Protein chips applications.

The aim of this work was the testing of a diagnostic tool for the detection of allergenic proteins from stored product mites/cockroaches and house dust mites. Species-specific antibodies were previously tested for specificity and cross-reactivity, and detection limit by ELISA method. In this work, protein chips were manufactured either manually, using a MicroCaster, or robotically, using a SpotArray. Proteins used were six recombinant Kunitz-type protease inhibitors (PI), two of group A (with anti-cathepsin D and anti-trypsin activity), two of group B (with anti-trypsin and anti-chymotrypsin activity) and two of group C (with anti-cathepsin B activity), and the soybean Bowman-Birk inhibitor. Proteins were immobilized onto epoxy activated glass slides. Protein chips were hybridised with serial dilutions of extracts from adult mites/insects and from spent growth media (SGM), in order to capture the allergenic proteases. Protein A was labelled with Alexa-555 and used for detection of polyclonal antibodies bound on the chip surface. The detection limit was 0.5µg protein extract, for Aleuroglyphus ovatus and 0.75µg in Tyrophagus putrescentiae specimens and protein extracts from the faeces fraction (SGM). By comparison, the detection limit of ELISA method was 2.5µg. The protease inhibitor-chips can allow rapid screening of proteases and their specificity by analysing the binding to each class of inhibitors. Results of protease binding to each KPI and recognition of allergenic proteases showed the practicability of protein microarrays as a diagnostic tool for detection of mite/insect contaminants in foods and work environment.