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Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device.

Sullivan L, Wall SJ, Carrington M, Ferguson MA - PLoS Negl Trop Dis (2013)

Bottom Line: While this test is successful, it is acknowledged that there may be room for improvement.The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods.These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT

Background: The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.

Methodology/principal findings: We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.

Conclusions/significance: Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

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Related in: MedlinePlus

Recombinant protein antigens used in this study.A generic representation of the ISGs is shown at the top and a representation of GRESAG4 is shown at the bottom. All have cleavable N-terminal signal peptides and internal transmembrane domains, typical of type-1 membrane proteins. The constructs prepared and expressed and the soluble proteins successfully purified, are indicated.
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pntd-0002087-g001: Recombinant protein antigens used in this study.A generic representation of the ISGs is shown at the top and a representation of GRESAG4 is shown at the bottom. All have cleavable N-terminal signal peptides and internal transmembrane domains, typical of type-1 membrane proteins. The constructs prepared and expressed and the soluble proteins successfully purified, are indicated.

Mentions: The amplified ISG gene segments were cloned into various pET bacterial expression plasmids that provide a His-tag fused either to the N-terminus or C-terminus of the protein, in some cases via a TEV protease cleavage site, as indicated in (Figure 1). Multiple constructs were designed for GRESAG4 encoding the predicted full-length extracellular domain and several small globular domains based on predictions from GLOBplot software [46] (Figure 1). These constructs were amplified from genomic DNA using the primers described in the Supporting Information (Table S1), cloned into TOPO-TA vector pCR2.1 and verified by DNA sequencing. The constructs were either cloned into the pET15bTEV vector, such that the proteins they encode are fused at the N-terminus to a His tag, or into a pGEX-TEV vector such that the protein is fused at its N-terminus to a glutathione S-transferase (GST) sequence via a TEV cleavage site (Figure 1). The details of protein expression in E. coli and subsequent purification are described in the Supporting Information (Text S1).


Proteomic selection of immunodiagnostic antigens for human African trypanosomiasis and generation of a prototype lateral flow immunodiagnostic device.

Sullivan L, Wall SJ, Carrington M, Ferguson MA - PLoS Negl Trop Dis (2013)

Recombinant protein antigens used in this study.A generic representation of the ISGs is shown at the top and a representation of GRESAG4 is shown at the bottom. All have cleavable N-terminal signal peptides and internal transmembrane domains, typical of type-1 membrane proteins. The constructs prepared and expressed and the soluble proteins successfully purified, are indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3584999&req=5

pntd-0002087-g001: Recombinant protein antigens used in this study.A generic representation of the ISGs is shown at the top and a representation of GRESAG4 is shown at the bottom. All have cleavable N-terminal signal peptides and internal transmembrane domains, typical of type-1 membrane proteins. The constructs prepared and expressed and the soluble proteins successfully purified, are indicated.
Mentions: The amplified ISG gene segments were cloned into various pET bacterial expression plasmids that provide a His-tag fused either to the N-terminus or C-terminus of the protein, in some cases via a TEV protease cleavage site, as indicated in (Figure 1). Multiple constructs were designed for GRESAG4 encoding the predicted full-length extracellular domain and several small globular domains based on predictions from GLOBplot software [46] (Figure 1). These constructs were amplified from genomic DNA using the primers described in the Supporting Information (Table S1), cloned into TOPO-TA vector pCR2.1 and verified by DNA sequencing. The constructs were either cloned into the pET15bTEV vector, such that the proteins they encode are fused at the N-terminus to a His tag, or into a pGEX-TEV vector such that the protein is fused at its N-terminus to a glutathione S-transferase (GST) sequence via a TEV cleavage site (Figure 1). The details of protein expression in E. coli and subsequent purification are described in the Supporting Information (Text S1).

Bottom Line: While this test is successful, it is acknowledged that there may be room for improvement.The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods.These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

View Article: PubMed Central - PubMed

Affiliation: College of Life Sciences, University of Dundee, Dundee, United Kingdom.

ABSTRACT

Background: The diagnosis of Human African Trypanosomiasis relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). While this test is successful, it is acknowledged that there may be room for improvement. Our aim was to develop a prototype lateral flow test based on the detection of antibodies to trypanosome antigens.

Methodology/principal findings: We took a non-biased approach to identify potential immunodiagnostic parasite protein antigens. The IgG fractions from the sera from Trypanosoma brucei gambiense infected and control patients were isolated using protein-G affinity chromatography and then immobilized on Sepharose beads. The IgG-beads were incubated with detergent lysates of trypanosomes and those proteins that bound were identified by mass spectrometry-based proteomic methods. This approach provided a list of twenty-four trypanosome proteins that selectively bound to the infection IgG fraction and that might, therefore, be considered as immunodiagnostic antigens. We selected four antigens from this list (ISG64, ISG65, ISG75 and GRESAG4) and performed protein expression trials in E. coli with twelve constructs. Seven soluble recombinant protein products (three for ISG64, two for ISG65 and one each for ISG75 and GRESAG4) were obtained and assessed for their immunodiagnostic potential by ELISA using individual and/or pooled patient sera. The ISG65 and ISG64 construct ELISAs performed well with respect to detecting T. b. gambiense infections, though less well for detecting T. b. rhodesiense infections, and the best performing ISG65 construct was used to develop a prototype lateral flow diagnostic device.

Conclusions/significance: Using a panel of eighty randomized T. b. gambiense infection and control sera, the prototype showed reasonable sensitivity (88%) and specificity (93%) using visual readout in detecting T. b. gambiense infections. These results provide encouragement to further develop and optimize the lateral flow device for clinical use.

Show MeSH
Related in: MedlinePlus