Jose Antonio Cespedes

Background: Immediate drug allergic reactions (IDAR) to betalactams are frequent, yet mislabelling remains common and negatively impacts clinical decisions. Conventional diagnostics such as STs and drug provocation are effective but limited by time, risk, and contraindications in severe cases. In vitro alternatives—sIgEquantification and basophil activation tests (BAT)—offer safer options, although performance may be affected by biological variability and suboptimal sensitivity with an important drawback for the latter in patients with non‐releaser basophils. This study aimed to evaluate a mast cell activation test (MAT) based on human CD34 ⁺ ‐derived mast cells (dMCs) for IDAR diagnosis to amoxicillin (AX) using both free AX and dendrimeric amoxicilloyl conjugates (G4/G5‐AXO). Methods: CD34 ⁺ cells were cultured for 10–12 weeks to generate dMCs. After passive sensitization with sera from AX‐allergic patients ( N = 28) or tolerant controls ( N = 11), dMCs were stimulated with free AX, G4‐AXO, or G5‐AXO. Activation was quantified by CD63 expression via flow cytometry. Diagnostic performance was compared with BAT and sIgE determination by ImmunoCAP. Results: MAT with free AX achieved 53.57% sensitivity, G4/G5‐AXO reached 46.43%, and all methods maintained 100% specificity. Combining results with AX or dendrimeric stimuli increased sensitivity to 75% while preserving specificity. Importantly, MAT identified positive cases among BAT non‐releasers and patients with undetectable sIgE. Conclusions: Under optimized conditions, MAT using dMCs and different AX determinants reached 75% sensitivity and 100% specificity. The complementary use of free AX and dendrimeric conjugates expands detection across heterogeneous IgE reactivity profiles from AX‐allergic patients, reinforcing the diagnostic value of advanced cellular models and engineered allergens.

In vitro cell activation through specific IgE bound to high-affinity receptors on the basophil surface is a widely used strategy for the evaluation of IgE-mediated immediate hypersensitivity reactions to betalactams. Cellular activation requires drug conjugation to a protein to form a large enough structure displaying a certain distance between haptens to allow the cross-linking of two IgE antibodies bound to the basophil’s surface, triggering their degranulation. However, no information about the size and composition of these conjugates is available. Routine in vitro diagnosis using the basophil activation test uses free amoxicillin, which is assumed to conjugate to a carrier present in blood. To standardize the methodology, we propose the use of well-controlled and defined nanomaterials functionalized with amoxicilloyl. Silica nanoparticles decorated with PAMAM–dendrimer–amoxicilloyl conjugates (NpDeAXO) of different sizes and amoxicilloyl densities (50–300 µmol amoxicilloyl/gram nanoparticle) have been prepared and chemically characterized. Two methods of synthesis were performed to ensure reproducibility and stability. Their functional effect on basophils was measured using an in-house basophil activation test (BAT) that determines CD63⁺ or CD203c^high activation markers. It was observed that NpDeAXO nanocomposites are not only able to specifically activate basophils but also do so in a more effective way than free amoxicillin, pointing to a translational potential diagnosis.

Background: Immediate drug hypersensitivity reactions (IDHRs) to clavulanic acid (CLV) have increased in the last decades due to a higher consumption alongside amoxicillin (AX). Due to its chemical instability, diagnostic procedures to evaluate IDHRs to CLV are difficult, and current in vitro assays do not have an optimal sensitivity. The inclusion of the specific metabolites after CLV degradation, which are efficiently recognised by the immune system, could help to improve sensitivity of in vitro tests.Methods: Recognition by dendritic cells (DCs) of CLV and the synthetic analogues of two of its hypothesised antigenic determinants (ADs) was evaluated by flow cytometry in 27 allergic patients (AP) and healthy controls (HC). Their ability to trigger the proliferation of T cells was also analysed by flow cytometry.Results: The inclusion of synthetic analogues of CLV ADs, significantly increased the expression of maturation markers on DCs from AP compared to HC. A different recognition pattern could be observed with each AD, and, therefore, the inclusion of both ADs achieves an improved sensitivity. The addition of synthetic ADs analogues increased the proliferative response of CD4⁺Th2 compared to the addition of native CLV. The combination of results from both ADs increased the sensitivity of proliferative assays from 19% to 65% with a specificity higher than 90%.Conclusions: Synthetic ADs from CLV are efficiently recognised by DCs with ability to activate CD4⁺Th2 cells from AP. The combination of analogues from both ADs, significantly increased the sensitivity of DC maturation and T‐cell proliferation compared to native CLV.