Figure 1.

Generation of Ct whole-proteome microarrays. (a) In order to generate expression constructs, gene specific primer pairs were designed for all 895 Ct ORFs, including adaptor sequences common to all forward (pink) and reverse primers (light green). Using genomic Ct DNA as template, all 895 genes were amplified, resulting in PCR products carrying the gene of interest (GOI) flanked by two adaptor sequences. These products served as templates for a second PCR, using common primers for all Ct genes. These primers contain transcriptional and translational elements, as well as sequences complementary to the adaptor sequences. After the second PCR, genes were therefore flanked by spacer sequences, regulatory sequences (T7 Promoter, untranslated region (UTR), ribosome binding site (RBS), start codon (ATG), T7 Terminator) and N-terminal 6xHis and C-terminal V5 fusion tags (red: sequences included in the forward primer; green: sequences included in the reverse primer). (b) The expression constructs were transferred onto Ni-NTA coated slides in a first spotting step. Subsequently, the expression mixture was spotted directly on top of the first spots, and proteins were expressed on the slide during incubation in a humidified environment. Efficiency of protein expression was determined using fluorescence-conjugated antibodies directed against the terminal fusion tags. Proteome Immunoassays were performed by incubation of serum on whole-proteome arrays. Antibodies present in the serum were able to bind to the immobilized antigens on the array, and quantified using a fluorescence-conjugated anti-human secondary antibody.