D2.12 - Resolving heterophile antibody interference in the ISAC multiplex assay using HBT (Scantibodies)

Multiplex IgE microarrays such as the Immuno Solid-phase Allergen Chip (ISAC) enable simultaneous assessment of sensitization to numerous molecular allergens. As antibody-based platforms, they remain vulnerable to immunological interference, particularly from heterophile antibodies, which can bind assay reagents nonspecifically and generate misleading signals. Although heterophile antibody interference is well recognized in several clinical immunoassays, it has not been described in multiplex allergy testing.

A routine ISAC test performed in June 2025 yielded an intense, homogeneous blue background across the entire microarray surface, precluding automated chip reading. A second serum sample obtained five months later reproduced the same artifact. Both samples were processed according to the standard ISAC protocol (Thermo Fisher Diagnostics, Uppsala). Attempts to mitigate the nonspecific signal by modifying analyzer reading parameters were unsuccessful.

Based on the suspected presence of heterophile antibody interference, the November 2025 sample was pretreated with a heterophile antibody–blocking reagent (HBT, Scantibodies Laboratory Inc.). The resulting supernatant was subsequently analyzed using the ISAC assay. To assess whether HBT pretreatment affected allergen recognition, specific IgE levels to all ISAC-positive allergens and to cross-reactive carbohydrate determinant (CCD) markers were measured before and after pretreatment using the Phadia 1000 platform.

HBT pretreatment completely eliminated the diffuse blue background artifact, restoring normal microarray fluorescence and enabling reliable automated interpretation. Specific IgE concentrations and sensitization profiles remained unchanged after HBT treatment, indicating that the pretreatment did not adversely affect analytical performance.

This report documents a previously unrecognized form of analytical interference in the ISAC assay attributable to heterophile antibodies. The characteristic homogeneous blue background, reproducible across two independent samples, rendered the microarray uninterpretable. HBT pretreatment proved highly effective in eliminating the interference without affecting true IgE reactivity.