D3.98 - An extended study for the multiple detection of IgE-mediated allergic reactions using microarray technology

Microarray technology allows small amounts of allergen extracts, natural and recombinant allergens to be spotted on different surfaces. In addition, small serum amounts can be used for specific Immunoglobulin E (sIgE) detection for a huge number of allergen specificities in a single step. The aim of this study was to identify specific IgE-mediated allergic reactions to different allergens using only two microtiter wells. For this purpose, the most important allergens from the group of molds, foods, mites, insect venoms, animal dander (skin, hair and feathers), occupational allergens and pollen were used.

The test is based on an Enzyme Linked Immuno Sorbent Assay combined with a Microarray technique. A set of 188 allergen extracts, natural and recombinant allergens were spotted in microtiter plate wells. The specific IgE from patient´s sample bind to the solid phase coupled allergens. After addition of anti-IgE-Enzyme-Conjugate a solid phase bound antigen/IgE/anti-IgE-HRP-complex is formed. The amount of bound antibody corresponds to the amount of horse radish peroxidase in the respective well and is detected by incubation with precipitating Tetramethylbenzidine (TMB) Substrate resulting in the development of blue colored allergen-spots. A calibration curve is run simultaneously with the serum samples, and the concentration of the allergen specific IgE antibodies is read by a special Microarray Reader. The concentrations are directly proportional to the intensities of the colored spots.

The results for all allergens were compared with two established assay methods: ImmunoCAP^® (Thermo Fisher) and ALLERG-O-LIQ (Dr. Fooke-Achterrath Laboratorien GmbH).

There was good agreement between the microarray results and both in vitro techniques. Area under the curve values were found to be > 0.85 compared to the other in vitro results. The Spearman correlation between commercially available in vitro systems and the microarray for all allergens tested shows a correlation coefficient of > 0.85.

For the detection of sIgE, the microarray shows comparable results to the other in vitro techniques. The microarray technique, which requires very small sample amounts, allows the detection of comprehensive IgE-mediated allergen responses for each patient by simultaneously testing 188 different allergens in two microtiter wells.