Isolation of Mal d 1 and Api g 1 - specific recombinant antibodies from mouse IgG Fab fragment libraries:Mal d 1-specific antibody exhibits cross-reactivity against Bet v 1

BACKGROUND: Around 3–5% of the population suffer from IgE-mediated food allergies in Western countries and the number of food-allergenic people is increasing. Individuals with certain pollen allergies may also suffer from a sensitisation to proteins in the food products. As an example a person sensitised to the major birch pollen allergen, Bet v 1, is often sensitised to its homologues, such as the major allergens of apple, Mal d 1, and celery, Api g 1, as well. Development of tools for the reliable, sensitive and quick detection of allergens present in various food products is essential for allergic persons to prevent the consumption of substances causing mild and even life-threatening immune responses. The use of monoclonal antibodies would ensure the specific detection of the harmful food content for a sensitised person. METHODS: Mouse IgG antibody libraries were constructed from immunised mice and specific recombinant antibodies for Mal d 1 and Api g 1 were isolated from the libraries by phage display. More detailed characterisation of the resulting antibodies was carried out using ELISA, SPR experiments and immunoprecipitation assays. RESULTS: The allergen-specific Fab fragments exhibited high affinity towards the target recombinant allergens. Furthermore, the Fab fragments also recognised native allergens from natural sources. Interestingly, isolated Mal d 1-specific antibody bound also to Bet v 1, the main allergen eliciting the cross-reactivity syndrome between the birch pollen and apple. Despite the similarities in Api g 1 and Bet v 1 tertiary structures, the isolated Api g 1-specific antibodies showed no cross-reactivity to Bet v 1. CONCLUSIONS: Here, high-affinity allergen-specific recombinant antibodies were isolated with interesting binding properties. With further development, these antibodies can be utilised as tools for the specific and reliable detection of allergens from different consumable products. This study gives new preliminary insights to elucidate the mechanism behind the pollen-food syndrome and to study the IgG epitope of the allergens

Crystals of HFBII (A), HFBI (B), and SDMT (C) produced with nanospheres.

<p>Crystals of HFBII (A), HFBI (B), and SDMT (C) produced with nanospheres.</p

Examples of the effects of the nanospheres on crystallization in the screening experiments of lysozyme (A–D), xylose isomerase (E, F), and xylanase (G, H) in combination with Crystal Screen HT.

<p>Crystals from control screens (A, C, D, G) and nanosphere screens (B, D, F, H) are shown. Precipitate due to nanosolution is present in B and F.</p

Summary of the effects of polystyrene nanospheres in the screening experiment with lysozyme, xylose isomerase, and xylanase in combination with Crystal Screen HT.

<p>Lysozyme experiment was repeated in order to assess the repeatability and therefore appears in the table twice. Numerical value indicates how many conditions produced crystals out of 96 possible conditions in Crystal Screen HT. Control experiment contained equal amount of water instead of nanosphere solution. ‘+’ indicates crystals in the nanosphere screen only, not in the control screen. ‘−’ indicates conditions that produced crystals in the control screen but not in the nanosphere screen.</p