References 1. Ames G.F., Kustu S.G., “Method for obtaining periplasmic proteins from bacterial cells using chloroform”. Patent Number: Assignee: The United States of America as represented by the Department of Health and Human Services, Washington, D.C. Material and Methods. Data acquisition and analysis The approach was based solely on data provided on-line by public databases such as the Brucella Bioinformatics Portal (Brucella Genome Data and the National Center for Biotechnology Information (NCBI- Predicted protein sequences were downloaded for all the available Brucella melitensis biotypes /strains. After obtaining the protein sequences the on-line software PSORTb (v.3.0.2) was used to determine their localization. The proteins were grouped according to their localization. The amino acid sequences of proteins belonging to Extracellular, Outer-Membrane and Periplasmic space were selected for further research. As an initial step BLASTp was used investigate the presence or absence of these proteins within the species of the Brucella genus. In the cases of unique proteins the amino acid sequence is further blasted against the protein databases of organisms that often cross react with anti-brucella antibodies. Further analysis of polymorphic sites and the impact of each polymorhism on the proteins structure and, subsequently, antigenicity was further tested using software such as PRED-TMBB, JPRED, PSIPRED and EpiC. Candidate would be isolated, analysed and tested for their antigenicity. Extraction and 2D analysis of periplasmic proteins Periplasmic proteins were extracting from Brucella melitensis cells using chloroform, according to Ames et al., Patent ml of bacterial cultures of Brucella melitensis field strains and the vaccine Rev1 strain were used for the analysis. 5-10μg of the extracted periplasmic proteins were analysed by 2D electrophoresis using ZOOM ® IPGRunner™ System (Life technologies). First dimension IEF was performed on Zoom ® Strip pH 3-10 and the second dimension was performed on a 10% SDS PAGE gel. The 2D gels were silver stained. Results. After determining the cellular localization of proteins from the Brucella spp, three subgroups gathering a total of proteins were investigated to identify candidate antigens that discriminate Rev.1 vaccinated from infected animals and cross-reactive antigens.; 170 of these proteins were extracellular, 308 were outer membrane proteins and 889 proteins were of periplasmic localization. Presence and homology within the genus of Brucella and throughout the cross- reactive species was investigated while for selected proteins In Silico antigenicity testing was performed. This filtering process resulted in the following proteins: Porin omp2a, TonB-dependent receptor protein, Leu/Ile/Val-binding protein homolog 2, Nitrous-oxide reductase, Periplasmic dipeptide transport protein, Outer membrane protein assembly factor (BamD), competence lipoprotein (Coml), Sugar ABC transporter and a probable sugar-binding periplasmic protein. Conclusion. In present work we characterized candidate protein antigens to be used for the differentiation of Brucella melitensis Rev1 and field strains. Field strains periplasmic proteins of Brucella melitensis presented a common unique pattern different from those of Rev1 strain. Introduction. In present work we characterized candidate protein antigens to be used for the differentiation of Brucella melitensis Rev1 and field strains. Bibliographically false-positive Brucella antibody tests can result due to cross-reactivity of antibodies from other Brucella spp. strains, Yersinia enterocolytica O:9, E. coli O:157, Salmonella group N (O:30), Pseudomonas spp, Francicella tularensis, Pasteurella spp, Moraxella phenylpuruvica and Orthrobactrum antropii. The identification of the potential candidate antigens was attempted using bioinformatic analyses. The main objective of this research was to identify proteins that could be used as standard markers for the discrimination of infected from cross-reactive animals but also from Rev.1 vaccinated animals, in an easy and low-cost method such as a western blot. Two candidate proteins were detected as potential target to discriminate Rev.1 vaccinated from non-vaccinated animals and cross-reactive antigens: the D0B7Y2 Sugar ABC transporter and the Q8YI0 Periplasmic dipeptide transport protein. Two additional proteins were identified as target to discriminate Brucellosis infected and non-infected animals: the Omp2a and the Q8YCE2, Sugar binding periplasmic protein. Finally, four additional candidate antigens were also identified to discriminate Brucella infected and animals’ carriers of cross-reactive antigens: the DNA-dependent RNA polymerase beta chain – rpoB, the Copper/Zinc superoxide dismutase IL gene – sodC, the 50S ribomosal proteins L7/L12 - rpIL gene and the ATP synthase subunit. The periplasmic proteins were isolated from bacterial cultures of Rev1 and 19 Brucella melitensis field strains. The 2D electrophoresis analysis of periplasmic proteins extracted from field strains showed a similar unique pattern different from those of Rev1 strain. As shown in Figure1, different protein spots between them are mainly detected in the region of pI range 4-5 and MW range 30-65kDa. According to bibliography the two periplasmic proteins (Q8YI0 Periplasmic dipeptide transport protein and Sugar binding periplasmic protein) identified by the bioinformatics analysis should be spoted in this area. Also, the region of pI range 9-10 and MW range kDa presented some different protein spots between Rev1 and field strains. Acknowledgments The work was funded by the General Secretariat for Research & Technology (GSRT) International S&T, Cooperation Directorate – European Union Division – GREECE as a part of an EU EMIDA ERA-NET project entitled “Brucella melitensis: biotyping and differential diagnostic- Brucmel” Figure 1. 2D gels of periplasmic proteins of Rev1 vaccine strain and three Brucella melitensis field strains. First dimension IEF was performed on Zoom ® Strip pH 3-10 and the second dimension was performed on a 10% SDS PAGE gel. The 2D gels were silver stained. The MW marker used is the BlueStar PLUS Prestained Protein Marker (NIPPON Genetics). The main differences of the 2D patterns of Rev1 and the field strains are marked.