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

Abstract

For detection of anti-C. trachomatis antibodies by serological assays, use of classical chlamydial antigens results in high cross-reactivity and poor sensitivity. Previously, we discovered 48 strongly reactive peptide antigens of C. trachomatis-specific B-cell epitopes from 21 immunodominant proteins, and individual testing and combined scoring of 5 to 11 peptide antigens provided highly sensitive and specific detection of anti-C. trachomatis antibodies in chemiluminescent ELISAs. To simplify this method, this study established a single-well labor-saving colorimetric ELISA using a mixture of 12 strongly reactive C. trachomatis peptide antigens (Ctr Mix1) for detection of anti-C. trachomatis antibodies. This Ctr Mix1 ELISA (94% sensitivity and 98% specificity) outperformed 4 commercial ELISAs (49% to 79% sensitivity and 98% specificity). This ELISA can be easily implemented and commercialized, with convenient setup for use in nonspecialized laboratories. Thus, this mixed peptide assay with superior specificity and sensitivity will improve serodiagnosis of C. trachomatis infections.

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

“…Overall, the Cpn peptide mixes showed high specificity similar to single peptide antigens, albeit the signal magnitude of mixed peptide assays was lower than that of the strongest component individual peptides, analogous to the earlier reported reactivities of C . trachomatis peptide mixes 47 .…”

“…C2 shows the consensus antibody status of the four commercial anti-Cpn IgG ELISAs, with manufacturer defined cutoffs. The consensus antibody status of Ctr mixed peptide assays at previously described cutoffs 47 is shown in C3, and in C4 the Ctr commercial ELISAs with manufacturer defined cutoffs. In C5, red or green indicates double positive or double-negative sera, respectively, for anti- C .…”

“…Previously, we have identified highly reactive and specific B cell epitopes 41–47 of immunodominant proteins of all Chlamydia species 48–51 . In extensive evaluation 46,47 , we also showed that the C . trachomatis -specific peptide antigens provide superior assays with high sensitivity and specificity.…”

“…Importantly, the high sensitivity of the peptide assays was mainly achieved by the use of multiple B-cell epitopes of several C . trachomatis immunodominant proteins 46,47 . Since antibody responses to individual B cell epitopes are stochastic 41–46 , only the combined use of multiple peptide antigens reliably measured host antibodies produced in response to C .…”

Mucosal Vaccination with UV-Inactivated Chlamydia suis in Pre-Exposed Outbred Pigs Decreases Pathogen Load and Induces CD4 T-Cell Maturation into IFN-γ+ Effector Memory Cells

“…Primary IgG antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-pig IgG-h+l cross-adsorbed antibody (A100-205P) (Bethyl Laboratories, Inc., Montgomery, TX, USA) by colorimetric enzyme-linked immunosorbent assay (ELISA) as described previously [ 27 ], with two modifications: (A) wash buffer contained 200 mM NaCl, 0.1% Tween-20, 0.005% Benzalkonium chloride, 20 mM Tris-HCl, and a pH of 7.4; (B) assay diluent and blocking buffer contained 10% chicken sera, 1% polyethylene glycol, 200 mM NaCl, 0.1% Tween-20, 0.005% Benzalkonium chloride, 50 mM Tris-HCl, and a pH of 7.4.…”