Comprehensive Molecular Serology of HumanChlamydia trachomatisInfections by Peptide Enzyme-Linked Immunosorbent Assays

Abstract

For detection of anti-Chlamydia trachomatis antibodies by serological assays, use of classical whole-organism chlamydial antigens results in high cross-reactivity. These antigens bind mainly antibodies against the major outer membrane protein (OmpA) and bind antibodies against other immunodominant non-OmpA proteins to a lesser extent, resulting in poor assay sensitivity. The specificity of C. trachomatis serology is also compromised by the high prevalence of cross-reactive anti-C. pneumoniae antibodies in human populations. We previously identified 48 highly specific C. trachomatis B cell epitope peptide antigens of 21 immunodominant proteins. This study validated peptide antigen-based novel ELISAs that provide highly specific and sensitive detection of anti-C. trachomatis antibodies. Compared to four commercial ELISAs that achieved only poor sensitivities (51.5% to 64.8%), the combined signals of 5 to 11 peptides provided high sensitivity (86.5% to 91.8%) at the same 98% specificity. Thus, by using multiple peptide antigens of immunodominant proteins, we created simple ELISAs with specificity and sensitivity superior to standard C. trachomatis serodiagnosis.

Mixed Chlamydia trachomatis Peptide Antigens Provide a Specific and Sensitive Single-Well Colorimetric Enzyme-Linked Immunosorbent Assay for Detection of Human Anti-C. trachomatis Antibodies

“…Previously, we identified highly reactive and specific B-cell epitopes of immunodominant proteins of all Chlamydia species ( 16 – 21 ). An important finding was that 16- to 30-amino-acid peptide antigens combined with an N-terminal hydrophilic serine-glycine-serine-glycine spacer were required for optimal signal strength ( 16 – 18 ).…”

“…Further testing of predicted peptide antigens with sera from C. trachomatis - infected women revealed additional human host-specific C. trachomatis B-cell epitopes that were recognized only by the natural human host ( 20 ) but did not react with sera from mice that were hyperimmunized to C. trachomatis ( 16 ). Subsequently, we comprehensively evaluated the utility of 11 top-ranked peptide antigens from 8 C. trachomatis proteins for use in human C. trachomatis serology ( 21 ). Results obtained for detection of antibodies against 4 commercial anti- C. trachomatis ELISA antigens and these 11 C. trachomatis peptide antigens showed that the peptide assays outperformed any of these commercial ELISAs both in specificity and sensitivity ( 21 ), a consequence of optimal peptide antigen design and ELISA protocol ( 16 – 21 ).…”

“…Subsequently, we comprehensively evaluated the utility of 11 top-ranked peptide antigens from 8 C. trachomatis proteins for use in human C. trachomatis serology ( 21 ). Results obtained for detection of antibodies against 4 commercial anti- C. trachomatis ELISA antigens and these 11 C. trachomatis peptide antigens showed that the peptide assays outperformed any of these commercial ELISAs both in specificity and sensitivity ( 21 ), a consequence of optimal peptide antigen design and ELISA protocol ( 16 – 21 ).…”

“…Importantly, the high sensitivity of the peptide assays was achieved mainly by the use of multiple B-cell epitopes of several C. trachomatis immunodominant proteins, including OmpA ( 21 ), compared to exclusive OmpA antigens used in commercial ELISAs. Given the stochasticity of antibody responses to individual B-cell epitopes ( 16 , 20 , 21 ), only the combined use of multiple peptide antigens can reliably measure host antibodies produced in response to C. trachomatis infection ( 21 ), similar to the quantitative results obtained with complex antigens. However, this requires single serum testing of individual peptide antigens in 11 separate microtiter wells ( 21 ).…”

“…Given the stochasticity of antibody responses to individual B-cell epitopes ( 16 , 20 , 21 ), only the combined use of multiple peptide antigens can reliably measure host antibodies produced in response to C. trachomatis infection ( 21 ), similar to the quantitative results obtained with complex antigens. However, this requires single serum testing of individual peptide antigens in 11 separate microtiter wells ( 21 ). In addition, the optimally sensitive chemiluminescent ELISA format is not commonly used by the diagnostic laboratory community, which typically prefers colorimetric ELISA formats.…”