Dendrimeric antigens for passive mast cell activation in the evaluation of amoxicillin allergy

Abstract

Background: Amoxicillin (AX) is frequently implicated in immediate IgE-mediated allergic reactions. Diagnosis is challenging,highlighting the need for new approaches enhancing in vitro sensitivity and specificity. Engineered nanostructures can mimicimmunological recognition of hapten-carrier conjugates, offering a strategy to improve diagnostic accuracy.Methods: Dendrimeric Antigens (DeAns), with controlled size (1st–5th generation) and multivalent AX determinants (8–128 units, respectively), were synthesized for in vitro immunological evaluation. In vitro IgE recognition was studied by com-petitive radio-immunoassay. Allergenic activity was evaluated in mouse bone marrow-derived mast cells (MC) sensitized withmouse anti-AX IgE monoclonal antibody and humanized RBL-2H3 (huRBL-2H3) and LUVA cells sensitized with sera fromβ-lactam-allergic subjects and tolerant controls, measuring degranulation in response to DeAns stimulation.Results: Five different DeAns were obtained as pure compounds. All DeAns were recognized by AX-sIgE. A clear size-dependent activation pattern was observed in the three cell models: lower-generation (1st–2nd) DeAns failed to induce degranu-lation, whereas DeAns of bigger size (3rd–5th generation) triggered significant, dose-dependent activation. Notably, no activationwas observed in tolerants and unsensitized cells or with blank dendrimers. In patient-sera assays, the passive MC activation test(pMAT) with DeAns provided complete diagnostic discrimination, with activation restricted to AX-allergic patients.Conclusions: DeAns are effective platforms for investigating effector cell activation in AX allergy. By fine-tuning structuralattributes—size and multivalence—we reveal the promising utility of DeAns in pMAT that leverage commercial cell lines andpatient sera. This approach could address key limitations of β-lactam allergy diagnostics, enabling more reliable and standard-ized in vitro testing.