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Cross-reactivity of classical chlamydial antigens compromises  Chlamydia (C . ) pneumoniae  serology. By testing with 185 human antisera, we expanded 18 previously discovered  C .  pneumoniae -specific B-cell epitopes to 48 peptide antigens from 12  C .  pneumoniae  immunodominant proteins. For specific detection of antibodies against  C .  pneumoniae , 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- C .  pneumoniae  antibody status of human sera, the top-performing CpnMixF12 peptide assay showed 91% sensitivity at 95% specificity, significantly higher than 4 commercial anti- C .  pneumoniae  IgG ELISAs (36-12% sensitivity at 95% specificity). Human  C .  pneumoniae  (Cpn) and  C .  trachomatis  (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  Chlamydia  spp., we confirm that commercial Cpn and Ctr ELISA antigens are cross-reactive among all  Chlamydia  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  C .  pneumoniae  infection.

Multi-peptide ELISAs overcome cross-reactivity and inadequate sensitivity of conventional Chlamydia pneumoniae serology