of the protein. One such extrinsic feature is the introduction of the protein in the presence of bacterial cell wall components. If a protein enters our body in the context of a bacterial infection, the innate immune response-induced cytokines (such as IL-12) usually skew the adaptive immune response towards the production of Th1-type cytokines (such as gamma-interferon) that are needed to efficiently fight the infection. Gamma-interferon prevents the production of Th2-type cytokines (particularly IL-4) that are needed for the switch to IgE. Regarding intrinsic features, the situation is not so clear-cut. While there is good evidence to support the claim that proteolytic activity may enhance the allergenicity of a protein (example: the mite allergen Der p 1), most allergens are not proteases. Some allergens have enzymatic activities that are unlikely to have an effect on human pathophysiology (example: pectinase activity of pollen allergens). Similarly, many allergens can bind small ligands, but the type of ligand varies considerably. It is not uncommon to find more than 10 different allergenic proteins in a single allergen source material (mite, pollen, peanut, shrimp, etc.). Although it seems contradictory to a basic function of the immune system, i.e., to react to non-self-proteins, it has been suggested that proteins having close homologues to human proteins are intrinsically more likely to be allergenic. Proteins of the lipocalin family are taken as an example.<sup>26</sup> It is clear that some proteins are more allergenic than others. Many factors are known to contribute to these differences, but prediction of the allergenicity (i.e., sensitization risk of a protein not cross-reactive with a known allergen) of a novel protein has thus far proven to be an
### Textbox 3 The exception: some allergens are NOT proteins

Examples of nonprotein allergens are drugs such as penicillin, chlorhexidine, and other pharmacological compounds such as rocuronium. It is generally assumed that these compounds depend for their allergenicity on a strong (covalent) interaction with a carrier protein, but this has not always been demonstrated convincingly. In fact, some smaller molecules, such as chlorhexidine or polyethylene glycols (PEGs), have two or more epitopes, which will allow for cross-linking of two or more receptor-bound IgE molecules on the surface of effector cells without help from an endogenous protein. How the IgE immune response to such small molecules may develop, has been much less studied, however. One explanation might be that a metabolite is the allergologically active substance. The above-mentioned allergic drug-protein complexes are often referred to as hapten-carrier complexes. Substances other than pharmacological compounds can also act as hapten. An important category of hapten-like structures are naturally occurring chains of simple sugars, referred to as glycans. The role of glycans as IgE-reactive structure is a source of some confusion. It is generally assumed that pure glycans are unable to induce IgE antibodies. This fits with the general scheme of IgE antibody production as a process that depends on signals provided by Th2 cells, as described above. Classical MHC-II molecules are very efficient at interacting with peptides but are unable to combine with pure glycans. However, for glycans coupled to a protein carrier, the situation is different. The cell-anchored antibody on some B cells can interact with the glycan. These B cells bind the glycoprotein via the antibody-glycan interaction. Next, the B cell ingests and digests the glycoprotein and presents the peptides in its MHC-II to the T cell. The T-cell receptor interacts with the peptide-MHC-II complex on the B cell, which results in activation of the T cell. The T cell activates the B cell, which results in differentiation of the B cell to an antibody-secreting plasma cell. The important point is that the conventional Th2 cell does not recognize the glycan and yet it can induce the B cell to produce antiglycan antibodies. It is possible that glycans can be allergenic not only as glycoprotein but also as glycolipid, potentially via sources of IL-4 other than Th2 cells. This is presumably mostly relevant for immune responses to invertebrate parasites such as helminths and ticks. Two prototypic glycans with well-established IgE-binding activity are known as CCD¹⁶,¹⁷ and the α-Gal epitope.¹⁷,¹⁸ CCD (cross-reactive carbohydrate determinant) refers to a group of related glycans that are characterized by a fucose and/or a xylose that are linked in a specific way to the core of the glycan. Such glycans are produced by invertebrates and plants but not by vertebrates. Because of the widespread presence of such structures in plant foods, patients with such IgE antibodies demonstrate cross-reactivity to virtually all plant foods.¹⁹ By contrast, α-Gal (in full: the Gal-α1-3Gal-β1-3GlcNAc epitope) is produced by nonprimate mammals but not by humans. This structure, now commonly referred to as α-Gal, has been associated with allergy to red meat.²⁰ For yet unknown reasons, the onset of systemic (skin) symptoms observed upon consumption of meat is not immediate but delayed. Where induction of IgE antibodies against CCD is thought to be driven by pollen exposure and/or insect stings, IgE antibodies against α-Gal most likely occur in response to tick bites or helminth infections.²¹ A convenient way to distinguish peptide-based epitopes from glycan-based epitopes is the use of proteolytic enzymes or glycan-destroying chemicals (periodate). Some IgE antibodies show dual recognition towards a glycoprotein allergen: their epitope consists of a combination of part of the glycan and part of the protein.²²

unsurmountable challenge. With the advent of algorithms based on machine learning and artificial intelligence, new potentially promising avenues may open up.²⁷ The allergenicity debate will undoubtedly go on (See also the Chapter on "Basic and theoretical aspects of allergens"). From a practical point of view, it is relevant that some source materials are more allergenic than others. Illustrative examples include allergens from cooked legumes that are less allergenic than those from roasted peanuts and the lipocalins from dogs (Can f 1, Can f 2 and Can f 4 as major allergens) being less allergenic than the major allergen from cats (Fel d 1), which is a NOT a lipocalin.

## 7 - Clinical relevance of individual allergenic proteins

Some inhalant allergen sources contain a single dominant major allergen. The clearest example is Bet v 1 in birch pollen, which is responsible for most of the IgE binding to the allergen source. For tree pollen-allergic patients in North-western and Central Europe, Bet v

1 is of decisive clinical importance because there is no "competing" major allergen. By contrast, multiple major allergens have been described for grass pollen (i.e., group 1 (e.g., Phl p 1) and group 5 (e.g., Phl p 5) and house dust mite group 1 (e.g., Der p 1) and group 2 (e.g., Der p 2)). Currently, it is assumed that allergens from both groups are of great clinical importance. Whether they play an individual role in determining clinical phenotypes is still largely unknown. In recent studies, first indications were found that specific individual (minor) house dust mite allergens (e.g., Der p 23 and Der p 20) are associated with more severe respiratory clinical presentations such as asthma.²⁸,²⁹ It has, however, also been proposed that such associations are not necessarily explained by the molecular properties of these allergens but by a high degree of sensitization characterized by broad recognition of a multitude of major and minor allergens.³⁰ For food allergy, individual allergen molecules have more clearly been associated with both defined clinical phenotypes and the severity of allergic symptoms.³¹ There are three ways one can become allergic to a specific food, (a) by direct exposure to that food via the oral
### Textbox 3 The exception: some allergens are NOT proteins

Examples of nonprotein allergens are drugs such as penicillin, chlorhexidine, and other pharmacological compounds such as rocuronium. It is generally assumed that these compounds depend for their allergenicity on a strong (covalent) interaction with a carrier protein, but this has not always been demonstrated convincingly. In fact, some smaller molecules, such as chlorhexidine or polyethylene glycols (PEGs), have two or more epitopes, which will allow for cross-linking of two or more receptor-bound IgE molecules on the surface of effector cells without help from an endogenous protein. How the IgE immune response to such small molecules may develop, has been much less studied, however. One explanation might be that a metabolite is the allergologically active substance. The above-mentioned allergic drug-protein complexes are often referred to as hapten-carrier complexes. Substances other than pharmacological compounds can also act as hapten. An important category of hapten-like structures are naturally occurring chains of simple sugars, referred to as glycans. The role of glycans as IgE-reactive structure is a source of some confusion. It is generally assumed that pure glycans are unable to induce IgE antibodies. This fits with the general scheme of IgE antibody production as a process that depends on signals provided by Th2 cells, as described above. Classical MHC-II molecules are very efficient at interacting with peptides but are unable to combine with pure glycans. However, for glycans coupled to a protein carrier, the situation is different. The cell-anchored antibody on some B cells can interact with the glycan. These B cells bind the glycoprotein via the antibody-glycan interaction. Next, the B cell ingests and digests the glycoprotein and presents the peptides in its MHC-II to the T cell. The T-cell receptor interacts with the peptide-MHC-II complex on the B cell, which results in activation of the T cell. The T cell activates the B cell, which results in differentiation of the B cell to an antibody-secreting plasma cell. The important point is that the conventional Th2 cell does not recognize the glycan and yet it can induce the B cell to produce antiglycan antibodies. It is possible that glycans can be allergenic not only as glycoprotein but also as glycolipid, potentially via sources of IL-4 other than Th2 cells. This is presumably mostly relevant for immune responses to invertebrate parasites such as helminths and ticks. Two prototypic glycans with well-established IgE-binding activity are known as CCD¹⁶,¹⁷ and the α-Gal epitope.¹⁷,¹⁸ CCD (cross-reactive carbohydrate determinant) refers to a group of related glycans that are characterized by a fucose and/or a xylose that are linked in a specific way to the core of the glycan. Such glycans are produced by invertebrates and plants but not by vertebrates. Because of the widespread presence of such structures in plant foods, patients with such IgE antibodies demonstrate cross-reactivity to virtually all plant foods.¹⁹ By contrast, α-Gal (in full: the Gal-α1-3Gal-β1-3GlcNAc epitope) is produced by nonprimate mammals but not by humans. This structure, now commonly referred to as α-Gal, has been associated with allergy to red meat.²⁰ For yet unknown reasons, the onset of systemic (skin) symptoms observed upon consumption of meat is not immediate but delayed. Where induction of IgE antibodies against CCD is thought to be driven by pollen exposure and/or insect stings, IgE antibodies against α-Gal most likely occur in response to tick bites or helminth infections.²¹ A convenient way to distinguish peptide-based epitopes from glycan-based epitopes is the use of proteolytic enzymes or glycan-destroying chemicals (periodate). Some IgE antibodies show dual recognition towards a glycoprotein allergen: their epitope consists of a combination of part of the glycan and part of the protein.²²

unsurmountable challenge. With the advent of algorithms based on machine learning and artificial intelligence, new potentially promising avenues may open up.²⁷ The allergenicity debate will undoubtedly go on (See also the Chapter on "Basic and theoretical aspects of allergens"). From a practical point of view, it is relevant that some source materials are more allergenic than others. Illustrative examples include allergens from cooked legumes that are less allergenic than those from roasted peanuts and the lipocalins from dogs (Can f 1, Can f 2 and Can f 4 as major allergens) being less allergenic than the major allergen from cats (Fel d 1), which is a NOT a lipocalin.

