The main goal of this project consisted on designing and setting up transferable technologies for the detection of the pathogenic bacteria Pseudomonas aeruginosa. With this aim, an immunoassay for the specific virulence factor, pyocyanin (PYO), was developed. This ELISA assay allowed to identify P. aeruginosa in approximately 2 hours rather than the 2-3 days that usually are required with the gold standard technique based on plate cultures. Thus, the project involved the production of monoclonal antibodies against PYO and the development of an ELISA assay with a very low limit of detection (LoD). Moreover, after its analytical characterization matrix effect studies were performed with culture media and were also started with sputum. Finally, the assay was validated as a diagnostic tool by assaying bacterial isolates from different patients presenting different infection stages.

The results obtained were promising since the microplate-based ELISA developed was able to achieve a LoD of 0.07 nM, which is much lower than the concentrations reported to be found in clinical samples. This assay allowed the characterization of the release kinetics of PYO and 1-OHphz (the main metabolite of PYO) of clinical isolates obtained from P. aeruginosa-infected patients and subsequently cultured in MH medium. In this regard, promising results were achieved since significant differences were identified between clinical isolates obtained from patients with an acute or a chronic infection. These data points out the real potential of PYO as a biomarker of P. aeruginosa infections and the posibility to use such virulence factor also as a biomarker for patient stratification and for an effective management of these kinds of infections.