Chlamydia trachomatis Whole-Proteome Microarray Analysis of The Netherlands Chlamydia Cohort Study.

Chlamydia trachomatis (Ct) whole-proteome microarrays were utilized to identify antibody patterns associated with infection; pelvic inflammatory disease (PID), tubal factor infertility, chronic pelvic pain (CPP) and ectopic pregnancy in a subsample of the Netherlands Chlamydia cohort study. Serum pools were analyzed on whole-proteome arrays. The 121 most reactive antigens identified during whole-proteome arrays were selected for further analysis with minimized microarrays that allowed for single sera analysis. From the 232 single sera; 145 (62.5%) serum samples were reactive for at least one antigen. To discriminate between positive and negative serum samples; we created a panel of in total 18 antigens which identified 96% of all microarray positive samples. Antigens CT_858; CT_813 and CT_142 were most reactive. Comparison of antibody reactivity's among women with and without Ct related sequelae revealed that the reactivity of CT_813 and CT_142 was less common among women with PID compared to women without (29.0% versus 58.6%, p = 0.005 and 25.8% versus 50.6%, p = 0.017 respectively). CT_858 was less common among CPP cases compared to controls (33.3% versus 58.6; p = 0.028). Using a whole-proteome array to select antigens for minimized arrays allows for the identification of novel informative antigens as general infection markers or disease associated antigens.

In situ, Cell-free Protein Expression on Microarrays and Their Use for the Detection of Immune Responses.

Until recently, whole-proteome microarrays for comprehensive studies of protein interactions were mostly produced by individual cloning and cellular expression of very many open reading frames, followed by protein isolation and purification as well as array production. To overcome this cumbersome process, we have developed a method to generate microarrays representing entire proteomes by a combination of multiple spotting and on-chip, cell-free protein expression. Here, we describe the protocol for the production of bacterial protein microarrays. With slight adaptations, however, the procedure can be applied to the proteome of any organism. Expression constructs of each gene are generated by PCR on bacterial genomic DNA followed by a common secondary amplification that is adding relevant regulative elements to either end of the constructs. The unpurified PCR-products are spotted onto the microarray surface. Full-length proteins are directly expressed in situ in a cell-free manner and stay attached to the surface without further action. As an example of a typical application, we describe here the proteome-wide analysis of the immune response to a bacterial infectious agent by characterizing the binding profiles of the antibodies in patient sera.