Sviluppo di un test LFIA semi-quantitativo per la rivelazione di anticorpi contro proteina Spike da SARS-CoV-2

Although years have passed since the beginning of the SARS-CoV-2 pandemic, the infection still appears to be a problem in several countries despite the massive vaccination campaign put in place. LFIA systems are heterogeneous immunochemical analytical assays, which belong to the class of so-called Point-of- Care Tests (POCT). POCTs are tests cheap, quick to perform, robust, and that are executable in situ by the patient. The purpose of this thesis work was to develop a semi-quantitative LFIA device for the measurement of the amount of antibody towards the Sars-CoV-2. In particular, the assay was intended to semi-quantify the antibodies contained in human serum samples and directed towards the SARS-COV- 2 spike protein. For this purpose, multiline LFIA devices were designed using different immunodiagnostic strategies, with the aim of correlating the number of colored test lines to the amount of the antibodies. Thus, the main objective is to verify that the strategy of using different reactive lines at different concentrations, allows to distinguish samples with different antibody titers according to the number of lines appeared, checking for the presence of statistically significant differences between the respective sample populations. The second objective posed is to understand which format performed better when used in semi-quantitative testing, between the one site immunometric assay (using Stapyloccoccal protein A, SpA as the detector reagent) or the double antigen immunometric assay. while also considering its ability to provide results that correlate as closely as possible with those of the reference CLIA assay. The third objective was to understand whether it was possible, using an in house produced RBD (Receptor Binding Domain) antigen, to achieve performance comparable to that obtained using a commercial antigen. Thus, a key part of the work was to set up a restricted experimental design useful for identifying the best working conditions for the two different RBDs used. To achieve these, experimental design, ROC curves and statistical analyses such as ANOVA on ranks tests or Spearmann correlation tests were employed. The immunometric format employing labeled SpA proved to be unable to provide results in agreement with those obtained from the reference CLIA test, while the double antigen format thanks to its high specificity, proved to be able to achieve the intended purpose. Three different devices were then created, differing in either the concentration of the species used to make the test lines or the type of RBD antigen used. These were used for the analysis of a set of 81 human serum samples and showed that it was possible to assign the antibody titer of the analyzed sample to a specific range, depending on the number of test lines that appeared.