*Article* **Threshold of Reactivity and Tolerance to Precautionary Allergen-Labelled Biscuits of Baked Milk- and Egg-Allergic Children**

**Vincenzo Fierro 1,†, Valeria Marzano 2,†, Linda Monaci <sup>3</sup> , Pamela Vernocchi <sup>2</sup> , Maurizio Mennini <sup>1</sup> , Rocco Valluzzi <sup>1</sup> , Stefano Levi Mortera <sup>2</sup> , Rosa Pilolli <sup>3</sup> , Lamia Dahdah <sup>1</sup> , Veronica Calandrelli <sup>1</sup> , Giorgia Bracaglia <sup>4</sup> , Stefania Arasi <sup>1</sup> , Carla Riccardi <sup>1</sup> , Alessandro Fiocchi 1,\* and Lorenza Putignani <sup>5</sup>**


**Abstract:** Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We evaluated by oral food challenge the reactivity to a brand of PAL-labelled milk- and egg-free biscuits of children with severe milk and egg allergy. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in such biscuits. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed using immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were undetectable with a limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Milk/egg content of the biscuits is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergens in foods, and can be used to help avoiding unnecessary PAL.

**Keywords:** labelling; food allergy; prevention; proteomics; mass spectrometry; cow's milk allergy
