International Journal of Microbiology and Immunology Research Vol. 3(5), pp. 064-075, August 2015 ISSN 2327-7769 ©2015 Academe Research Journals

Full Length Research Paper

Identification and evaluation of immunogenic Salmonella Typhi proteins in sera of acute typhoid fever patients using two protein separation methods

Chin KL, Prabha B and Phua KK*

Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia.

*Corresponding author. E-mail: kkphua7@gmail.com. Tel: +604-6534853; Fax: +604-6534803.

Accepted 22 July, 2015

Abstract

Pooled sera from typhoid fever patients and normal subjects were subjected to protein fractionation using 2 different methods: 1) affinity chromatography by removing albumin and immunoglobulin, or 2) ultrafiltration membrane by removing proteins above 100 kDa; in order to unmask low abundance and low molecular weight proteins. Protein preparations from both separation methods were subjected to Western blot analysis. The results showed that different separation methods gave different antigen-antibody binding profiles inherent of the technology used. A total of five immunoprecipitin bands were found to be specific for typhoid fever. Mass spectrometry and bioinformatic analyses indicated the presence of Salmonella Typhi (S. Typhi) proteins in bands 12, 15 and 50 kDa; and bands 18, 25 and 50 kDa contained human proteins. S. Typhi proteins, namely: blue copper oxidase (CueO), outer membrane protein C (OmpC), and flagellar hook-associated protein 1 (HAP1), were selected and cloned using Escherichia coli expression system. Using indirect ELISA, the individual recombinant proteins gave diagnostic sensitivities of <50%, whereas combining all 3 proteins in a single test gave a higher sensitivity of 70% and specificity of 93%. This study has shown that serum fractionation can uncover immunogenic proteins unique to the pathogen, which can be used as biomarkers for specific diagnosis of the disease.

Key words: Typhoid fever, affinity chromatography, ultrafiltration membrane, blue copper oxidase (CueO), outer membrane protein C (OmpC), flagellar hook-associated protein 1 (HAP1), in vivo immunogenic proteins.