Abstract
Using Chlamydia trachomatis (Ct) as a complex model organism, we describe a method to generate bacterial whole-proteome microarrays using cell-free, on-chip protein expression. Expression constructs were generated by two successive PCRs directly from bacterial genomic DNA. Bacterial proteins expressed on microarrays display antigenic epitopes, thereby providing an efficient method for immunoprofiling of patients and allowing de novo identification of disease-related serum antibodies. Through comparison of antibody reactivity patterns, we newly identified antigens recognized by known Ct-seropositive samples, and antigens reacting only with samples from cervical cancer (CxCa) patients. Large-scale validation experiments using high-throughput suspension bead array serology confirmed their significance as markers for either general Ct infection or CxCa, supporting an association of Ct infection with CxCa. In conclusion, we introduce a method for generation of fast and efficient proteome immunoassays which can be easily adapted for other microorganisms in all areas of infection research.
Highlights
Success of protein expression on the microarray was determined by incubation with fluorescence-conjugated antibodies directed against the N- and C-terminal fusion tags
While the planar microarray permits the screening of relatively few samples for informative antibodies to an entire bacterial proteome, multiplex serology allows screening thousands of serum samples for relatively few antigens
Using our whole-proteome approach, we were able to confirm 20 of these antigens. Sixteen of these antigens were associated with general Ct infection in our study
Summary
Success of protein expression on the microarray was determined by incubation with fluorescence-conjugated antibodies directed against the N- and C-terminal fusion tags. Slides mounted in single chamber frames were blocked with 2 ml SuperBlock blocking buffer (Thermo Scientific) in ProPlate Slide Modules (Grace Bio-Labs) on an orbital shaker at room temperature for 45 min. They were washed twice for 5 min with 2 ml phosphate-buffered saline containing 0.05% Tween[20] (PBST) on a shaker. A protein was considered to be expressed if its signal intensity generated by the labeled antibodies to either the 6xHis or the V5 tag was higher than the mean plus five standard deviations of 20 NC1 replicat
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
