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By testing with 185 human antisera, we expanded 18 previously discovered <jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic>-specific B-cell epitopes to 48 peptide antigens from 12\u2009<jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic> immunodominant proteins. For specific detection of antibodies against <jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic>, we developed novel ELISAs with strongly reactive individual peptide antigens and mixtures of these peptides. By comparison to a composite reference standard (CRS) for anti-<jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic> antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti-<jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic> IgG ELISAs (36-12% sensitivity at 95% specificity). Human <jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic> (Cpn) and <jats:italic>C<\/jats:italic>. <jats:italic>trachomatis<\/jats:italic> (Ctr) seroreactivity was 54% biased towards co-positivity in commercial Cpn and Ctr ELISAs, but unbiased in Cpn and Ctr peptide antibody assays, suggesting severe cross-reactivity of commercial ELISAs. Using hyperimmune mouse sera against each of 11 <jats:italic>Chlamydia<\/jats:italic> spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all <jats:italic>Chlamydia<\/jats:italic> spp., but Cpn and Ctr peptide antigens react only with antisera against the cognate chlamydial species. With simultaneously high specificity and sensitivity, and convenient use for non-specialized laboratories, these ELISAs have the potential to improve serodiagnosis of <jats:italic>C<\/jats:italic>. <jats:italic>pneumoniae<\/jats:italic> infection.<\/jats:p>","DOI":"10.1038\/s41598-019-51501-5","type":"journal-article","created":{"date-parts":[[2019,10,21]],"date-time":"2019-10-21T12:11:10Z","timestamp":1571659870000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":13,"title":["Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology"],"prefix":"10.1038","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7243-8501","authenticated-orcid":false,"given":"Kh Shamsur","family":"Rahman","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bernhard","family":"Kaltenboeck","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2019,10,21]]},"reference":[{"key":"51501_CR1","unstructured":"Kuo, C. 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