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Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection.

ABSTRACT: The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB patient sera. Of 409 TB proteins produced, ninety-two yielded seropositive reactions, fourteen including eight novel proteins showed strong immunoreactivity. Further, six were selected and constructed as a multiple-antigen combination set through analysis of various combinations. A comparative study of the multiple-antigen combination set and a commercially available kit revealed that the combination set showed 66.3% (95% CI 60.5-71.8) sensitivity, which was significantly higher than that of the commercial kit [31.6% (95% CI 26.3-37.3)]. The specificity of both methods was similar at 89.6% (95% CI 83.3-95.4) and 90.6% (95% CI 83.0-95.6), respectively. This study provides a set of novel diagnostic protein markers with great potential for the development of novel diagnostic tools for active TB.

SUBMITTER: Zhou F

PROVIDER: S-EPMC4642701 | biostudies-other | 2015

REPOSITORIES: biostudies-other

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Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection.

Zhou Fangbin F Xu Xindong X Wu Sijia S Cui Xiaobing X Fan Lin L Pan Weiqing W

Scientific reports 20151020

The lack of effective and accurate diagnostic tools contributes to the high prevalence of tuberculosis (TB) worldwide. The current serodiagnostics for TB are inadequate mainly due to lack of TB-specific antigens with highly accurate diagnosis. In the current study, we aimed to identify novel diagnostic antigens using glutathione S-transferase (GST)-fusion protein technique. We determined the reactivity of these recombinant proteins arrayed in solution and on GSH-immobilized microplates with TB p ...[more]

PMID: 26481294

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Project description:Johne's disease, a chronic gastrointestinal inflammatory disease caused by Mycobacterium avium subspecies paratuberculosis, is endemic in dairy cattle and other ruminants worldwide and remains a challenge to diagnose using traditional serological methods. Given the close phylogenetic relationship between M. avium subsp. paratuberculosis and the human pathogen Mycobacterium tuberculosis, here, we applied a whole-proteome M. tuberculosis protein array to identify seroreactive and diagnostic M. avium subsp. paratuberculosis antigens. A genome-scale pairwise analysis of amino acid identity levels between orthologous proteins in M. avium subsp. paratuberculosis and M. tuberculosis showed an average of 62% identity, with more than half the orthologous proteins sharing >75% identity. Analysis of the M. tuberculosis protein array probed with sera from M. avium subsp. paratuberculosis-infected cattle showed antibody binding to 729 M. tuberculosis proteins, with 58% of them having ?70% identity to M. avium subsp. paratuberculosis orthologs. The results showed that only 4 of the top 40 seroreactive M. tuberculosis antigens were orthologs of previously reported M. avium subsp. paratuberculosis antigens, revealing the existence of a large number of previously unrecognized candidate diagnostic antigens. Enzyme-linked immunosorbent assay (ELISA) testing of 20 M. avium subsp. paratuberculosis recombinant proteins, representing reactive and nonreactive M. tuberculosis orthologs, further confirmed that the M. tuberculosis array has utility as a screening tool for identifying candidate antigens for Johne's disease diagnostics. Additional ELISA testing of field serum samples collected from dairy herds around the United States revealed that MAP2942c had the strongest seroreactivity with Johne's disease-positive samples. Collectively, our studies have considerably expanded the number of candidate M. avium subsp. paratuberculosis proteins with potential utility in the next generation of rationally designed Johne's disease diagnostic assays.

Project description:Discovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value-assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC-MS/MS to analyse MTB culture filtrate proteins (MTB-CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB-CFPs - including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R-squared value ranged from 0.64 to 0.79. The only exception was ESAT-6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI-assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.

Dataset Information

Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection.

Publications

Protein array identification of protein markers for serodiagnosis of Mycobacterium tuberculosis infection.

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