## 7 - Clinical relevance of individual allergenic proteins

Some inhalant allergen sources contain a single dominant major allergen. The clearest example is Bet v 1 in birch pollen, which is responsible for most of the IgE binding to the allergen source. For tree pollen-allergic patients in North-western and Central Europe, Bet v

1 is of decisive clinical importance because there is no "competing" major allergen. By contrast, multiple major allergens have been described for grass pollen (i.e., group 1 (e.g., Phl p 1) and group 5 (e.g., Phl p 5) and house dust mite group 1 (e.g., Der p 1) and group 2 (e.g., Der p 2)). Currently, it is assumed that allergens from both groups are of great clinical importance. Whether they play an individual role in determining clinical phenotypes is still largely unknown. In recent studies, first indications were found that specific individual (minor) house dust mite allergens (e.g., Der p 23 and Der p 20) are associated with more severe respiratory clinical presentations such as asthma.²⁸,²⁹ It has, however, also been proposed that such associations are not necessarily explained by the molecular properties of these allergens but by a high degree of sensitization characterized by broad recognition of a multitude of major and minor allergens.³⁰ For food allergy, individual allergen molecules have more clearly been associated with both defined clinical phenotypes and the severity of allergic symptoms.³¹ There are three ways one can become allergic to a specific food, (a) by direct exposure to that food via the oral
### Textbox 4 Allergens have to get into our body to sensitize and to do harm

Allergenic proteins have to be in solution in order to get into our tissues, both for the sensitization phase (the interaction reaction with a professional antigen-presenting cell, T cell and B cell) and for elicitation phase (the interaction with the IgE antibody on the surface of the mast cell or basophilic leukocyte). For inhaled allergens, the allergen will be contained in or attached to a particle (pollen grain, mould spore, mite faecal particle, a skin flake, hair, a textile fibre, or a fluid droplet) from which it is extracted upon deposition on the mucous membranes. The size of the allergen-carrying particle (typically 5–20 µm) is important, because this determines the most likely site of deposition (upper or lower airways). The allergen has to be released into the mucosal fluid to pass through the airway epithelial barrier. For this passage also a size limit exists. The diameter of a typical globular allergen molecule is some 1000 times smaller than the allergen-carrying particle (2–10 nm, in molecular mass units: 5–50 kDa). It cannot be ruled out that sensitization to inhaled allergens can also occur via the skin, in particular when the skin barrier is impaired, but also in that case, moisture is needed to solubilize the allergen, and size limits to allow it to cross the skin barrier will be similar. For food allergens the biophysical requirements for allergenicity are different in at least 2 ways. Firstly, food processing can substantially change the solubility of some proteins and in some cases also change allergenicity. In addition to the destructive effects of cooking on many allergens, another well-known example is the loss of allergenicity following the mincing of apples, due to oxidative browning of apple polyphenols. This results in denaturation of apple proteins by the tannin-like structures. Secondly, the digestive system could increase allergenicity by releasing small soluble allergenic fragments from poorly soluble conglomerates, or decrease allergenicity by more extensive fragmentation. In addition to proteases, also the low pH in the stomach and the detergent action of bile salts are important in modifying the allergenicity of ingested proteins. The resistance to proteolytic digestion is obviously of no relevance if sensitization to food occurs via the skin. The question why 2S albumins are excellent primary sensitizers while Bet v 1-like allergens are not, will most likely be explained by the abundance of the respective proteins in the environment.

route, or increasingly likely (also) via the skin, (b) by cross-reactivity between foods, and (c) by cross-reactivity between respiratory allergen sources and foods. The best-known example of the latter is fruit and tree nut allergy as comorbidity with birch pollen allergy.³²

### Textbox 5 What are major and minor allergens?

Officially, a major allergen is an allergen that is recognized by IgE antibodies of >50% of patients allergic to the allergen source, and a minor by <50% of patients. This old definition has increasingly been challenged as being too simplistic.²⁴ In most cases, major allergens also bind a large fraction of the allergen source-specific IgE and are therefore (most likely) of dominant clinical importance. Similarly, minor allergens usually bind only a small fraction of the overall IgE response against the allergen source. The designation major allergen should therefore most likely take the importance of the allergen in the overall IgE response against the source into account. Most major allergens occupy the lower numbers in the nomenclature system, simply because researchers tended to identify the most dominant allergens first.

The mechanism behind this association is cross-reactivity of Bet v 1-specific IgE with structurally homologous allergens in foods like apple, peach, hazelnut and peanut (see Textbox 7). The clinical phenotype observed in such patients is characterized by mild-to-moderate symptoms restricted to the oral cavity. The explanation for the lack of (severe) systemic symptoms is thought to reside in the protease-sensitive nature of the Bet v 1-related food allergens that are readily digested in the gastrointestinal tract.³³ This characteristic also explains why sensitization to these Bet v 1-related food allergens is never seen in patients without birch pollen allergy. Apple Mal d 1 or peach Pru p 1, the Bet v 1 homologues of these fruits, are completely digested before they can directly sensitize. Moreover, Bet v 1-like allergens are not abundantly expressed in fruits, making the chance of exposure to sufficient protein in the digestive tract or on the skin even more unlikely. This does not mean that a fruit like peach cannot directly sensitize atopic subjects. Until recently, this is mainly reported for patients living around the Mediterranean Sea, but it is increasingly also observed beyond those areas.³⁴,³⁵ The implicated allergen for peach allergy, originally mostly reported in countries like Spain, Italy, and Greece, is the non-specific lipid transfer protein (LTP), that is, Pru p 3. IgE antibodies against Pru p 3 are associated with an increased risk for severe systemic reactions,³⁶ and they can cross-react quite broadly to other fruits, as well as to tree nuts, legumes, and some vegetables.³⁷ This more "dangerous" profile of LTPs has been attributed to their high degree of protease (and food-processing) resistance. Another factor that may contribute to their higher risk profile is that they go into solution effectively at low pH only, i.e., in the stomach, resulting in the absence of an early oral warning signal. In addition to Bet v 1-related allergens and LTPs, tree nuts, legumes, and seeds contain far more abundant seed storage proteins, such as 2S albumins, and 7S and 11S globulins. These proteins are involved in direct sensitization, which often occurs at younger ages. As reported for LTPs, these seed storage
### Textbox 4 Allergens have to get into our body to sensitize and to do harm

Allergenic proteins have to be in solution in order to get into our tissues, both for the sensitization phase (the interaction reaction with a professional antigen-presenting cell, T cell and B cell) and for elicitation phase (the interaction with the IgE antibody on the surface of the mast cell or basophilic leukocyte). For inhaled allergens, the allergen will be contained in or attached to a particle (pollen grain, mould spore, mite faecal particle, a skin flake, hair, a textile fibre, or a fluid droplet) from which it is extracted upon deposition on the mucous membranes. The size of the allergen-carrying particle (typically 5-20 µm) is important, because this determines the most likely site of deposition (upper or lower airways). The allergen has to be released into the mucosal fluid to pass through the airway epithelial barrier. For this passage also a size limit exists. The diameter of a typical globular allergen molecule is some 1000 times smaller than the allergen-carrying particle (2-10 nm, in molecular mass units: 5-50 kDa). It cannot be ruled out that sensitization to inhaled allergens can also occur via the skin, in particular when the skin barrier is impaired, but also in that case, moisture is needed to solubilize the allergen, and size limits to allow it to cross the skin barrier will be similar. For food allergens the biophysical requirements for allergenicity are different in at least 2 ways. Firstly, food processing can substantially change the solubility of some proteins and in some cases also change allergenicity. In addition to the destructive effects of cooking on many allergens, another well-known example is the loss of allergenicity following the mincing of apples, due to oxidative browning of apple polyphenols. This results in denaturation of apple proteins by the tannin-like structures. Secondly, the digestive system could increase allergenicity by releasing small soluble allergenic fragments from poorly soluble conglomerates, or decrease allergenicity by more extensive fragmentation. In addition to proteases, also the low pH in the stomach and the detergent action of bile salts are important in modifying the allergenicity of ingested proteins. The resistance to proteolytic digestion is obviously of no relevance if sensitization to food occurs via the skin. The question why 2S albumins are excellent primary sensitizers while Bet v 1-like allergens are not, will most likely be explained by the abundance of the respective proteins in the environment.

route, or increasingly likely (also) via the skin, (b) by cross-reactivity between foods, and (c) by cross-reactivity between respiratory allergen sources and foods. The best-known example of the latter is fruit and tree nut allergy as comorbidity with birch pollen allergy.³²

### Textbox 5 What are major and minor allergens?

Officially, a major allergen is an allergen that is recognized by IgE antibodies of >50% of patients allergic to the allergen source, and a minor by <50% of patients. This old definition has increasingly been challenged as being too simplistic.²⁴ In most cases, major allergens also bind a large fraction of the allergen source-specific IgE and are therefore (most likely) of dominant clinical importance. Similarly, minor allergens usually bind only a small fraction of the overall IgE response against the allergen source. The designation major allergen should therefore most likely take the importance of the allergen in the overall IgE response against the source into account. Most major allergens occupy the lower numbers in the nomenclature system, simply because researchers tended to identify the most dominant allergens first.

The mechanism behind this association is cross-reactivity of Bet v 1-specific IgE with structurally homologous allergens in foods like apple, peach, hazelnut and peanut (see Textbox 7). The clinical phenotype observed in such patients is characterized by mild-to-moderate symptoms restricted to the oral cavity. The explanation for the lack of (severe) systemic symptoms is thought to reside in the protease-sensitive nature of the Bet v 1-related food allergens that are readily digested in the gastrointestinal tract.³³ This characteristic also explains why sensitization to these Bet v 1-related food allergens is never seen in patients without birch pollen allergy. Apple Mal d 1 or peach Pru p 1, the Bet v 1 homologues of these fruits, are completely digested before they can directly sensitize. Moreover, Bet v 1-like allergens are not abundantly expressed in fruits, making the chance of exposure to sufficient protein in the digestive tract or on the skin even more unlikely. This does not mean that a fruit like peach cannot directly sensitize atopic subjects. Until recently, this is mainly reported for patients living around the Mediterranean Sea, but it is increasingly also observed beyond those areas.³⁴,³⁵ The implicated allergen for peach allergy, originally mostly reported in countries like Spain, Italy, and Greece, is the non-specific lipid transfer protein (LTP), that is, Pru p 3. IgE antibodies against Pru p 3 are associated with an increased risk for severe systemic reactions,³⁶ and they can cross-react quite broadly to other fruits, as well as to tree nuts, legumes, and some vegetables.³⁷ This more "dangerous" profile of LTPs has been attributed to their high degree of protease (and food-processing) resistance. Another factor that may contribute to their higher risk profile is that they go into solution effectively at low pH only, i.e., in the stomach, resulting in the absence of an early oral warning signal. In addition to Bet v 1-related allergens and LTPs, tree nuts, legumes, and seeds contain far more abundant seed storage proteins, such as 2S albumins, and 7S and 11S globulins. These proteins are involved in direct sensitization, which often occurs at younger ages. As reported for LTPs, these seed storage
### Textbox 6 Short introduction on the production of IgE antibodies to conventional protein allergens

A cardinal feature of an allergen is the ability to induce the production of IgE antibodies. The first step to initial IgE antibody production is the activation and expansion of naïve allergen-reactive IgM-producing B cells. This process depends on the interaction between various cells (in particular dendritic antigen presenting cells and T helper cells) and the production of many cytokines. IgE antibody production is a process that depends on signals provided by T-helper cells that are skewed by innate signals to become Th2 cells. Properties of allergen molecules can contribute to the innate process driving towards Th2 immune responses.³⁸⁻⁴⁰ Major Histocompatibility Complex-II (MHC-II) molecules present peptides derived from the protein to Th2 cells. The T cell receptor interacts with the peptide-MHC-II complex, which results in activation of the T cell. This activated T cell can activate B cells, but only if these B cells have the same peptide in their surface-anchored MHC-II. In contrast to the antigen-presenting cell, the B cell can only ingest antigens if the antigen binds to the surface-anchored unique antibody of that B cell. These B cells ingest and digest the protein and present the peptides in its MHC-II to the T cell. The B cell further activates the T cell, which results in the production of cytokines by the T cell. The T cell activates the B cell, which results in differentiation of the B cell to an antibody-secreting plasma cell. The activated IgM-producing B cells can differentiate to change their isotype by a process called class-switch recombination (which results in a change in isotype production from IgM to IgG1, IgG4, IgA1, etc., and sometimes to IgE) and to increase their affinity by a process called somatic hypermutation. Both processes result in irreversible changes in the DNA of the B cell. The discovery of the crucial role of IL-4 produced by Th2-cells (and other cells) in the generation of IgE responses is a milestone in the history of allergy.⁴¹,⁴²

proteins, in particular the 2S albumins, are remarkably stable and IgE antibodies against them proved to be better markers for predicting a positive double-blind placebo-controlled food challenge, that is, for clinical allergy. They are also associated with more severe symptoms. This was first demonstrated convincingly for peanut Ara h 2,³¹ but hazelnut 2S (Cor a 14) and 11S (Cor a 9) play such a role as well⁴³,⁴⁴; however, for hazelnut allergy as a whole, they would not qualify as major allergens because birch pollen-associated hazelnut allergy is the dominant phenotype.⁴⁵ This illustrates that minor allergens can be of major clinical relevance. Based on these and other observations, molecular diagnosis is increasingly used for attempts to reliably assess the risk of patients to experience severe symptoms. In

some more recent papers, models combining molecular recognition profiles and clinical and demographic background have been proposed to improve severity risk assessments.⁴⁵,⁴⁶

