y the presence of antibodies against the muscle components of the neuromuscular junction. The main antigen targets are the AChR, MuSK and LRP4. Antibodies against these three antigens are detected in 80-85%, 5-7% and ~2-3% of the MG patients, respectively. Despite the extensive research that has been done in the field of MG, there is a significant number of patients that do not have any detectable antibodies against the known antigens. These patients are known as seronegative MG patients (SN-MG). SN-MG presents a serious gap in the understanding of the etiology and pathogenic mechanisms of the disease. This is likely due to the presence of unknown antigens and / or the lack of sensitivity of the commercially available diagnostic assays, unable to detect antibodies at low concentrations.The aim of this thesis was to develop very sensitive diagnostic assays for the detection of anti-LRP4 and anti-MuSK antibodies among the previously SN-MG patients, for the minimization of SN-MG. The detection of anti-LRP4 antibodies is currently undertaken only by the sensitive but qualitative CBA technique. The development of a quantitative technique (RIPA, ELISA) for the quantification of these antibodies is essential for disease monitoring. Regarding the detection of anti-MuSK antibodies, the development of a more sensitive technique, from those used until now (RIPA), is necessary. Α diagnostic assay in which MuSK will be in native form (as in CBA), will give us the opportunity to detect not only low concentration anti-MuSK antibodies but also antibodies which require the native structured protein. The development of a quantitative and sensitive diagnostic method detecting anti-LRP4 antibodies requires the expression, isolation and purification of an intact antigen (LRP4 in our case). Human LRP4 is a large protein (212 kDa) with many post-transcriptional modifications. Because of the complexity of LRP4, the over-expression of this protein in heterologous expression systems is particularly difficult. For this reason, in addition to the intact LRP4, constructs of the whole extracellular domain (ECD) as well as of functional domains of LRP4-ECD were designed to be expressed in different expression systems. In order to improve the quantity and quality of the expressed recombinant proteins we used various expression systems the selection of which was based on the post-transcription mechanisms available as well as the ability to express large amount of recombinant proteins. More specifically, for the expression of the high complexity intact LRP4 (1905aa) and LRP4-ECD (21-1725aa) proteins, the eukaryotic baculovirus expression system and HEK293 mammalian cells were used, respectively. These expression systems are capable of expressing functional and properly structured proteins but usually in poor yields. On the other hand, for the expression of the smaller and less complex functional domains of LRP4-ECD, the P. pastoris and E. Coli expression systems were used. The latter are able to express large amounts of target proteins but lacking the complicated post-transcription mechanisms. Only few of the designed constructs were expressed at an acceptable level of yield in order to be used for the development of a diagnostic assay. More specifically, efforts were made to develop an indirect ELISA using the LRP4-ECD as an immobilized antigen. Using this technique was achieved the identification of only 14% of the anti-LRP4 positive MG sera (identified by CBA). In addition, the expressed in E. Coli domains of LRP4 (785-1093aa, 1093-1439aa, 1004-1306aa) used for the development of another indirect ELISA, identified the ~10% of LRP4-MG. However, using the expressed in P. pastoris LRP4 domain (21-737aa), a RIPA technique was developed identifying the 41.2% of LRP4-CBA positive MG sera. From the developed assays, RIPA seems to be the most sensitive one. Nevertheless efforts focused on the sensitivity improvements of the developed quantitative assays continue, aiming the quantification of the anti-LRP4 antibodies in LRP4-MG sera.In the second part of this thesis we applied a CBA for the detection of MuSK antibodies undetectable by RIPA. We tested 633 triple-SN-MG patients' sera from 13 countries, and detected 13% of these sera as MuSK-positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of patients with other neuroimmune diseases. Interestingly, we also detected a significant number of double positives (AChR/MuSK-MG, LRP4/MuSK-MG), suggesting their overall rate is more frequent than previously described. Moreover, the clinical data of the newly diagnosed MuSK-MG patients were collected and evaluated. Importantly, we found that 27% of SN-MG patients with ocular MG (i.e. MG with only ocular symptoms present) were MuSK antibody positive, whereas 23% of the newly identified MuSK-MG patients had thymic hyperplasia suggesting thymic abnormalities; these percentages are much higher than those described earlier for classical MuSK-MG. The treatment of MG depends on the type of the circulating autoantibodies. Moreover, the course of the disease is associated with the antibody titer. Therefore, the development of diagnostic tools like these mentioned above can lead to a faster and better disease treatment, improving the quality of MG patients’ life.υστήματα έκφρασης του ζυμομύκητα P. pastoris και των βακτηρίων E.Coli (BL21). Χαρακτηριστικό των συγκεκριμένων συστημάτων έκφρασης είναι οι μεγάλες ποσότητες πρωτεΐνης
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