Ara h 9 is a major allergen in peanut-allergic patients from the Mediterranean area. Ara h 9 is capable of inducing histamine release from basophils, but to a lesser extent than Pru p 3.

Several hazelnut allergens with different clinical relevance and crossreactive properties have been identified and characterized so far. The aim of this study was to develop protocols for producing relatively large amounts of three recombinant hazelnut allergens Cor a 1.04, Cor a 2, and Cor a 8 in a folded and immunologically active form. The availability of well-characterized, pure recombinant allergens will improve diagnostic in vitro tests for food allergy, by allowing a highly sensitive component resolved diagnosis. Depending on the individual hazelnut allergen, protocols for heterologous production - either as fusion or nonfusion protein - were developed to obtain homogenous protein batches. The resulting proteins were purified by a two-step FPLC method and their IgE antibody reactivity was verified. Identity was verified by N-terminal sequencing and MALDI-TOF-MS analysis. Their secondary and tertiary structure was controlled by circular dichroism (CD)-spectroscopy and NMR analysis. Decisions on the strategies for expression and purification of allergens on a large scale were made on a case by case basis: Preparation of rCor a 1.04 and rCor a 2 as fusion proteins in E. coli from inclusion bodies resulted in approximately 10 mg pure protein per liter whereas rCor a 8 expression in yeast as nonfusion protein yielded 30 mg/L.

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Allergen microarrays are under development for a component-resolved diagnosis of Type I (IgE-mediated) allergies. Here we report an improved microarray coupled to microfluidics for the detection of allergen specific immunoglobulin E (IgE). The signal intensity for IgE detection in serum has been improved by using glass slides coated with a novel poly[DMA-co-NAS] brush copolymer which is able to immobilize allergens in their native conformation and by carrying out the incubation step in dynamic conditions. The assay, fully automated, was performed in a microcell, using a software-controlled fluidic processor, to bring assay reagents on the surface of the array. Microfluidics turns the binding between serum immunoglobulins and immobilized allergens from a diffusion-limited to a kinetic-limited process by ensuring an efficient mixing of serum samples on the surface of the microarray. As a result of this, the binding of high affinity IgE antibodies is enhanced whereas that of low affinity IgG antibodies, which are present at higher concentration, is impaired paving the way to more accurate and sensitive results.

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