## 8 – The allergenic risk spectrum

Some allergens are considered more dangerous than others in that they elicit more severe allergic symptoms. By contrast, some IgE inducers are not really allergens at all because they do not induce symptoms. The prevalence of such cases has often been underestimated because they are rarely detected in the doctor's office. They used to be identified mostly in population surveys and birth cohort studies. This situation is changing, particularly since the introduction of the large allergen microarrays, as discussed in Chapter A04. Peanut is a prototypic example of an allergenic source material to which many people have IgE antibodies, but they can often freely consume peanuts. Finding IgE to peanuts in peanut-tolerant subjects is particularly common among pollen-sensitized patients.⁵¹ This association is due to IgE cross-reactivity between allergens from pollen and (glyco-) proteins in peanut and many other vegetable sources. Bet v 1-related cross-reactivity has been discussed above. Peanut contains a cross-reactive homologue, Ara h 8. However, cross-reactivity has also been evident between peanut and grasses, which do not have a cross-reactive Bet v 1 homologue. Profilin and CCD have been found to be the most likely additional cross-reactive substances.¹⁷,⁵² It has been convincingly demonstrated that CCD-specific IgE antibodies are of limited if any clinical relevance.⁵³ It is tempting to assume that all nonsensitizing cross-reactive allergens are relatively safe. While this is true in many cases, severe reactions caused by exposure to such presumed nonsensitizing cross-reactive allergens have been reported. A more recent addition to the spectrum of highly cross-reactive structures that clearly demonstrates this are the nonprimate mammalian α-Gal sugar moieties present in red meat that are associated with delayed severe reactions.²⁰ In general, however, the relatively low biologic activity of cross-reacting allergens may reflect lower epitope density and lower affinity of the IgE-allergen interaction, but it has been disappointingly difficult to predict biologic activity on the basis of immunochemical characteristics in individual cases. The use of a bioassay such as the basophil activation test is a promising alternative.⁵⁴ In addition, other experimental assays will further help to define the biological activity of individual IgE-binding molecules.⁵⁵ Allergen sources are complex, heterogeneous mixtures of proteins. They contain harmless IgE-binding structures such as CCD, molecules that induce mild symptoms only and molecules that are associated with severe symptoms including food or insect venom-induced anaphylaxis. Moreover, molecules can inform us about the origin and route of sensitization, sometimes reflected in clear geographic differences. Dissection of these molecular characteristics of allergen sources is of the utmost importance to improve allergy diagnosis, prevention, and therapy.
### Textbox 6 Short introduction on the production of IgE antibodies to conventional protein allergens

A cardinal feature of an allergen is the ability to induce the production of IgE antibodies. The first step to initial IgE antibody production is the activation and expansion of naïve allergen-reactive IgM-producing B cells. This process depends on the interaction between various cells (in particular dendritic antigen presenting cells and T helper cells) and the production of many cytokines. IgE antibody production is a process that depends on signals provided by T-helper cells that are skewed by innate signals to become Th2 cells. Properties of allergen molecules can contribute to the innate process driving towards Th2 immune responses.³⁸⁻⁴⁰ Major Histocompatibility Complex-II (MHC-II) molecules present peptides derived from the protein to Th2 cells. The T cell receptor interacts with the peptide-MHC-II complex, which results in activation of the T cell. This activated T cell can activate B cells, but only if these B cells have the same peptide in their surface-anchored MHC-II. In contrast to the antigen-presenting cell, the B cell can only ingest antigens if the antigen binds to the surface-anchored unique antibody of that B cell. These B cells ingest and digest the protein and present the peptides in its MHC-II to the T cell. The B cell further activates the T cell, which results in the production of cytokines by the T cell. The T cell activates the B cell, which results in differentiation of the B cell to an antibody-secreting plasma cell. The activated IgM-producing B cells can differentiate to change their isotype by a process called class-switch recombination (which results in a change in isotype production from IgM to IgG1, IgG4, IgA1, etc., and sometimes to IgE) and to increase their affinity by a process called somatic hypermutation. Both processes result in irreversible changes in the DNA of the B cell. The discovery of the crucial role of IL-4 produced by Th2-cells (and other cells) in the generation of IgE responses is a milestone in the history of allergy.⁴¹,⁴²

proteins, in particular the 2S albumins, are remarkably stable and IgE antibodies against them proved to be better markers for predicting a positive double-blind placebo-controlled food challenge, that is, for clinical allergy. They are also associated with more severe symptoms. This was first demonstrated convincingly for peanut Ara h 2,³¹ but hazelnut 2S (Cor a 14) and 11S (Cor a 9) play such a role as well⁴³,⁴⁴; however, for hazelnut allergy as a whole, they would not qualify as major allergens because birch pollen-associated hazelnut allergy is the dominant phenotype.⁴⁵ This illustrates that minor allergens can be of major clinical relevance. Based on these and other observations, molecular diagnosis is increasingly used for attempts to reliably assess the risk of patients to experience severe symptoms. In

some more recent papers, models combining molecular recognition profiles and clinical and demographic background have been proposed to improve severity risk assessments.⁴⁵,⁴⁶

## 8 – The allergenic risk spectrum

Some allergens are considered more dangerous than others in that they elicit more severe allergic symptoms. By contrast, some IgE inducers are not really allergens at all because they do not induce symptoms. The prevalence of such cases has often been underestimated because they are rarely detected in the doctor's office. They used to be identified mostly in population surveys and birth cohort studies. This situation is changing, particularly since the introduction of the large allergen microarrays, as discussed in Chapter A04. Peanut is a prototypic example of an allergenic source material to which many people have IgE antibodies, but they can often freely consume peanuts. Finding IgE to peanuts in peanut-tolerant subjects is particularly common among pollen-sensitized patients.⁵¹ This association is due to IgE cross-reactivity between allergens from pollen and (glyco-) proteins in peanut and many other vegetable sources. Bet v 1-related cross-reactivity has been discussed above. Peanut contains a cross-reactive homologue, Ara h 8. However, cross-reactivity has also been evident between peanut and grasses, which do not have a cross-reactive Bet v 1 homologue. Profilin and CCD have been found to be the most likely additional cross-reactive substances.¹⁷,⁵² It has been convincingly demonstrated that CCD-specific IgE antibodies are of limited if any clinical relevance.⁵³ It is tempting to assume that all nonsensitizing cross-reactive allergens are relatively safe. While this is true in many cases, severe reactions caused by exposure to such presumed nonsensitizing cross-reactive allergens have been reported. A more recent addition to the spectrum of highly cross-reactive structures that clearly demonstrates this are the nonprimate mammalian α-Gal sugar moieties present in red meat that are associated with delayed severe reactions.²⁰ In general, however, the relatively low biologic activity of cross-reacting allergens may reflect lower epitope density and lower affinity of the IgE-allergen interaction, but it has been disappointingly difficult to predict biologic activity on the basis of immunochemical characteristics in individual cases. The use of a bioassay such as the basophil activation test is a promising alternative.⁵⁴ In addition, other experimental assays will further help to define the biological activity of individual IgE-binding molecules.⁵⁵ Allergen sources are complex, heterogeneous mixtures of proteins. They contain harmless IgE-binding structures such as CCD, molecules that induce mild symptoms only and molecules that are associated with severe symptoms including food or insect venom-induced anaphylaxis. Moreover, molecules can inform us about the origin and route of sensitization, sometimes reflected in clear geographic differences. Dissection of these molecular characteristics of allergen sources is of the utmost importance to improve allergy diagnosis, prevention, and therapy.
### Textbox 7 Allergen cross-reactivity and its assessment

Two allergens are cross-reactive if antibodies exist that recognize both allergens. The antibody will usually have a preference for one allergen over the other. This preferential recognition provides a clue as to identify of the more relevant of the two allergens. A single allergen molecule has several IgE-binding regions (called epitopes). Among IgE antibodies to the birch allergen, Bet v 1, that are induced by inhaling birch pollen, two types of antibody populations can be distinguished based on their reactivity with Mal d 1, the homologous protein of apple. Some IgE anti-Bet v 1 antibodies will not react with Mal d 1, because they are directed to a nonconserved part of Bet v 1. Other IgE antibodies will react not only with Bet v 1 but also with Mal d 1. The latter reaction will usually be of lower affinity. In this example, Bet v 1 can be shown to be a more complete allergen than Mal d 1. Grass pollen extract does not at all inhibit the binding between IgE anti Bet v 1 and Mal d 1 (because grass pollen extract does not contain a cross-reactive Bet v 1 homologue). In this way, it is possible to rank allergen source materials (such as birch, apple, celery, and peanut) in a cross-reactivity hierarchy. This is most reliably done by using a quantitative bi-directional cross-inhibition protocol,⁴⁷ but less demanding protocols may also be informative. Some cross-reactions are relatively restricted (example: cross-reactivity among grasses). Others are broader (example: Bet v 1/Mal d 1, with much lower cross-reactivity to the homologous protein in peanut, celery and potato and no cross-reactivity with grasses). Others cross wider phylogenetic barriers (examples: cross-reactivity between pollen from birch and grass due to profilin⁴⁸a,⁴⁸b and cross-reactivity between shrimp and mites due to tropomyosin)⁴⁹. Among the glycan epitopes, CCDs tend to be even more cross-reactive (example: cross-reactivity between bee venom and potato).¹⁶ Glycan-based cross-reactivity is different from protein-based cross-reactivity because the degree and fine structure of glycosylation is variable among glycoproteins, even at the single-cell level.⁵⁰a It is not unusual to find that 2 allergen source materials share several distinct cross-reactive molecules. An example is the cross-reactivity among birch pollen, vegetables and fruits, which was found to be due to at least 3 cross-reactive structures.⁵⁰b

## 9 – Molecular sensitization profiles: biomarkers for disease progression?

A relatively unexplored area is whether IgE recognition profiles have predictive value for disease progression. This field will most likely develop rapidly in the near future. Some evidence from the field of food allergy suggests that recognition profiles of specific epitopes on major food allergens, using short synthetic peptides, can predict outgrowth or persistence.⁵⁶ Another study reported that persistence of peanut allergy is associated with the number of peanut allergens recognized.⁵⁷ More recently, IgE against Der p 1 and in particular Der p 23 at young age was shown to be predictive for development of asthma later in life²⁸. These studies are just the beginning and illustrate the importance of carefully dissecting molecular composition of allergen sources.

