Detection of allergen-specific IgE in sera from pediatric patients with food allergy using AlphaCL

Detection of serum allergen-specific IgE antibodies (sIgE) that can cross-link the high-affinity IgE receptor (FcεRI) is crucial for diagnosing IgE-mediated food allergy (FA). We previously developed an amplified luminescence proximity homogeneous assay involving cross-linking (AlphaCL) to detect functionally relevant sIgE. This study was intended to enhance analytical sensitivity of AlphaCL and evaluate its utility in detecting functional allergen-sIgE in sera from rats and IgE-mediated FA patients.

Serum samples were collected from ovalbumin (OVA)-sensitized rats and from 10 patients each with allergies to ovomucoid (OVM), ω5-gliadin (ω5G), or casein. To enhance sensitivity of conventional AlphaCL, allergens were desthiobiotinylated and immobilized on streptavidin plates, allowing serum components to be washed out before assaying. sIgE levels were measured using AlphaCL (in both rats and patients), ELISA (rats), and ImmunoCAP™ (patients). AlphaCL signals were compared with those from unsensitized controls and analyzed the correlation with ImmunoCAP™ results.

The improved AlphaCL showed significantly higher sensitivity than the conventional method and retained 66% of the signal observed in serum-free samples. AlphaCL signals were elevated in all OVA-sensitized rats, all patients with OVM or ω5G allergy, and 8 of 10 with casein allergy. Strong correlations were observed between AlphaCL and ImmunoCAP™ for OVM (r_s = 0.98), ω5G (r_s = 0.93) and casein (r_s = 0.86). AlphaCL detected allergen-sIgE corresponding to ImmunoCAP™ Class ≥ 3 for OVM and ω5G, and Class ≥ 4 for casein.

The improved AlphaCL can detect functional allergen-sIgE with FcεRIα-cross-linking ability, supporting its potential clinical utility in diagnosing IgE-mediated FA.