Dendrimeric Antigens for Passive Mast Cell Activation in the Evaluation of Amoxicillin Allergy

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 mimic immunological 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 competitive radio-immunoassay. Allergenic activity was evaluated in mouse bone marrow-derived mast cells (MC) sensitized with mouse anti-AX IgE monoclonal antibody and humanized RBL-2H3 (huRBL-2H3) and LUVA cells sensitized with sera from β-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 degranulation, whereas DeAns of bigger size (3rd-5th generation) triggered significant, dose-dependent activation. Notably, no activation was 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 structural attributes-size and multivalence-we reveal the promising utility of DeAns in pMAT that leverage commercial cell lines and patient sera. This approach could address key limitations of β-lactam allergy diagnostics, enabling more reliable and standardized in vitro testing.