## 10 – Allergen extracts: imperfect but not yet obsolete

Both allergy diagnostics and allergen immunotherapy (AIT) still heavily depend on extracts of the allergen sources. In particular in diagnostics, molecular approaches are gaining ground⁵⁸ rapidly, but extracts can certainly not be dismissed.⁵⁹ Usually, allergen extracts are simple aqueous extracts of the crude allergen source. In most cases, extraction is carried out at neutral or close to neutral pH, followed by a defatting step, and dialysis. In the case of food extracts, the source material may sometimes be partially processed before extraction, for example, peanut meal of mildly roasted peanuts. What are the potential shortcomings of allergen extracts? Allergen sources are biologic products with inherent variability of composition. Extraction with aqueous buffers at neutral pH may not optimally extract all possible allergens, especially those that are lipid-soluble. This is particularly relevant for food extracts because the natural route of exposure through the stomach includes exposure to low pH. A good example of a food allergen that is not optimally extracted at neutral pH is LTP from legumes such as peanut and lentils.⁶⁰ This phenomenon may also be the explanation for the huge variability in LTP content reported for a series of commercially available skin test reagents for hazelnut allergy.⁶¹ Extraction at low pH has proven to be the solution. Another problem encountered when preparing diagnostic food extracts, in particular of fruits and vegetables, is that enzymatic oxidative processes are initiated when the food tissue is disrupted. In particular, Bet v 1-related food allergens such as Mal d 1 in apple, Pru p 1 in peach, or Cor a 1 in hazelnut are sensitive to these processes and they lose their IgE-binding capacity. Finally, the defatting step has been implicated in loss of lipophilic allergens such as oleosins in legumes, nuts, and seeds.⁶² Together, these
### Textbox 7 Allergen cross-reactivity and its assessment

Two allergens are cross-reactive if antibodies exist that recognize both allergens. The antibody will usually have a preference for one allergen over the other. This preferential recognition provides a clue as to identify of the more relevant of the two allergens. A single allergen molecule has several IgE-binding regions (called epitopes). Among IgE antibodies to the birch allergen, Bet v 1, that are induced by inhaling birch pollen, two types of antibody populations can be distinguished based on their reactivity with Mal d 1, the homologous protein of apple. Some IgE anti-Bet v 1 antibodies will not react with Mal d 1, because they are directed to a nonconserved part of Bet v 1. Other IgE antibodies will react not only with Bet v 1 but also with Mal d 1. The latter reaction will usually be of lower affinity. In this example, Bet v 1 can be shown to be a more complete allergen than Mal d 1. Grass pollen extract does not at all inhibit the binding between IgE anti Bet v 1 and Mal d 1 (because grass pollen extract does not contain a cross-reactive Bet v 1 homologue). In this way, it is possible to rank allergen source materials (such as birch, apple, celery, and peanut) in a cross-reactivity hierarchy. This is most reliably done by using a quantitative bi-directional cross-inhibition protocol,⁴⁷ but less demanding protocols may also be informative. Some cross-reactions are relatively restricted (example: cross-reactivity among grasses). Others are broader (example: Bet v 1/Mal d 1, with much lower cross-reactivity to the homologous protein in peanut, celery and potato and no cross-reactivity with grasses). Others cross wider phylogenetic barriers (examples: cross-reactivity between pollen from birch and grass due to profilin⁴⁸a,⁴⁸b and cross-reactivity between shrimp and mites due to tropomyosin)⁴⁹. Among the glycan epitopes, CCDs tend to be even more cross-reactive (example: cross-reactivity between bee venom and potato).¹⁶ Glycan-based cross-reactivity is different from protein-based cross-reactivity because the degree and fine structure of glycosylation is variable among glycoproteins, even at the single-cell level.⁵⁰a It is not unusual to find that 2 allergen source materials share several distinct cross-reactive molecules. An example is the cross-reactivity among birch pollen, vegetables and fruits, which was found to be due to at least 3 cross-reactive structures.⁵⁰b

## 9 – Molecular sensitization profiles: biomarkers for disease progression?

A relatively unexplored area is whether IgE recognition profiles have predictive value for disease progression. This field will most likely develop rapidly in the near future. Some evidence from the field of food allergy suggests that recognition profiles of specific epitopes on major food allergens, using short synthetic peptides, can predict outgrowth or persistence.⁵⁶ Another study reported that persistence of peanut allergy is associated with the number of peanut allergens recognized.⁵⁷ More recently, IgE against Der p 1 and in particular Der p 23 at young age was shown to be predictive for development of asthma later in life²⁸. These studies are just the beginning and illustrate the importance of carefully dissecting molecular composition of allergen sources.

## 10 – Allergen extracts: imperfect but not yet obsolete

Both allergy diagnostics and allergen immunotherapy (AIT) still heavily depend on extracts of the allergen sources. In particular in diagnostics, molecular approaches are gaining ground⁵⁸ rapidly, but extracts can certainly not be dismissed.⁵⁹ Usually, allergen extracts are simple aqueous extracts of the crude allergen source. In most cases, extraction is carried out at neutral or close to neutral pH, followed by a defatting step, and dialysis. In the case of food extracts, the source material may sometimes be partially processed before extraction, for example, peanut meal of mildly roasted peanuts. What are the potential shortcomings of allergen extracts? Allergen sources are biologic products with inherent variability of composition. Extraction with aqueous buffers at neutral pH may not optimally extract all possible allergens, especially those that are lipid-soluble. This is particularly relevant for food extracts because the natural route of exposure through the stomach includes exposure to low pH. A good example of a food allergen that is not optimally extracted at neutral pH is LTP from legumes such as peanut and lentils.⁶⁰ This phenomenon may also be the explanation for the huge variability in LTP content reported for a series of commercially available skin test reagents for hazelnut allergy.⁶¹ Extraction at low pH has proven to be the solution. Another problem encountered when preparing diagnostic food extracts, in particular of fruits and vegetables, is that enzymatic oxidative processes are initiated when the food tissue is disrupted. In particular, Bet v 1-related food allergens such as Mal d 1 in apple, Pru p 1 in peach, or Cor a 1 in hazelnut are sensitive to these processes and they lose their IgE-binding capacity. Finally, the defatting step has been implicated in loss of lipophilic allergens such as oleosins in legumes, nuts, and seeds.⁶² Together, these
shortcomings are probably the main reason that skin testing for many
foods is performed using the poorly standardized but more sensitive
prick-to-prick method with fresh foods. To overcome the loss of sen-
sitivity for detecting IgE antibodies against hazelnut Cor a 1, the ex-
tract can effectively be improved by spiking with recombinant Cor a 1.
Although Good Manufacturing Practices (GMP) requirements do not
facilitate broad in vivo application of recombinant allergens, for exam-
ple, recombinant Mal d 1 has been successfully used in skin testing,
oral challenges and sublingual immunotherapy.⁶³⁻⁶⁵

11 – Conclusion

Overall, one can conclude that the multitude of factors influenc-
ing extract composition results in batch-to-batch and company-to-
company differences, which may lead to differences in the diagnostic
and therapeutic management of patients. For several reasons, this is

particularly true for skin test reagents. Traditionally, these products
are provided for free by allergen manufacturers to support the selec-
tion and subsequent sales of immunotherapy products. Regulatory
pressure now requires skin test reagents to be registered. This de-
velopment has resulted in many "less important" allergen specifici-
ties being removed from the market, because they would require too
big an investment in documentation of their clinical performance.
Potential solutions such as spiking with recombinant allergens are
not really an option either, because recombinant allergens used in
vivo need to be produced under GMP conditions and tested in toxic-
ity studies. Again, this is too large an investment. In the future the
number of skin test reagents available will therefore be rather limited,
and extracts for *in vitro* diagnosis will continue to be improved by the
use of different extraction methods and/or spiking. Increasingly, mo-
lecular diagnostics will supplement and partly replace extract-based
tests, to overcome the imperfections of extracts and facilitate im-
proved risk assessment and subsequent advice to patients.
shortcomings are probably the main reason that skin testing for many
foods is performed using the poorly standardized but more sensitive
prick-to-prick method with fresh foods. To overcome the loss of sen-
sitivity for detecting IgE antibodies against hazelnut Cor a 1, the ex-
tract can effectively be improved by spiking with recombinant Cor a 1.
Although Good Manufacturing Practices (GMP) requirements do not
facilitate broad in vivo application of recombinant allergens, for exam-
ple, recombinant Mal d 1 has been successfully used in skin testing,
oral challenges and sublingual immunotherapy.⁶³⁻⁶⁵

11 – Conclusion

Overall, one can conclude that the multitude of factors influenc-
ing extract composition results in batch-to-batch and company-to-
company differences, which may lead to differences in the diagnostic
and therapeutic management of patients. For several reasons, this is

particularly true for skin test reagents. Traditionally, these products
are provided for free by allergen manufacturers to support the selec-
tion and subsequent sales of immunotherapy products. Regulatory
pressure now requires skin test reagents to be registered. This de-
velopment has resulted in many "less important" allergen specifici-
ties being removed from the market, because they would require too
big an investment in documentation of their clinical performance.
Potential solutions such as spiking with recombinant allergens are
not really an option either, because recombinant allergens used in
vivo need to be produced under GMP conditions and tested in toxic-
ity studies. Again, this is too large an investment. In the future the
number of skin test reagents available will therefore be rather limited,
and extracts for *in vitro* diagnosis will continue to be improved by the
use of different extraction methods and/or spiking. Increasingly, mo-
lecular diagnostics will supplement and partly replace extract-based
tests, to overcome the imperfections of extracts and facilitate im-
proved risk assessment and subsequent advice to patients.
# A03 - Molecular allergy diagnostics in clinical practice

Thilo Jakob, Paolo M Matricardi, Olga Luengo and Jörg Kleine-Tebbe

## Highlights

* Diagnostic work-up for IgE-mediated allergic reactions/diseases starts with the history, followed by sensitization tests (skin, IgE, and basophil tests) and optional challenge tests.

* Molecular allergens for IgE testing provide additional information, particularly in polysensitized patients and with allergens of low abundance, low stability, or associated risks.

* IgE-reactivity to members of the same allergen family reflects the degree of protein homology and IgE cross-reactivity. If it is high, the relevance needs to be sorted out. In case it is low, selected IgE testing of other family members can provide additional information.

* Proper interpretation should complete diagnostic testing: positive sensitizations to allergen extracts or molecules are only clinically relevant in case of corresponding symptoms.

Conventional allergy diagnostics are based on detecting specific IgE antibodies in the blood or skin with reactivity for allergen extracts obtained from various allergen sources such as pollen grains, house dust mite, or cat dander. These extracts contain many components (glycosylated and nonglycosylated proteins, lipids, etc.), the majority of which are irrelevant for the allergic reaction and allergy diagnostics. Progress in molecular biology over the last 3 decades has allowed us to identify and characterize single allergens in detail at a molecular level. Large allergen data banks have been established (e.g., www.allergen.org, www.allergome.org) in which information on identified allergens is accessible for the scientific and medical community. Currently as of December 2022, more than 4900 different allergens (plus approx. 1500 isoforms) have been described (www.allergome.org), almost 1500 of which have been expressed as recombinant proteins. Many of these allergens have already and will become available for in vitro allergy diagnostics, either as highly purified native or recombinant proteins. The use of single allergenic molecules (instead of extracts) has introduced a new area of high-resolution molecular allergy diagnostics (also designated "component resolved diagnostics," CRD⁶⁶) and changed our understanding of sensitization profiles and cross-reactivity.⁶⁷ Daily routine molecular allergy diagnostics offers a number of benefits that give us a higher diagnostic precision and allow for better management of the patient. To utilize the full potential in clinical practice, an in-depth general knowledge of molecular allergology and a clear rationale for their use are needed as it relates to when and how allergenic molecules are to be used for diagnostic purposes ("always think molecular—use molecules, when needed"). This section

* summarizes general considerations for the diagnostic workup of our allergy patients in the age of molecular allergology,

* provides a number of universal reasons to utilize molecular diagnostics, and

* describes the rationale behind different approaches ("from symptoms to molecules"; "from molecules to symptoms") that allow us to make the optimal use of molecular allergy diagnostics in clinical practice.

## 1 - General considerations for the diagnostic work-up of allergy patients

In patients with suspected IgE-mediated reactions and/or diseases, the diagnostic algorithm should include the following sequential steps (Figures 1 and 2):

### A. Clinical evaluation and examination

a. allergy-related history including information on comorbidities, differential diagnoses.

b. clinical examination.

