General concept and aims of the EuroPrevall Food Allergen Library

Diagnosis of food allergy comprises convincing case history, in vitro diagnosis and in vivo tests (skin prick tests and food challenges). All in vitro tests are based on the detection of allergen specific IgE and the specificity and sensitivity of the individual test depends on the quality of the analyte. Although total extracts of the suspected food are routinely used, the concept of component resolved diagnostic assays has gained rising interest in the recent past. However, identification, purification and authentication of proteins from various food sources of either animal or plant origin represents a challenge for the allergologist. Within the EC funded IP Europrevall a food allergen library was established, comprising the already identified allergens from various food sources as well as newly identified ones according to changing dietary habits. The aim of the allergen library is to collect and improve existing allergen purification protocols and to evaluate expression strategies for producing recombinant allergens. Subsequently, authentication of the highly pure protein batches is performed using state of the art methods including mass spectrometry, MALDI-TOF and N-terminal sequencing. Secondary structure is evaluated by NMR maps of low molecular weight proteins and CD spectroscopy. Allergenic activity is characterized using a panel of selected sera in ELISA, immunoblots as well as in histamine release cell assays. In the first round 31 allergens from 10 foods also listed in the EC labelling directive (apple, peach, hazelnut, peanut, celery, cow’s milk, goats milk, hen’s egg, fish and shrimp) were entered into the library. As a follow up 11 allergens from walnut, sesame seed, soybean, mustard and wheat were provided to the library and will be evaluated accordingly. In the course of the characterisation of 31 food allergens relevant new data on their physicochemical properties were obtained which might be useful for designing appropriate food processing strategies with reduced allergen load. For example recombinant Pru p 1, the Bet v 1 homologue from peach is regarded as a labile protein. However, under neutral pH the protein refolds after heating up to 95°C as analysed by CD spectroscopy. In contrast, Pru p 3, the lipid transfer protein from peach is heat resistant and stable at acidic pH. However, when changing to neutral pH the protein is affected by heat treatment and unable to refold when cooled down. Regarding the allergens from peanuts the complex mixture of subunits and isoforms was analysed for Ara h 1, Ara h 2, Ara h 6, and Ara h 3/ 4 and differential IgE binding capacity of the basic and acidic subunits of the seed protein allergens was observed. In conclusion this allergen library concept should provide harmonised quality criteria for the production of either natural or recombinant food allergens to be used for component resolved diagnosis taking their different origin into account. These allergen batches will be used in the CAP and CHIP format to screen a significant number of food allergic patients sera and to compare its performance with existing in vitro diagnostics. Furthermore, this detailed analysis should provide actual data on the prevalence of individual food allergies across Europe and help to discriminate between major and minor allergens.