matrixscience.com/. In general, proteins with the highest sequence coverage and Mascot score were selected as candidate antigens. The proteome pattern of B. henselae selleck screening library was resolved on a 2-D gel and conserved over the pI range of pH 3–10 and MW 10–120 kDa. An average of 288 protein spots were detected on the 2-D gels and all immunoreactive discriminate spots were manually excised from the gels, which
corresponds to 12 distinct proteins encoded by chromosome and one protein named Pap31 encoded by phage (Fig. 2, Table S1) (Alsmark et al., 2004). Sera obtained from B. henselae-infected patients showed an immune reaction to numerous proteins for almost all the immunoblots analyzed. However, the immunoreactivity obtained for patients with Selleckchem JAK inhibitor IE due to B. henselae was greater than that for those with CSD. This can be explained by more systemic infection occurring in patients with IE, in whom the massive infiltration of bacteria may be present. The peptides obtained by antigen processing are present to the actors of HLA system; thus, numbered protein spots are highly reactive on Western blot. Thus, for all patients, we have obtained a reproducible pattern
of reactivity with IE. The immunoreactive proteins were clustered on the zone of the gel showing pI 4.0–6.0. Some spots were found beyond this zone pattern of immunoreactive spots. Moreover, the sensitivity of the ECL reaction was greater than that in the silver-stained gel, hampering the analysis of immunoblots. Clearly, this zone of the gel was hardly accessible for manual spot-picking and we have not focused on these spots due to technical limitations (Kowalczewska et al., 2008). Our aim was to identify the most discriminate spots that are easy to match with any immunoblot performed with clinical sample from patients
with IE due to B. henselae. The choice of a reference gel was very important to show the reproducibility of the results as well as the similarity of the immunoreactive patterns within patients with IE and finally the best coverage of matching spots. However, PCA analysis has not clearly demonstrated the homogeneity of this IE group, because, considering two independent matchings Fossariinae with two different reference gels, we could observe more heterogeneity among cases with IE (Figs 1, 3 and 4). It is important to underline that two cases with IE showed an immunoreactivity pattern similar to those from CSD (Fig. 3). They colocalized with CSD and BD immunoblots in the PCA analysis. Several spots widely distributed in 2-D gel were immunoreactive with sera of patients with CSD (Figs 3 and 4). In general, the large spots were immunoreactive. The majority of these spots corresponded to the spots found in a very reactive zone of patients with IE. However, a consistent reactivity to a single spot by all sera was not observed.