2.3. Laboratory Analysis

The Pan-DEC microarray was created by ADI as previously described [29] and represents a new analytical tool that complements an earlier array focused on ETEC [30] comprising 800 potential surface proteins and an additional 4168 gene features. These features were identified through a comparative analysis of 207 clinical ETEC isolates. The selected ETEC genes were found in more than 40% of the ETEC isolates and were absent in the genomes of three common E. coli commensal strains. Similarly, following a comparative genomic analysis of non-ETEC pathogenic E. coli species, 2168 protein-coding genes were chosen to represent EHEC (730 features), EIEC (452 features), EPEC (456 features), EAEC (288 features) and ExPEC (242 features). These genes were specific to each species, not found in commensal E. coli, and were not previously represented in the described ETEC proteome chip. To specifically focus on DEC, the analysis of ExPEC gene features was excluded in the final analysis. The controls included in the DEC microarray consisted of blank IVTT reactions serving as negative controls for protein expression, IgG and IgA antibodies for secondary antibody quality control and blank buffers. These selected genomic features were expressed into proteins using a cell-free in vitro transcription–translation (IVTT) system with each of the proteins containing a 5′ polyhistidine (HIS) epitope and a 3′ haemaglutinin (HA) epitope. These proteins were then printed onto nitrocellulose-coated glass slides using a robotic printer and validated before utilization.