B. sensitization test(s) with allergen extracts, i.e., skin prick tests SPT,⁶⁸ slgE tests,⁶⁹ basophil activation tests,¹⁵⁶ providing information on allergic sensitization, i.e., the "risk for allergy."

B1. indirect or direct evidence of allergen-specific IgE

B3. interpretation of sensitization test result(s) (clinical relevance or not?).

B2. Sensitization test with allergenic molecules (applying allergen-specific IgE tests).

B2. direct evidence of present or absent allergen-specific IgE to defined allergens? *(slgE to the complete extract is needed to interpret the result of slgE to single allergenic molecules).

B3. interpretation of sensitisation test result(s) (clinical relevance or not?)

C. Challenge test (optional, depending on the allergen source in question).

a. demonstration of clinical symptoms upon allergen exposure.

b. interpretation (qualitative conclusion: positive or negative?

quantitative conclusion: ratio component slgE/whole extract-slgE allows to determine the extent to which a given allergenic component is responsible for sensitization to a whole extract⁷⁰).

For each of these steps certain general considerations may be helpful to make the best out of our expanding knowledge of the molecular nature of allergens. These considerations are listed below and combined with examples from clinical practice (small italic font).

### A. Some history-related information might immediately suggest certain underlying allergenic molecules ("think molecular"), due to

* temporal relationship of symptoms with particular exposures (i.e., pollen, furry animals, house dust, certain foods).

* patient's observation of certain triggers representing particular pattern (related triggers, i.e., indicative selection of foods),

* degree and variety of symptoms indicating involvement of certain molecules (either mild oropharyngeal or severe systemic
# A03 - Molecular allergy diagnostics in clinical practice

Thilo Jakob, Paolo M Matricardi, Olga Luengo and Jörg Kleine-Tebbe

## Highlights

* Diagnostic work-up for IgE-mediated allergic reactions/diseases starts with the history, followed by sensitization tests (skin, IgE, and basophil tests) and optional challenge tests.

* Molecular allergens for IgE testing provide additional information, particularly in polysensitized patients and with allergens of low abundance, low stability, or associated risks.

* IgE-reactivity to members of the same allergen family reflects the degree of protein homology and IgE cross-reactivity. If it is high, the relevance needs to be sorted out. In case it is low, selected IgE testing of other family members can provide additional information.

* Proper interpretation should complete diagnostic testing: positive sensitizations to allergen extracts or molecules are only clinically relevant in case of corresponding symptoms.

Conventional allergy diagnostics are based on detecting specific IgE antibodies in the blood or skin with reactivity for allergen extracts obtained from various allergen sources such as pollen grains, house dust mite, or cat dander. These extracts contain many components (glycosylated and nonglycosylated proteins, lipids, etc.), the majority of which are irrelevant for the allergic reaction and allergy diagnostics. Progress in molecular biology over the last 3 decades has allowed us to identify and characterize single allergens in detail at a molecular level. Large allergen data banks have been established (e.g., www.allergen.org, www.allergome.org) in which information on identified allergens is accessible for the scientific and medical community. Currently as of December 2022, more than 4900 different allergens (plus approx. 1500 isoforms) have been described (www.allergome.org), almost 1500 of which have been expressed as recombinant proteins. Many of these allergens have already and will become available for in vitro allergy diagnostics, either as highly purified native or recombinant proteins. The use of single allergenic molecules (instead of extracts) has introduced a new area of high-resolution molecular allergy diagnostics (also designated "component resolved diagnostics," CRD⁶⁶) and changed our understanding of sensitization profiles and cross-reactivity.⁶⁷ Daily routine molecular allergy diagnostics offers a number of benefits that give us a higher diagnostic precision and allow for better management of the patient. To utilize the full potential in clinical practice, an in-depth general knowledge of molecular allergology and a clear rationale for their use are needed as it relates to when and how allergenic molecules are to be used for diagnostic purposes ("always think molecular—use molecules, when needed"). This section

* summarizes general considerations for the diagnostic workup of our allergy patients in the age of molecular allergology,

* provides a number of universal reasons to utilize molecular diagnostics, and

* describes the rationale behind different approaches ("from symptoms to molecules"; "from molecules to symptoms") that allow us to make the optimal use of molecular allergy diagnostics in clinical practice.

## 1 - General considerations for the diagnostic work-up of allergy patients

In patients with suspected IgE-mediated reactions and/or diseases, the diagnostic algorithm should include the following sequential steps (Figures 1 and 2):

### A. Clinical evaluation and examination

a. allergy-related history including information on comorbidities, differential diagnoses.

b. clinical examination.

B. sensitization test(s) with allergen extracts, i.e., skin prick tests SPT,⁶⁸ slgE tests,⁶⁹ basophil activation tests,¹⁵⁶ providing information on allergic sensitization, i.e., the "risk for allergy."

B1. indirect or direct evidence of allergen-specific IgE

B3. interpretation of sensitization test result(s) (clinical relevance or not?).

B2. Sensitization test with allergenic molecules (applying allergen-specific IgE tests).

B2. direct evidence of present or absent allergen-specific IgE to defined allergens? *(slgE to the complete extract is needed to interpret the result of slgE to single allergenic molecules).

B3. interpretation of sensitisation test result(s) (clinical relevance or not?)

C. Challenge test (optional, depending on the allergen source in question).

a. demonstration of clinical symptoms upon allergen exposure.

b. interpretation (qualitative conclusion: positive or negative?

quantitative conclusion: ratio component slgE/whole extract-slgE allows to determine the extent to which a given allergenic component is responsible for sensitization to a whole extract⁷⁰).

For each of these steps certain general considerations may be helpful to make the best out of our expanding knowledge of the molecular nature of allergens. These considerations are listed below and combined with examples from clinical practice (small italic font).

### A. Some history-related information might immediately suggest certain underlying allergenic molecules ("think molecular"), due to

* temporal relationship of symptoms with particular exposures (i.e., pollen, furry animals, house dust, certain foods).

* patient's observation of certain triggers representing particular pattern (related triggers, i.e., indicative selection of foods),

* degree and variety of symptoms indicating involvement of certain molecules (either mild oropharyngeal or severe systemic
<figure><img src="image_12.png" /><figcaption>FIGURE 1 General diagnostic work-up of IgE-mediated allergic reactions and diseases. After collecting the allergy history and performing a physical examination (A) appropriate sensitization tests are applied (B1). IgE sensitizations are directly demonstrated by serological allergen-specific IgE determination⁶⁹ and/or indirectly by skin prick tests (SPT)⁶⁸ or basophil activation tests (BAT),¹⁵⁶ if indicated. Sensitization tests should be completed by careful interpretation (B1), validating the agreement with the history to ultimately evaluate the clinical relevance of the obtained results. In case of uncertainty (B1), i.e., due to a nonconclusive history, challenge tests are applied (C) to induce allergic symptoms under controlled conditions.⁷⁰,⁷¹ A clear outcome will support the decision on the clinical relevance of suspected allergen triggers and provide the basis for potential therapeutic consequences (D).</figcaption></figure>

symptoms to, i.e., legumes, tree nuts or seeds) (potential marker allergens).

Examples:

Oropharyngeal symptoms after eating raw apples, hazelnuts, carrots and/or soy and/or symptoms of allergic rhinoconjunctivitis during the birch pollen season: Suggestive for the presence of IgE to major birch pollen allergen Bet v 1-specific with subsequent serological (and clinical) cross-reactions (primary inhalant sensitization, but due to structural similarity of Bet v 1 and its homologues secondary symptoms in the oral cavity upon exposure).

Oropharyngeal symptoms after eating various (nonrelated) fruits and vegetables such as melon, citrus fruits, banana, avocado AND symptoms of allergic rhinoconjunctivitis during the grass pollen season: Suggestive for IgE to minor (grass) pollen allergen profilin (i.e., timothy grass profilin Phl p 12) with subsequent serological (and clinical?) cross-reactions. (nota bene: profilin sensitization is variable depending on the geographical region and more prevalent in Central and Southern Europe or other geographical areas with similar climate and vegetation)

Anaphylaxis in the context of exercise after consumption of wheat-containing food, which is suggestive for IgE to omega-5 gliadin Tri a 19.

B1. Some sensitization test results with extracts, either by SPT or serology, might immediately suggest certain underlying allergenic molecules, i.e.:

* particular pattern of sensitization, pointing to cross-reactive molecules

* unusual pattern or magnitude of sensitization test results (i.e. to non-related allergen sources)

Examples:

Positive reactions to fagales tree (hazel, alder, birch, beech, oak) pollen, potentially with symptoms during the tree pollen season:

Suggestive for the presence of IgE to the major birch pollen allergen Bet v 1 with subsequent serological (and potential clinical) cross-reactions to related fagales trees.

Positive reactions to nonrelated pollen plants, sometimes all pollen sources, with various, not necessarily corresponding symptoms: Suggestive for the presence of IgE to pan-pollen allergens (profilins, i.e., Bet v 2 or Phl p 12 and/or polcalcins, i.e., Bet v 4 or Phl p 7) with subsequent serological (and clinical?) cross-reactions to profilin-containing pollen and plant foods as well as polcalcin-containing pollen. In case of double sensitization to
<figure><img src="image_12.png" /><figcaption>FIGURE 1 General diagnostic work-up of IgE-mediated allergic reactions and diseases. After collecting the allergy history and performing a physical examination (A) appropriate sensitization tests are applied (B1). IgE sensitizations are directly demonstrated by serological allergen-specific IgE determination⁶⁹ and/or indirectly by skin prick tests (SPT)⁶⁸ or basophil activation tests (BAT),¹⁵⁶ if indicated. Sensitization tests should be completed by careful interpretation (B1), validating the agreement with the history to ultimately evaluate the clinical relevance of the obtained results. In case of uncertainty (B1), i.e., due to a nonconclusive history, challenge tests are applied (C) to induce allergic symptoms under controlled conditions.⁷⁰,⁷¹ A clear outcome will support the decision on the clinical relevance of suspected allergen triggers and provide the basis for potential therapeutic consequences (D).</figcaption></figure>

symptoms to, i.e., legumes, tree nuts or seeds) (potential marker allergens).

Examples:

Oropharyngeal symptoms after eating raw apples, hazelnuts, carrots and/or soy and/or symptoms of allergic rhinoconjunctivitis during the birch pollen season: Suggestive for the presence of IgE to major birch pollen allergen Bet v 1-specific with subsequent serological (and clinical) cross-reactions (primary inhalant sensitization, but due to structural similarity of Bet v 1 and its homologues secondary symptoms in the oral cavity upon exposure).

Oropharyngeal symptoms after eating various (nonrelated) fruits and vegetables such as melon, citrus fruits, banana, avocado AND symptoms of allergic rhinoconjunctivitis during the grass pollen season: Suggestive for IgE to minor (grass) pollen allergen profilin (i.e., timothy grass profilin Phl p 12) with subsequent serological (and clinical?) cross-reactions. (nota bene: profilin sensitization is variable depending on the geographical region and more prevalent in Central and Southern Europe or other geographical areas with similar climate and vegetation)

Anaphylaxis in the context of exercise after consumption of wheat-containing food, which is suggestive for IgE to omega-5 gliadin Tri a 19.

B1. Some sensitization test results with extracts, either by SPT or serology, might immediately suggest certain underlying allergenic molecules, i.e.:

* particular pattern of sensitization, pointing to cross-reactive molecules

* unusual pattern or magnitude of sensitization test results (i.e. to non-related allergen sources)

Examples:

Positive reactions to fagales tree (hazel, alder, birch, beech, oak) pollen, potentially with symptoms during the tree pollen season:

Suggestive for the presence of IgE to the major birch pollen allergen Bet v 1 with subsequent serological (and potential clinical) cross-reactions to related fagales trees.

