Near 100 aged-matched, gender-balanced amoxicillin allergic and tolerant subjects, were recruited for this project, after agreeing to participate and signing the informed consent, as required by the national legislation. A series of nanoparticles of different sizes (20nm, 30nm or 50nm), containing different amount of amoxicillin (80, 100, 300 and 600 nM AX) were developed and evaluated for their capacity to better activate sample basophils than free amoxicillin. Basophil activation was measured by detection of CD63 and CD203c basophil activation markers. Parallel testing was done with free amoxicillin and nanoparticle-associated amoxicillin. We found that only bigger nanoparticles displaying the highest amount of amoxicillin (NP-50/600AX) were able to activate human basophils in the blood samples. Importantly, only allergic patient-derived basophils were activated by the nanoparticle, meaning that they retained specificity. In fact, NP-50/600AX was near 20 times more potent than free amoxicillin, yet conserving specificity to activate only basophils from allergic subjects and not from healthy controls. However, to achieve enough sensitivity very high amounts of NP-50/600AX were needed, affecting their stability and impeding a correct flow-cytometry measurements. In parallel, other optimization strategies were tested, like the addition of bacterial components to the sample with the aim to "prepare" the basophils before incubation with amoxicillin to obtain a higher degree of activation. All known bacterial derived molecules were tested as potential "co-factors" for the basophil activation test. Only bacterial lippopolisaccharyde (LPS) showed a significant increase in basophil activation upon incubation with amoxicillin in the basophil activation test, which was evident only in alergic subjects. The level of improvement in sensitivity raised from 46% to 68%, while specificity was kept at 95%, meaning that this can be an improvement that can be transferred to the clinic after a proper validation. In this line, a prospective study is being conducted to estimate the power of this optimization as well as the suitability to implement it into the clinical practice. In this sense, we developed a series of assays to standardize the basophil activation test, according to the European regulation for in vitro devices. Flow cytometers are the core instruments of the BAT. A risk-based approach was carried out to determine the best comparators for correlation studies among instruments. We found that %CD63 and %CD203c were the best comparators, to calculate analytical correlation, and clinical agreement between instruments. This qualification studies are part of the verifications required by the current european IVD regulation and are key to conduct multicentric studies, often important to measure reproducibility of the technique. In addition, we aim to study basophil interaction with the nanoparticles and with LPS, and for this we set up a rapid, medium throughput basophil isolation method, which gave highly pure, viable and functional basophils.The results of the nanoparticles, LPS, standardization were published in high impact journals, and disseminated through international meetings and activities to reach the general public such as the European researchers night or press articles and social media. Importantly, new nano structures displaying amoxicillin and LPS are planned to be developed soon, thanks to a national grant for the next three years.
