Flow Cytometry in Human Cells Exposed to Tick Salivary Proteins.

The Indirect basophil activation test (iBAT) is a flow cytometry-based assay in which basophils are first passively sensitized with patient-derived IgE and subsequently stimulated with the allergen of interest to assess activation markers on the cell surface. This method was implemented by using the human KU812 cell line to investigate basophil activation mechanisms triggered by tick-derived biomolecules. Unlike the traditional BAT, the iBAT approach avoids variability from non-responder patients and donor-dependent differences, providing a stable and reproducible in vitro model. KU812 cells were differentiated with IL-4, primed with serum from a human patient diagnosed with α-Gal syndrome (AGS), and stimulated with increasing concentrations of tick salivary proteins, including metalloprotease (A0A0K8RCY8) and antigen p23 (A0A0K8RKR7). Activation was assessed by flow cytometry through CD63 and CD203c expression. Dose-response curves were analyzed to determine CDmax, EC₅₀, and AUC, enabling comparison of allergenic potency and overall activation. This iBAT model offers a controlled and reproducible method to explore IgE-mediated mechanisms relevant to tick exposure and the pathophysiology of AGS.