Positive reactions to nonrelated pollen plants, sometimes all pollen sources, with various, not necessarily corresponding symptoms: Suggestive for the presence of IgE to pan-pollen allergens (profilins, i.e., Bet v 2 or Phl p 12 and/or polcalcins, i.e., Bet v 4 or Phl p 7) with subsequent serological (and clinical?) cross-reactions to profilin-containing pollen and plant foods as well as polcalcin-containing pollen. In case of double sensitization to
<figure><img src="image_13.png" /><figcaption>FIGURE 2 Diagnostic work-up with targeted (singleplex) molecular-based IgE testing. After sensitization testing (B1) with allergen extracts, more detailed information regarding the IgE repertoire is obtained with molecular-based testing (B2). This diagnostic approach, coined "top-down" is followed by a thorough interpretation (B3) including challenge tests (C), if needed.</figcaption></figure>

profilin AND polcalcin: commonly positive reactions to ALL pollen species can be expected, and it is not possible to define the precise sensitization specificity with allergen extracts.

Multiple sensitizations to different furry animals, which is suggestive of IgE reactivity to the animal pan allergen serum albumin or certain lipocalins.

B2. Tests for allergen-specific IgE to molecules (Figure 2) can be applied with one reagent as single test (singleplex)⁶⁹ or with many reagents (multiplex)⁷¹ for, i.e., screening purposes (see chapter A04 for more technical information). Reasons for molecular IgE testing, either singleplex or multiplex, will be summarized below (see also paragraph 5 of this chapter). Following options will be extensively discussed in the following sections:

* "Classical" diagnostic work-up (Figure 1) with extract-based sensitization test(s), before employing allergen-specific IgE testing with molecules (Figure 2) ("top-down approach," paragraph 3 of this chapter).

* Novel diagnostic work-up with primarily allergen molecule-related information, i.e., allergen-specific IgE to a panel of related molecules to explain diverse clinical reactions or diseases ("bottom-up approach," paragraph 4 of this chapter).

* Integrated use of both approaches, first working from the history, applying extract-based sensitization tests (SPT, IgE) before exploring the entire individual IgE repertoire with an extended panel of allergen molecules ("U-shape approach," paragraph 5 of this chapter).

C. Finally, if the information provided by the patient's history and/or the sensitization test results is inconclusive and does not allow for a clear decision on the clinical relevance of the suspected allergen source, additional challenge tests should be applied. They should ultimately demonstrate or rule out clinical symptoms following allergen exposure.

In case of inhalants (pollen, mites, molds, furry animals) standardized extracts of the suspected allergen source are applied on the mucosal surface (i.e., conjunctiva, nose).⁷² In case of food allergy (plant foods, i.e., fruits, vegetables, legumes) increasing doses of the suspected allergen are given orally, ideally in a double-blind, placebo-controlled fashion.⁷³,⁷⁴

Subsequent immediate or delayed (i.e., exacerbation of an atopic eczema) type allergic symptoms would prove current clinical relevance; by contrast, a lack of any objective clinical reaction would rule out a previously suspected allergy (provided that no additional co-factors are required to elicit the allergic reaction, as exemplified by all forms of food-dependent, exercise induced anaphylaxis.⁷⁵
<figure><img src="image_13.png" /><figcaption>FIGURE 2 Diagnostic work-up with targeted (singleplex) molecular-based IgE testing. After sensitization testing (B1) with allergen extracts, more detailed information regarding the IgE repertoire is obtained with molecular-based testing (B2). This diagnostic approach, coined "top-down" is followed by a thorough interpretation (B3) including challenge tests (C), if needed.</figcaption></figure>

profilin AND polcalcin: commonly positive reactions to ALL pollen species can be expected, and it is not possible to define the precise sensitization specificity with allergen extracts.

Multiple sensitizations to different furry animals, which is suggestive of IgE reactivity to the animal pan allergen serum albumin or certain lipocalins.

B2. Tests for allergen-specific IgE to molecules (Figure 2) can be applied with one reagent as single test (singleplex)⁶⁹ or with many reagents (multiplex)⁷¹ for, i.e., screening purposes (see chapter A04 for more technical information). Reasons for molecular IgE testing, either singleplex or multiplex, will be summarized below (see also paragraph 5 of this chapter). Following options will be extensively discussed in the following sections:

* "Classical" diagnostic work-up (Figure 1) with extract-based sensitization test(s), before employing allergen-specific IgE testing with molecules (Figure 2) ("top-down approach," paragraph 3 of this chapter).

* Novel diagnostic work-up with primarily allergen molecule-related information, i.e., allergen-specific IgE to a panel of related molecules to explain diverse clinical reactions or diseases ("bottom-up approach," paragraph 4 of this chapter).

* Integrated use of both approaches, first working from the history, applying extract-based sensitization tests (SPT, IgE) before exploring the entire individual IgE repertoire with an extended panel of allergen molecules ("U-shape approach," paragraph 5 of this chapter).

C. Finally, if the information provided by the patient's history and/or the sensitization test results is inconclusive and does not allow for a clear decision on the clinical relevance of the suspected allergen source, additional challenge tests should be applied. They should ultimately demonstrate or rule out clinical symptoms following allergen exposure.

In case of inhalants (pollen, mites, molds, furry animals) standardized extracts of the suspected allergen source are applied on the mucosal surface (i.e., conjunctiva, nose).⁷² In case of food allergy (plant foods, i.e., fruits, vegetables, legumes) increasing doses of the suspected allergen are given orally, ideally in a double-blind, placebo-controlled fashion.⁷³,⁷⁴

Subsequent immediate or delayed (i.e., exacerbation of an atopic eczema) type allergic symptoms would prove current clinical relevance; by contrast, a lack of any objective clinical reaction would rule out a previously suspected allergy (provided that no additional co-factors are required to elicit the allergic reaction, as exemplified by all forms of food-dependent, exercise induced anaphylaxis.⁷⁵
Subsequently, interpretation needs to be integral part of any sus-
pected sensitization (i.e., demonstrated by SPT, IgE, BAT) as well of a
challenge test outcome (Figure 1).

* A positive test result is only clinically relevant in the case of corresponding allergic symptoms that are temporally associated with a defined allergen exposure.

* A negative test (i.e., allergen-specific IgE) result against one recombinant allergen molecule or a mixture of natural isoforms of one single allergen can indicate exclusion of allergic sensitization or risk of allergy to the allergen in question (see paragraph 5 of this chapter for details), provided that

a. the total IgE is high enough (i.e., > 20 kU/l)

b. the allergen reagent is of sufficient abundance, fully intact, and presenting all its epitopes

c. the analytical performance of the IgE antibody assay has been optimized for a low limit of quantitation (i.e., 0.1 kU/L).

Electronic clinical diaries (e-Diaries) have been recently proposed as an additional diagnostic tool to establish clinical relevance of IgE sensitization to major allergenic molecules of pollen.⁷⁶ The use of e-Diaries has been integrated in a novel diagnostic algorithm, combining CRD with digital health, for etiological diagnosis and allergen immunotherapy (AIT) prescription (@IT.2020). The implementation of this algorithm has been tested in a multicenter study in several southern European countries, where polysensitization to cross-reactive pollen and overlapping pollen season make the etiological diagnosis difficult.⁷⁷

In conclusion, the clinical relevance of an allergic sensitization (i.e., presence of allergen-specific IgE, independent of the use of alergen extracts or molecules for diagnostic purposes) can ultimately only be determined by the physician and not by the test. Therefore, the complete diagnostic results of sensitizations and challenge test results will always have to be interpreted within the clinical context and on the basis of the individual's case history.

2 – Common reasons to utilize molecular diagnostics and their limitations

There are several general reasons that speak in favor of using sin-
gle allergens as compared to allergen extracts (Figure 3). They are
principally related to an improved assay performance (i.e., assay
sensitivity and analytical specificity) and/or to additional levels of
interpretation such as risk assessment or differentiation between
genuine ("primary") sensitization and cross-reactivity, particularly
in presumed polysensitizations. While these arguments clearly
support the use of single allergens in clinical routine, we need to
be careful not to over interpret results of molecular allergy diag-
nostics, which have clear limitations when it comes to predicting
clinical outcomes. slgE test results—regardless of using extracts or
single molecules—only reflect the status of sensitization and al-
ways must be interpreted in the context of the clinical data. The
benefits and limitations of molecular allergy diagnostics are out-
lined below.

**FIGURE 3** Utility of allergen extracts and allergenic molecules for diagnostic work-up. Mono/limited oligo sensitizations (A) and/or minor clinical risks (B) as well as high abundance allergen molecules in the allergen source suspected (C) and/or allergens of high stability (D) indicate suitability of allergen extracts for proper diagnostic work-up. In case of polysensitizations and/or allergen triggers associated with high clinical risks as well as low abundant and/or labile allergenic molecules in the extract, the diagnostic work-up should consider the use of molecular components for IgE detection.

<figure><img src="image_14.png" /></figure>
Subsequently, interpretation needs to be integral part of any sus-
pected sensitization (i.e., demonstrated by SPT, IgE, BAT) as well of a
challenge test outcome (Figure 1).

* A positive test result is only clinically relevant in the case of corresponding allergic symptoms that are temporally associated with a defined allergen exposure.

* A negative test (i.e., allergen-specific IgE) result against one recombinant allergen molecule or a mixture of natural isoforms of one single allergen can indicate exclusion of allergic sensitization or risk of allergy to the allergen in question (see paragraph 5 of this chapter for details), provided that

a. the total IgE is high enough (i.e., > 20 kU/l)

b. the allergen reagent is of sufficient abundance, fully intact, and presenting all its epitopes

c. the analytical performance of the IgE antibody assay has been optimized for a low limit of quantitation (i.e., 0.1 kU/L).

Electronic clinical diaries (e-Diaries) have been recently proposed as an additional diagnostic tool to establish clinical relevance of IgE sensitization to major allergenic molecules of pollen.⁷⁶ The use of e-Diaries has been integrated in a novel diagnostic algorithm, combining CRD with digital health, for etiological diagnosis and allergen immunotherapy (AIT) prescription (@IT.2020). The implementation of this algorithm has been tested in a multicenter study in several southern European countries, where polysensitization to cross-reactive pollen and overlapping pollen season make the etiological diagnosis difficult.⁷⁷

In conclusion, the clinical relevance of an allergic sensitization (i.e., presence of allergen-specific IgE, independent of the use of alergen extracts or molecules for diagnostic purposes) can ultimately only be determined by the physician and not by the test. Therefore, the complete diagnostic results of sensitizations and challenge test results will always have to be interpreted within the clinical context and on the basis of the individual's case history.

2 – Common reasons to utilize molecular diagnostics and their limitations

There are several general reasons that speak in favor of using single allergens as compared to allergen extracts (Figure 3). They are principally related to an improved assay performance (i.e., assay sensitivity and analytical specificity) and/or to additional levels of interpretation such as risk assessment or differentiation between genuine ("primary") sensitization and cross-reactivity, particularly in presumed polysensitizations. While these arguments clearly support the use of single allergens in clinical routine, we need to be careful not to over interpret results of molecular allergy diagnostics, which have clear limitations when it comes to predicting clinical outcomes. slgE test results—regardless of using extracts or single molecules—only reflect the status of sensitization and always must be interpreted in the context of the clinical data. The benefits and limitations of molecular allergy diagnostics are outlined below.

**FIGURE 3** Utility of allergen extracts and allergenic molecules for diagnostic work-up. Mono/limited oligo sensitizations (A) and/or minor clinical risks (B) as well as high abundance allergen molecules in the allergen source suspected (C) and/or allergens of high stability (D) indicate suitability of allergen extracts for proper diagnostic work-up. In case of polysensitizations and/or allergen triggers associated with high clinical risks as well as low abundant and/or labile allergenic molecules in the extract, the diagnostic work-up should consider the use of molecular components for IgE detection.

