conformational epitopes was now reduced to for IgG1. when presented to the immune system in its native state. In contrast to BLG, a huge reduction in the importance of conformational epitopes were seen when ALA were administered by the oral route and thereby presented to the rat immune system after exposure to the acidic and proteolytic environment in the GI tract. When administered orally, conformational epitopes were still found to be in excess, however the approximate ratio of linear vs. conformational epitopes was now reduced to for IgG1. This indicates that for ALA the epitope recognition profile is greatly influenced by the administration route. In contrast to BLG and ALA, em /em -casein showed a much greater importance of linear epitopes (Figure? 5 C). The antibody reactivity against the native and denatured em /em -casein differed only by one titre value, indicating that approximately half of all em /em -casein specific antibodies were raised against conformational epitopes and the other half against linear epitopes. Similarly to RAF1 BLG, the importance of conformational epitopes seemed not to differ between the two administration routes showing an approximate ratio of linear vs. conformational epitopes of em 1:1 /em . The proportion of antibodies directed against linear and conformational epitopes in individual rats differed greatly demonstrating the heterogeneity of antibody responses in rats. Whereas all rats, independent on the administration route, raised the greatest amount of antibodies against conformational BLG epitopes compared to linear BLG epitopes, a single rat recognised C25-140 the denatured ALA better than the native ALA when administered by oral route, and for em /em -casein several rats reacted with the greatest response against the denatured em /em -casein compared to native em C25-140 /em -casein, demonstrating a C25-140 greater importance of just linear epitopes compared to conformational epitopes for these rats (data not shown). Discussion When studying food allergens, characterisation of the specific antibody response is important. To understand how allergenic potential relates to the protein structure we C25-140 have examined the amount and types of antibodies raised against three cows milk allergens, with reference to their reactivity to the native and denatured form of the allergen. Animal models have been used to study the immunogenicity and allergenicity of various food allergens, using various routes of administration with purified proteins or whole foods, with or without the use of adjuvant [22,23]. In this study we used BN rats. The BN rat is a high Ig, particularly IgE-responder strain which to a certain degree resembles atopic humans in their predisposition to develop IgE-mediated allergy [24]. Moreover, it is an animal strain regarded as a useful model for studying sensitisation to food proteins [25,26], since it generates IgG and IgE antibodies of similar protein specificity [24,25] as well as similar epitope specificity C25-140 [27,28] to those produced in humans. The inherent capacity of the three cows milk allergens to induce a specific IgG1 and IgE response following systemic (i.p.) administration showed only minor differences. All three allergens induced high antibody titres, though em /em -casein induced a little lower mean antibody titre than BLG and ALA, due to a large variance in its immunogenicity and sensitising capacity between individual rats. In the context of food allergy, oral administration is the most appropriate route of exposure as this is considered to be the primary route of sensitisation [18,22,29,30]. A greater variation in the immunogenicity between the three cows milk allergens was seen upon oral administration compared to i.p. administration. Whereas BLG was able to induce a specific IgG1 response in all rats, only 6 out of 10 rats responded to ALA and 5 out of the 10 rats to em /em -casein after oral administration. This could indicate that the inherent immunogenic capacity of food allergens may be modulated by the modifications resulting from the acidic and proteolytic environment in the GI tract and the way in which they are presented to the immune system. The specific antibody responses upon oral administration correlate well with the susceptibility of the three cows milk allergens to digestion. BLG has been demonstrated to be a protein resistant to digestion in contrast to both ALA and em /em -casein that are easily digestible proteins [18], probably resulting in a lower chance for ALA and em /em -casein than BLG to survive the digestion process in the GI tract and survive in a form retaining enough structure and size to be recognised by.
