Validation of a Multiplex Molecular Macroarray for the Determination of Allergen-Specific IgE Sensitizations in Dogs

Abstract

Detecting IgE sensitizations in the serum of allergic dogs is commonly performed using allergen extracts, but these are difficult to standardize. This article details the development and validation of the Pet Allergy Xplorer (PAX; Nextmune, Stockholm, Sweden), the first multiplex macroarray for the detection of IgE sensitization in dogs using allergen extracts and molecular components; the PAX is derived from the Allergy Xplorer (ALEX²; MacroArray Diagnostics, Vienna, Austria). The selection of allergens, cartridge processing, strategy for identifying and blocking IgE directed against cross-reactive carbohydrate determinants (CCDs), and the method used for determining the positivity threshold are described. The validation of the PAX included evaluations of the specificity of its anti-IgE monoclonal antibody, specificity of IgE binding to target allergens, assay precision, and internal consistency. Additionally, the influence of possible confounding factors, such as sample type, the influence of hemolysis, lipemia, bilirubinemia, and elevated CCD-IgE, was tested. Finally, the sensitization rates of 23,858 European dogs to 145 environmental and Hymenoptera venom allergens were summarized. The PAX is accurate and reproducible and has a unique CCD-detection and blocking strategy; its molecular allergens offer a unique window on allergen cross-reactivity.

Keywords: IgE; allergen; allergy; atopic dermatitis; dog; molecular allergology; serodiagnosis; serology.

Figure 1

Representation of an ALEX²/PAX cartridge, which enables the simultaneous multiplex testing of up to 300 allergens and their controls. Photo courtesy of MacroArray Diagnostics, Vienna, Austria.

Figure 2

PAX CCD-blocking strategy.

Figure 3

ELISA with the 5.91 anti-dog IgE monoclonal antibody: this antibody recognized dog IgE but not dog IgG.

Figure 4

PAX internal consistency: there is a strong positive correlation between the IgE values against the cross-reactive allergens Der f 2 and Der p 2 (a), but a negative correlation between those of two non-cross-reactive allergen pairs, Der f 2 and Tyr p 2 (b), or Der f 2 and Par j 2 (c).

Figure 5

Comparison of Der f 2 and Der f-specific IgE values in 1283 dogs: In most dogs, the IgE levels against Der f 2 exceeded those against the Der f extract.