Although C. pneumoniae-specific antibody responses have been characterized by immunoblotting, only few major surface proteins (MOMP, Omp2, and CrpA; Iijima et al., 1994; Klein et al., JAK inhibitor 2003; Mygind et al., 1998) and some Inc proteins (Cpj0146, Cpj0147, and Cpj0308) have been detected (Hongliang et al., 2010). However, these antigens have yielded variable results with respect to the consistency and accuracy of C. pneumoniae identification. Taken together, very little information is available regarding specific detection of C. pneumoniae. We determined the sequence of the whole genome of C. pneumoniae J138 isolated

in Japan (Shirai et al., 2000) and found that this strain features putative protein coding from its 1069 open reading frames (ORFs). A comprehensive bioinformatics approach was applied for annotation taxonomy, and about half of the predicted genes were found to encode proteins without any known functions. To identify novel specific antigens from C. pneumoniae, we screened 455 genes without any known functions. A fusion protein expression library of C. pneumoniae was constructed in Saccharomyces cerevisiae. Protein extracts of the recombinant yeast cells expressing the green fluorescent protein (GFP)-tagged C. pneumoniae proteins were subjected to Western blot analysis using serum samples from C. pneumoniae-infected patients as the primary

antibodies. This study sought to identify specific and highly immunodominant antigens, which are required for the development of new serodiagnostic assays, and hopefully, vaccines, in the future. Thirteen serum samples were collected from eight patients HSP90 (age: range, 4–11 years; see more Table 1), who had been clinically diagnosed with primary acute C. pneumoniae infection. The levels of C. pneumoniae-specific immunoglobulin (Ig) IgA, IgG, and IgM in these patients were evaluated using two different

ELISA kits: (1) HITAZYME C. pneumoniae kits for IgA, IgG, and IgM that utilize the soluble elementary body (EB)-outer membrane complex, without the lipopolysaccharide, as the antigen (Hitachi Chemical, Japan) and (2) C. pneumoniae-ELISA plus Medac kits for IgA and IgG and C. pneumoniae-sELISA Medac kit for IgM, which utilize the purified cell wall membrane proteins as the antigen (Medac Diagnostika, Germany). Eight serum samples from 0-year-old healthy children were used as negative controls. Chlamydophila pneumoniae genomic DNA was obtained from the EBs of the C. pneumoniae J138-infected HEp-2 cells (Miura et al., 2001). We used a gene expression system controlled by a Tet-off promoter in S. cerevisiae. The ORFs of 455 genes from C. pneumoniae J138, including genes of unknown function (Supporting Information, Table S1), were cloned into a pMT830 vector, which was constructed as previously described (Tabuchi et al., 2009). This vector system allows a protein of interest to be expressed with GFP fused to the C-terminus.