Immune profiling with a Salmonella Typhi antigen microarray identifies new diagnostic biomarkers of human typhoid

Abstract

Current serological diagnostic assays for typhoid fever are based on detecting antibodies against Salmonella LPS or flagellum, resulting in a high false-positive rate. Here we used a protein microarray containing 2,724 Salmonella enterica serovar Typhi antigens (>63% of proteome) and identified antibodies against 16 IgG antigens and 77 IgM antigens that were differentially reactive among acute typhoid patients and healthy controls. The IgG target antigens produced a sensitivity of 97% and specificity of 80%, whereas the IgM target antigens produced 97% and 91% sensitivity and specificity, respectively. Our analyses indicated certain features such as membrane association, secretion, and protein expression were significant enriching features of the reactive antigens. About 72% of the serodiagnostic antigens were within the top 25% of the ranked antigen list using a Naïve bayes classifier. These data provide an important resource for improved diagnostics, therapeutics and vaccine development against an important human pathogen.

Serodiagnosis of Acute Typhoid Fever in Nigerian Pediatric Cases by Detection of Serum IgA and IgG Against Hemolysin E and Lipopolysaccharide

“…We therefore printed purified t1477 and four different sources of purified LPS in parallel lines on nitrocellulose membrane using a noncontact jet printer, and the membrane was dried and cut into immunostrips. Antigens t3710 and t1459 described in a previous study 10 were also printed, but were unreactive (not shown). Immunostrips were then probed with 56 randomized serum samples from the U.S. control, Nigerian healthy control, "No Growth," "Growth, S. Typhi," and "Growth, NTS" groups.…”

“…In previous studies, we described the production and use of a S. Typhi partial proteome microarray (∼63%) for profiling antibodies in human adult typhoid cases from Malawi, 9 Vietnam, 10 and Bangladesh, 18 and in the mouse S. Typhimurium model.…”

“…Full S. Typhi proteome microarrays (Antigen Discovery Inc., Irvine, CA) were manufactured according to published methods. 9,10 The content of the arrays comprised an ORFeome library cloned previously with ∼63% coverage of the S. Typhi proteome supplemented with the additional S. Typhi open reading frames (ORFs) to provide full coverage. In addition, LPSs from Salmonella typhosa (S. Typhi), purified by gel filtration (L2387; Sigma-Aldrich, Dorset, United Kingdom), 11 was printed in four 3-fold serial dilutions from 0.1 to 0.003 mg/mL.…”

“…Serum samples were shipped to UCI on dry ice and were probed onto proteome arrays as described previously. 9,10 Arrays were scanned in a GenePix 4200AL (Molecular Devices, Sunnyvale, CA), and captured TIFF files were quantified using ScanArray Express software (Perkin Elmer, Waltham, MA). Median spot pixel intensities subtracted of local background were imported into Microsoft Excel (Microsoft Corp., Redmond, WA).…”

“…S. Typhi proteins were purified and printed on immunostrips as described previously. 10 In brief, proteins were expressed from the plasmids used for protein microarrays in Escherichia coli BL-21 cell, and were extracted using BugBuster (Novagen, Billerica, MA). Inclusion body preparations were obtained by centrifugation, solubilized in urea, and dialyzed against phosphate-buffered saline to remove urea.…”