In order to address this aspect, in this work we characterised the frequency of self- and poly-reactive EX 527 circulating na?��ve, unswitched memory and switched memory B cells from patients with SS at the single cell level through the cloning and in vitro expression of complete rmAbs which bear identical specificity to the original B cells. To test the reactivity U0126 side effects against different allo- and auto-antigens, first supernatants were tested for polyreactivity against double and single-stranded DNA, lipopolysaccharide and insulin by ELISA as previously reported. Antibodies that reacted against at least two structurally diverse selfand non-self-antigens were defined as polyreactive. Internal controls for polyreactivity were kindly provided by Prof Hedda Wardemann at Max Planck Institute for Infection Biology and added on each plate consisting of the rmAbs mGO53, JB40, and ED38 as previously reported. Second, to analyse the autoreactivity profile of the cloned antibodies, purified IgG were screened for selfreactivity against Hep-2 cells using the indirect-immunofluorescence assay. For the polyreactivity ELISAs, antibodies were tested at 1 mg/ml and the cut-off optical density at which all antibodies were considered reactive was determined for each experiment based on the mean OD plus 2 standard deviations of the mGO53 control antibody as previously described. Finally, for detection of ANA using Hep-2 coated slides as substrate, purified antibodies were incubated at 10 mg/ml. Alexa Fluor 488-conjugated goat antihuman IgG was used as detection antibody. Hep-2 staining patterns were visualised using an Olympus BX60 microscope and digital images acquired using identical exposure times throughout. ANA were scored independently by 3 trained observers and considered positive in case of concordance by at least 2 observers. Antibodies expressed at a concentration below 1 mg/ml were excluded from the analysis. Mutational analysis is normally used to study whether a B cell has experienced a positive antigen selection. Normally, non-synonymous mutations occur less frequently in the FRs in order to maintain the right Ig fold, whereas is positively selected in the CDRs to increase the antigen affinity of the Ig. Thus, the comparison of the silent to replacement ratios in the FR and CDR regions is used to study the presence of antigen selection. S/R ratio calculation showed a significant difference comparing CDRs and FRs of the IgH V genes in both memory unswitched and memory switched SS B cells. Additionally, we adopted the binomial distribution method recently described by Chang and Casali using the IgTA software in order to calculate the probability of antigen selection based on the somatic mutation analysis in the Ig repertoire from memory unswitched and switched B cells.