Simultaneous detection of total and allergenspecific IgE by using purified allergens in a fluorescent multiplex array

Background: Testing serum samples for total and allergen-specific IgE requires separate testing for each antibody and allergen specificity. Objective: To apply fluorescent suspension array technology to allow simultaneous detection of total and allergen-specific IgE in serum in a single quantitative test. Methods: A 7-plex suspension array for the simultaneous detection of total IgE and IgE specific to Der p 1, Der p 2, Fel d 1, Can f 1, Bet v 1, and Phl p 5 was developed, using mAb or purified allergens covalently coupled to fluorescent microspheres. The multiplex array was validated by comparing total and allergen-specific IgE levels in serum from patients with allergy with results obtained by enzyme immunoassays. Results: There was a highly significant correlation between total IgE levels measured by multiplex array and fluorescent enzyme immunoassay (r = 0.97; P <.001; n = 63). Total and allergen-specific IgE levels also correlated with enzyme-linked and fluorescent enzyme immunoassay results (r = 0.44-0.94; n 95 or 106). The multiplex array was reproducible (r 0.86-0.99; mean coefficient of variance percentage, 12% to 25%). The sample volume required for a 7-plex assay was <20 mu L per sample, compared with >400 mu L in current immunoassays. Conclusion: The multiplex array is a high-throughput system that allows simultaneous quantification of allergen-specific and total IgE. Clinical implications: Our results suggest that fluorescent multiplex technology will facilitate large-scale epiderniologic studies of allergic sensitization. The reduced serum volume is an advantage for pediatric studies.