Because antigen recognition
may vary greatly among patients, we examined in detail the reactivity of individual serum samples to each antigen. For this analysis, we selected the clones for the 58 ORFs of C. pneumoniae that exhibited positive signals in the initial immunoscreening; the serum samples that contained the highest titers in the ELISA assays were used as primary antibodies. The selected serum samples are indicated BIBW2992 ic50 by an asterisk in Table 1. A great variability was noted in the number of antigens detected using various combinations of individual serum samples as the primary antibody and isotype-specific anti-human immunoglobulins as the secondary antibodies (Fig. 3). Among the 58 ORFs tested, positive signals were detected for the antigens in a total of 39 ORFs by the combination selleck kinase inhibitor of at least one patient’s serum sample as the primary antibody and one of the isotype-specific anti-IgA, anti-IgG, or anti-IgM as the secondary antibody. Although anti-C. pneumoniae IgA in No. 4-3 serum, anti-C. pneumoniae IgG in No. 3-2 and 5-2, and anti-C. pneumoniae
IgM in No. 6 and 8 produced negative results in both the ELISA tests, some ORFs were clearly recognized as antigens. These results indicated that the serum sample definitely contains IgA, IgG, and IgM antibodies against the proteins encoded by some ORFs. We summarized the data for positive ORFs and have listed their orthologs and homologs from C. trachomatis in Fig. 3b. Among the 39 ORFs, we identified 11 ORFs as antigens (Cpj0147, Cpj0159, Cpj0178, Cpj0186, Cpj0268, Cpj0308, Cpj0472, Cpj0677, Cpj0678, Cpj1056, and Cpj1070) that do not have orthologs in the C. trachomatis genome. Among the other 19 ORFs, which were not detected by any individual serum sample (Fig. 3a and b), but were detected by pooled serum sample (Fig. 2), nine ORFs (Cpj0067, Cpj0181, Cpj0214, Cpj0224, Cpj0225, Cpj0339, Cpj0355, Cpj0356, and Cpj0457) do not have orthologs in the C. trachomatis genome (Fig. 3b). We believe that these 20 ORFs without orthologs in the C. trachomatis
genome represent strongly immunogenic antigens that are highly Inositol monophosphatase 1 specific to C. pneumoniae. In this study, we intended to identify novel C. pneumoniae-specific antigens by screening the C. pneumoniae genome. We applied a bioinformatics approach for annotation taxonomy that allowed us to concentrate on a subset of proteins with unknown functions. To identify the antigens recognized by the antibodies in the patients with primary C. pneumoniae infection, we designed a screening system to use patients’ serum samples as immunological probes for the genomic screening of a C. pneumoniae-ORF expression library. We measured the titers of the isotype-specific immunoglobulins using the commercially available ELISA kits HITAZYME and Medac. These kits gave both negative and positive results for antibody titers of IgA, IgG, and IgM.