<figure><img src="image_14.png" /></figure>
**2a Factors improving assay performance are able to warrant the use of allergenic molecules instead of extracts:**

**Molecules of low abundance and/or weak stability**

If allergen molecules, being of low abundance or missing in the ex-
tract, can improve the assay's analytical sensitivity (LoQ) of an IgE
test, their use is meaningful and important. (i.e., Gly m 4 vs soy ex-
tract, omega-5-gliadin vs wheat extract).

**Risk- or severity-associated molecules**

If allergen molecules provide improved analytical specificity ("selec-
tivity") and allow additional clinical assumption(s) (i.e., increased risk
association, clinical severity, or other associated clinical features of an
IgE sensitization), their use is a gain meaningful and recommended (i.e.,
storage proteins Ara h 1, 2, 3, 6 vs whole peanut extract).

**Indicator of cross-reactivity**

Certain allergen molecules can serve as indicators for serological
cross-sensitizations through the binding of cross-reactive IgE. In
case of a positive result, they can demonstrate the lack of analytical
specificity of an IgE test with allergen extracts (in affected subjects
with potential cross-reactions) (e.g., profilin or polcalcin, members of
plant pan-allergen families).

**Marker of genuine (species-specific) sensitization**

Particular allergen molecules (often major allergens) can serve as mark-
ers for a primary, "genuine," family- or species-specific sensitization.
They provide improved analytical specificity compared with allergen
extracts (particularly in affected subjects with potential cross-reactions)
(e.g., marker allergens Ves v 1 and Ves v 5 from yellow jacket venom and
marker allergens Api m 1, Api m 3, Api m 10 from honey bee venom vs hy-
menoptera whole venom preparations from the corresponding species). The
above rationale is primarily based on the status of sensitization (pres-
ence or absence of IgE antibody) and not on the clinical manifestations
of the subjects. Examples are given in more detail below.

**2b Limitations in improving predictions on clinical outcome from (isolated) molecule-based sensitization test results**

In contrast to parameters that describe the analytical assay per-
formance (analytical sensitivity, analytical specificity, see above),
clinical diagnostic criteria are required when it comes to making pre-
dictions on the clinical outcome. General clinical diagnostic criteria
in the field of (molecular) allergology include:

I diagnostic sensitivity (proportion of positive IgE antibody tests in patients with allergic symptoms/disease).

II diagnostic specificity (proportion of negative IgE antibody tests in asymptomatic/healthy individuals).

III indicator of clinical cross-reactivity (allergic symptoms to aller-
genic sources that did not elicit the primary sensitization).

IV prediction of clinical reactions (positive predictive value, PPV, neg-
ative predictive value, NPV, thresholds, likelihood ratio etc.).

All of the above criteria require a thorough individual inter-
pretation of each test result based on the previous history and if

needed additional proof of reproducible and objective symptoms
in the affected allergic subject upon exposure (i.e., challenge test).
Subsequently, these clinical criteria will go beyond the essential
("raw") assay result of an allergen-specific IgE test (IgE sensitization
in question: yes or no). In general, clinical diagnostic criteria are:

* less suitable and sometimes misleading for a proper assay evaluation of sensitization tests

* largely not needed to demonstrate the advantage of single allergic molecules in IgE assays and

* often burdened with unsatisfactory study results due to the per se limited and imperfect prediction of clinical outcomes (clinical reactivity) by sensitization tests.

In conclusion, the above listed advantages of molecular allergy
diagnostics mostly refer to an improved detection and discrimination
of allergic sensitization. Molecular allergy diagnostics, however, have
clear limitations for improving predictions of the clinical outcome.
After all, the detection of slgE is primarily an indicator of "sensitiza-
tion" and—despite various attempts to integrate clinical data and re-
sults of challenge tests—not a decisive predictor of clinical reactivity.

**3 – From symptoms to molecules: the “top-down approach”**

Based on the experience that detection of allergen-specific IgE does not equal clinical relevance, current guidelines on allergy diagnostics recommend that the diagnostic workup should be primarily guided by the clinical symptoms. Random screening for IgE sensitization is discouraged since the number of positive IgE results to a certain allergen source usually exceeds by far the number of clinically relevant allergies.⁷⁸ This “top-down” approach—from the symptoms to the allergen source also applies to molecular allergy diagnostics and can be defined as follows:

*Definition:* Diagnostic work-up from symptoms to molecules ("top-down approach," Figure 2) aims for more detailed characterization of the IgE repertoire unfolding important molecular IgE sensitizations that provide information beyond the extract-based test results.

In practice, taking the case history and performing a symptom
guided diagnostic work-up with extract-based SPT and/or IgE test-
ing usually allows the identification (or exclusion) of IgE sensitiza-
tions to potentially involved allergen sources (Figure 1). Here two
main scenarios are usually encountered:

A. Limited numbers of positive extract-based sensitization test results

In case of rather restricted IgE antibody responses with only few
positive results to inhalants like tree or grass or weed pollen, cer-
tain molds, one or two furry animals, only a single insect venom
(bee or wasp venom) or only selected food items, the analytical
specificity of an extract-based sensitization test might be suffi-
cient to identify the underlying allergen source. No further testing
would be required, if the extract-based sensitization test permits
**2a Factors improving assay performance are able to warrant the use of allergenic molecules instead of extracts:**

**Molecules of low abundance and/or weak stability**

If allergen molecules, being of low abundance or missing in the ex-
tract, can improve the assay's analytical sensitivity (LoQ) of an IgE
test, their use is meaningful and important. (i.e., Gly m 4 vs soy ex-
tract, omega-5-gliadin vs wheat extract).

**Risk- or severity-associated molecules**

If allergen molecules provide improved analytical specificity ("selec-
tivity") and allow additional clinical assumption(s) (i.e., increased risk
association, clinical severity, or other associated clinical features of an
IgE sensitization), their use is a gain meaningful and recommended (i.e.,
storage proteins Ara h 1, 2, 3, 6 vs whole peanut extract).

**Indicator of cross-reactivity**

Certain allergen molecules can serve as indicators for serological
cross-sensitizations through the binding of cross-reactive IgE. In
case of a positive result, they can demonstrate the lack of analytical
specificity of an IgE test with allergen extracts (in affected subjects
with potential cross-reactions) (e.g., profilin or polcalcin, members of
plant pan-allergen families).

**Marker of genuine (species-specific) sensitization**

Particular allergen molecules (often major allergens) can serve as mark-
ers for a primary, "genuine," family- or species-specific sensitization.
They provide improved analytical specificity compared with allergen
extracts (particularly in affected subjects with potential cross-reactions)
(e.g., marker allergens Ves v 1 and Ves v 5 from yellow jacket venom and
marker allergens Api m 1, Api m 3, Api m 10 from honey bee venom vs hy-
menoptera whole venom preparations from the corresponding species). The
above rationale is primarily based on the status of sensitization (pres-
ence or absence of IgE antibody) and not on the clinical manifestations
of the subjects. Examples are given in more detail below.

**2b Limitations in improving predictions on clinical outcome from (isolated) molecule-based sensitization test results**

In contrast to parameters that describe the analytical assay per-
formance (analytical sensitivity, analytical specificity, see above),
clinical diagnostic criteria are required when it comes to making pre-
dictions on the clinical outcome. General clinical diagnostic criteria
in the field of (molecular) allergology include:

I diagnostic sensitivity (proportion of positive IgE antibody tests in patients with allergic symptoms/disease).

II diagnostic specificity (proportion of negative IgE antibody tests in asymptomatic/healthy individuals).

III indicator of clinical cross-reactivity (allergic symptoms to aller-
genic sources that did not elicit the primary sensitization).

IV prediction of clinical reactions (positive predictive value, PPV, neg-
ative predictive value, NPV, thresholds, likelihood ratio etc.).

All of the above criteria require a thorough individual inter-
pretation of each test result based on the previous history and if

needed additional proof of reproducible and objective symptoms
in the affected allergic subject upon exposure (i.e., challenge test).
Subsequently, these clinical criteria will go beyond the essential
("raw") assay result of an allergen-specific IgE test (IgE sensitization
in question: yes or no). In general, clinical diagnostic criteria are:

* less suitable and sometimes misleading for a proper assay evaluation of sensitization tests

* largely not needed to demonstrate the advantage of single allergenic molecules in IgE assays and

* often burdened with unsatisfactory study results due to the per se limited and imperfect prediction of clinical outcomes (clinical reactivity) by sensitization tests.

In conclusion, the above listed advantages of molecular allergy
diagnostics mostly refer to an improved detection and discrimination
of allergic sensitization. Molecular allergy diagnostics, however, have
clear limitations for improving predictions of the clinical outcome.
After all, the detection of slgE is primarily an indicator of "sensitiza-
tion" and—despite various attempts to integrate clinical data and re-
sults of challenge tests—not a decisive predictor of clinical reactivity.

**3 – From symptoms to molecules: the “top-down approach”**

Based on the experience that detection of allergen-specific IgE does not equal clinical relevance, current guidelines on allergy diagnostics recommend that the diagnostic workup should be primarily guided by the clinical symptoms. Random screening for IgE sensitization is discouraged since the number of positive IgE results to a certain allergen source usually exceeds by far the number of clinically relevant allergies.⁷⁸ This “top-down” approach—from the symptoms to the allergen source also applies to molecular allergy diagnostics and can be defined as follows:

*Definition:* Diagnostic work-up from symptoms to molecules ("top-down approach," Figure 2) aims for more detailed characterization of the IgE repertoire unfolding important molecular IgE sensitizations that provide information beyond the extract-based test results.

In practice, taking the case history and performing a symptom
guided diagnostic work-up with extract-based SPT and/or IgE test-
ing usually allows the identification (or exclusion) of IgE sensitiza-
tions to potentially involved allergen sources (Figure 1). Here two
main scenarios are usually encountered:

A. Limited numbers of positive extract-based sensitization test results

In case of rather restricted IgE antibody responses with only few
positive results to inhalants like tree or grass or weed pollen, cer-
tain molds, one or two furry animals, only a single insect venom
(bee or wasp venom) or only selected food items, the analytical
specificity of an extract-based sensitization test might be suffi-
cient to identify the underlying allergen source. No further testing
would be required, if the extract-based sensitization test permits
a proper and specific diagnostic work-up. The exceptions are potentially false negative sensitization tests in case of underrepresented or unstable single allergens.

B Broad panel of positive extract-based sensitization test results More frequently we encounter the scenario in which rather broad IgE antibody responses occur with many positive results to extracts from inhalants or reported symptoms to many (plant) foods. This indicates possible cross-reactivities and a lack of analytical specificity of the extract-based test approach.

In this setting, further work-up ("top-down approach," Figure 2) with allergen molecules may allow a more detailed and meaningful characterization of the IgE repertoire, identifying important molecular IgE sensitizations. Examples of situations in which molecular allergy diagnostics provide additional information beyond the extract-based tests are listed below:

### 3.1 Examples of situations for further molecular diagnostic work-up

3.1.a. Allergen source with potentially competing clinically relevant allergen sources

* Multiple sensitizations to (nonrelated) pollen species (i.e., from trees, grasses, weeds) with overlapping seasons

Examples: pollinating plants (trees, grasses, weeds) with overlapping seasons. Here the use of marker allergens and pan allergens allows discrimination between genuine sensitization and cross-reactivity.

* Symptoms to multiple (nonrelated) plant foods due to potential cross-reactivity.

