Characterization of meat allergens for improved diagnosis of meat allergy

30 % of the population in industrialized countries suffer from IgE-mediated allergies. Allergic reactions to food belong to the most important and most dangerous type I allergies, since they are often associated with life- threatening anaphylactic reactions. So far there is only limited knowledge on allergic reactions caused by meat. The main focus of this project is the identification and characterization of meat allergens from various meat sources (poultry, beef, pork and lamb). The identified allergens will be expressed in bacterial expression systems, to generate recombinant molecules. These recombinant meat allergens will first be characterized regarding their biochemical and structural features and then be evaluated regarding their IgE binding capacity and allergenicity. The recombinant allergens, which show immunological characteristics comparable to their natural counterparts, represent well-defined single components, which can be applied for improved diagnosis of meat allergy. A further goal is to use the recombinant meat allergens for the generation of meat allergen specific rabbit antisera and to use the rabbit antisera for the development of sandwich ELISA assay systems, which will represent useful tools for the evaluation of commercially available diagnostic meat extracts and for the detection of meat allergens in food, even in a high through-put format. Furthermore, this project will also lead to the development of strategies for the treatment of meat allergic patients.

Food allergies are regarded as very dangerous diseases, because they are often associated with life-threatening anaphylactic reactions. About 0.5% to 8% of food allergies are caused by various meat sources. So far, there was only limited knowledge about the allergenic molecules involved meat allergy. Within this project we succeeded in identifying several allergenic molecules from chicken meat (myosin light chain 1, designated Gal d 7; myosin light chain 3; parvalbumin) and from mammalian meat (myosin light chain 1 and myosin light chain 3 from beef and pork; parvalbumin from pork). We produced these molecules in a bacterial expression system in recombinant form, purified them and saw that they all represent IgE reactive molecules, which are now available as tools for improved diagnosis of meat allergic individuals. Since pork is the most widely consumed meat in the world and a major part of pork meat is sold in a processed form, another focus of the project was to investigate whether proteins present in pork meat products can also induce allergic reactions. Using sera from pork meat allergic individuals we analyzed different pork meat products as well as raw, boiled and grilled pork meat for the presence of allergens. The sera recognized allergens of different molecular weight in raw and grilled pork, in salami, prosciutto, speck, extra sausage and ham, but not in boiled pork, in blood sausage and in liver sausage. Mass spectrometry analysis allowed the identification of the IgE reactive proteins as serum albumin, myosin light chain 1 and parvalbumin. These experiments showed that also processed meat products can contain allergens. In one type of meat allergy allergic symptoms are not caused by proteins, but by a carbohydrate moiety called alpha-Gal, which can be present on glycoproteins, but also on glycolipids. Allergic reactions in this type of meat allergy are characterized by a delayed onset of symptoms 3-6 hours after consumption of red meat. It has already been suggested that this unusual delay in symptoms might be due to a delay in the appearance of alpha-Gal in the circulation. To see whether this might be true, we compared the transport of alpha-Gal containing glycolipids and glycoproteins across human intestinal epithelial Caco-2 cells. We saw that only alpha-Gal bound to lipids, but not to proteins, can be transported across the epithelial cells and is in this way able to induce allergic reactions. As a matter of fact it is known that it takes 4 - 5 hours for dietary lipids to reach the circulation, whereas peak levels of digested proteins are found in the blood already 1 - 2 hours post meal. We thus provided evidence that the delay in symptoms after consumption of red meat might be caused by alpha-Gal carrying glycolipids.