Examples:

a. apples, hazelnuts, cherries, plums, peaches, carrots, soy (suggesting Bet v 1-cluster, predominantly in the northern hemisphere with birch trees)

b. melon, banana, apples, nuts, peanut, citrus and others (suggesting profilin-cluster, often due to high regional grass pollen exposure)

c. peach, apple, lettuce, green bean, tree nuts, peanut and others (suggesting LTP-cluster, mainly in the Mediterranean region).

- Multiple sensitizations to furry animals (with potential clinical consequences) Examples: i.e., cats, dogs, horses, small furry animals (suggesting serum albumin or certain lipocalins as cross-reacting allergens).

3.1.b. Allergen source with a variety of different single allergens, either resembling cross-reactive or genuine molecules.

* Anaphylactic Hymenoptera sting reaction and sensitization to both honey bee and yellow jacket venom.

* Both allergen sources contain potentially cross-reactive allergens such as Api m 2, Ves v 2 (hyaluronidases), Api m 5, Ves v 3 (dipeptidyl peptidases), Api m 12, Ves v 6 (vitellogenins), and marker allergens that are specific for honey bee venom (Api m 1, Api m 3, Api m 4, Api m 10) or yellow jacket venom (Ves v 1, Ves v 5). Use of marker allergens allows discrimination between genuine sensitization and cross-reactivity and may be important to prevent potential adverse reactions from immunotherapy or predict lack of allergen immunotherapy (AIT) efficacy

* Variable symptoms to certain plant foods

* Examples: fruits, vegetable

* Severe reactions to plant foods Examples: peanut, soy, tree nuts, seeds.

#### 3.1.c. Immunotherapy prescription?

In the case of specific immunotherapy prescription it may also be relevant to assess if the sensitization to the allergenic source is mostly at the expense of the major allergen quantified and standardized in the commercial extract.

### 3.2. Criteria for selecting appropriate molecules (from an allergen source) (Figure 5). The general reasons given in section 2.a provide criteria to select certain molecules for further diagnostic work-up:

Bold letters indicate availability as reagents mainly non-USA, (e.g., Europe, Japan); regular letters: not (yet) available as reagents (see also section 2.a for explanations).

#### Molecules of low abundance and/or weak stability

Examples: use major birch pollen allergen Bet v 1 as a representative to demonstrate potential cross-reactivities to low abundant, labile Bet v 1-homologues, i.e., Cor a 1.04 (hazelnut), Act d 8 (kiwi), Pru p 1 (peach), Gly m 4 (soy), and others. Other examples of presently not well represented allergens are the peanut allergens Ara h 10, Ara h 11, Ara h 14, Ara h 15 (oleosins), the wheat allergen Tri a 19 (omega-5-gliadin), and natural rubber latex allergen (Hev b 5 (acidic structural protein)).

#### Risk- or severity-associated molecules

Examples: 2S albumins, i.e., Ara h 2, Ara h 6/7 (peanut), Cor a 14 (hazelnut), Gly m 8 (soy), other seed storage proteins Ara h 1, Ara h 3, Cor a 9, Cor a 11, Gly m 5, Gly m 6, nsLTP, i.e., Pru p 3 (representative LTP marker allergen in peach, Mediterranean), Cor a 8 (hazelnut, Mediterranean), Ara h 9 (peanut, Mediterranean), other examples: alpha-Gal (delayed type red meat allergy).

#### Indicators of cross-reactivity

Examples: Fel d 2, Can f 3, Equ c 3 (serum albumins); Bet v 1, Act d 8, Ara h 8, Pru p 1, (Bet v 1-homologues); Amb a 8, Ara h 5, Art v 4, Bet v 2, Ole e 2*, Phl p 12, Pru p 4 (profilins, pan-allergen in pollen and plant foods); Amb a 10, Art v 5, Bet v 4, Ole e 3, Phl p 7 (polcalcins, pan-allergen in pollen); CCD (cross-reactive carbohydrate determinants)
a proper and specific diagnostic work-up. The exceptions are potentially false negative sensitization tests in case of underrepresented or unstable single allergens.

B Broad panel of positive extract-based sensitization test results More frequently we encounter the scenario in which rather broad IgE antibody responses occur with many positive results to extracts from inhalants or reported symptoms to many (plant) foods. This indicates possible cross-reactivities and a lack of analytical specificity of the extract-based test approach.

In this setting, further work-up ("top-down approach," Figure 2) with allergen molecules may allow a more detailed and meaningful characterization of the IgE repertoire, identifying important molecular IgE sensitizations. Examples of situations in which molecular allergy diagnostics provide additional information beyond the extract-based tests are listed below:

### 3.1 Examples of situations for further molecular diagnostic work-up

3.1.a. Allergen source with potentially competing clinically relevant allergen sources

* Multiple sensitizations to (nonrelated) pollen species (i.e., from trees, grasses, weeds) with overlapping seasons

Examples: pollinating plants (trees, grasses, weeds) with overlapping seasons. Here the use of marker allergens and pan allergens allows discrimination between genuine sensitization and cross-reactivity.

* Symptoms to multiple (nonrelated) plant foods due to potential cross-reactivity.

Examples:

a. apples, hazelnuts, cherries, plums, peaches, carrots, soy (suggesting Bet v 1-cluster, predominantly in the northern hemisphere with birch trees)

b. melon, banana, apples, nuts, peanut, citrus and others (suggesting profilin-cluster, often due to high regional grass pollen exposure)

c. peach, apple, lettuce, green bean, tree nuts, peanut and others (suggesting LTP-cluster, mainly in the Mediterranean region).

- Multiple sensitizations to furry animals (with potential clinical consequences) Examples: i.e., cats, dogs, horses, small furry animals (suggesting serum albumin or certain lipocalins as cross-reacting allergens).

3.1.b. Allergen source with a variety of different single allergens, either resembling cross-reactive or genuine molecules.

* Anaphylactic Hymenoptera sting reaction and sensitization to both honey bee and yellow jacket venom.

* Both allergen sources contain potentially cross-reactive allergens such as Api m 2, Ves v 2 (hyaluronidases), Api m 5, Ves v 3 (dipeptidyl peptidases), Api m 12, Ves v 6 (vitellogenins), and marker allergens that are specific for honey bee venom (Api m 1, Api m 3, Api m 4, Api m 10) or yellow jacket venom (Ves v 1, Ves v 5). Use of marker allergens allows discrimination between genuine sensitization and cross-reactivity and may be important to prevent potential adverse reactions from immunotherapy or predict lack of allergen immunotherapy (AIT) efficacy

* Variable symptoms to certain plant foods

* Examples: fruits, vegetable

* Severe reactions to plant foods Examples: peanut, soy, tree nuts, seeds.

#### 3.1.c. Immunotherapy prescription?

In the case of specific immunotherapy prescription it may also be relevant to assess if the sensitization to the allergenic source is mostly at the expense of the major allergen quantified and standardized in the commercial extract.

### 3.2. Criteria for selecting appropriate molecules (from an allergen source) (Figure 5). The general reasons given in section 2.a provide criteria to select certain molecules for further diagnostic work-up:

Bold letters indicate availability as reagents mainly non-USA, (e.g., Europe, Japan); regular letters: not (yet) available as reagents (see also section 2.a for explanations).

#### Molecules of low abundance and/or weak stability

Examples: use major birch pollen allergen Bet v 1 as a representative to demonstrate potential cross-reactivities to low abundant, labile Bet v 1-homologues, i.e., Cor a 1.04 (hazelnut), Act d 8 (kiwi), Pru p 1 (peach), Gly m 4 (soy), and others. Other examples of presently not well represented allergens are the peanut allergens Ara h 10, Ara h 11, Ara h 14, Ara h 15 (oleosins), the wheat allergen Tri a 19 (omega-5-gliadin), and natural rubber latex allergen (Hev b 5 (acidic structural protein)).

#### Risk- or severity-associated molecules

Examples: 2S albumins, i.e., Ara h 2, Ara h 6/7 (peanut), Cor a 14 (hazelnut), Gly m 8 (soy), other seed storage proteins Ara h 1, Ara h 3, Cor a 9, Cor a 11, Gly m 5, Gly m 6, nsLTP, i.e., Pru p 3 (representative LTP marker allergen in peach, Mediterranean), Cor a 8 (hazelnut, Mediterranean), Ara h 9 (peanut, Mediterranean), other examples: alpha-Gal (delayed type red meat allergy).

#### Indicators of cross-reactivity

Examples: Fel d 2, Can f 3, Equ c 3 (serum albumins); Bet v 1, Act d 8, Ara h 8, Pru p 1, (Bet v 1-homologues); Amb a 8, Ara h 5, Art v 4, Bet v 2, Ole e 2*, Phl p 12, Pru p 4 (profilins, pan-allergen in pollen and plant foods); Amb a 10, Art v 5, Bet v 4, Ole e 3, Phl p 7 (polcalcins, pan-allergen in pollen); CCD (cross-reactive carbohydrate determinants)
## Markers of genuine (species-specific) sensitization

Examples: Fel d 1 (cat), Api m 1, Api m 3, Api m 4, Api m 10 (honey bee venom), Ves v 1, Ves v 5 (Vespula species), Bet v 1 (fagales), Ole e 1 (olive tree, plantane), Phl p 1, Phl p 5 (grass) Art v 1 (mugwort), Amb a 1 (ragweed), Par j 2 (pellitory).

In summary, after taking the history and performing extract-based sensitization tests a diagnostic work-up including specific IgE to allergenic molecules is useful to increase assay sensitivity for single allergens of low abundance (in extracts) or weak stability. An increased analytical specificity will help to identify risk- or severity-associated allergens, indicators for cross-reactivity and marker allergens of genuine (primary) sensitization.

Interpretation is an integral part of each sensitization test: Positive results are only clinically relevant in case of corresponding symptoms; negative results can ideally rule out an allergic sensitization and subsequent clinical reaction to the tested allergen.

## 4 – From molecules to symptoms: the “bottom-up approach”

Instead of performing symptom-oriented focused molecular allergy diagnostics (“top-down approach,” see above), one can simply turn this approach around and start from the bottom, i.e., with the molecules.⁷⁹ In an ideal scenario, diagnostic tools would allow us to characterize the entire IgE repertoire to all potential allergens that

a patient has been exposed to. It would then be conceivable that we first analyze the entire IgE repertoire and then start talking to the patient to find out which of the detected IgE sensitizations are clinically relevant. This would be a “broad bottom-up approach,” i.e., turning the diagnostic pyramid upside down. However, a number of reasons suggest that in real life this is not appropriate: (a) We are far from being able to characterize the entire IgE repertoire, i.e., the individual IgE response to the entire allergome—currently only approx. 200 of the 3000 known allergens are available for diagnostic purposes. (b) An entire IgE repertoire characterization would be exceedingly expensive and yield enormous amounts of information that require processing and interpretation. (c) At present molecular allergy research attempts and multiplex technologies still depend on the availability of allergens for diagnosis. Many research projects have so far focused on certain molecules, i.e., Bet v 1-homologous proteins in various sources, leading to a broad spectrum of available proteins. However, this does not mean that this group is more relevant than other allergens to which less attention was paid in the past or which are more difficult to be produced as recombinant allergens. (d) Finally, the number of positive IgE results to a certain allergen source usually exceeds by far the number of clinically relevant allergies. Screening the IgE response to the entire allergome thus would most likely result in generation of large proportions of positive test results that have no clinical relevance (as it would be the case of performing extensive extract-based skin prick tests without the guidance of a previous thorough clinical history).

<figure><img src="image_15.png" /><figcaption>FIGURE 4 Diagnostic work-up with broad (multiplex) molecular-based IgE testing: In complex cases and/or inconclusive diagnostic outcomes after previous testing (A) a panel of molecular allergens might be applied for subsequent (multiplex) IgE testing (B2). After final interpretation (B3) with an optional challenge (C) this approach, coined “bottom-up,” might facilitate improved decisions on therapeutic consequences.</figcaption></figure>