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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

Immuno-affinity chromatography and identification of potential diagnostic antigens.(A) Schematic representation of the preparation of IgG-Sepharose from T. b. gambiense infection and non-infection (control) sera, the immune-affinity capture of trypanosome antigens from a whole detergent lysate and their subsequent elution and concentration by ethanol precipitation. (B) Colloidal Comassie blue stained SDS-PAGE gel of the proteins eluted from infection IgG-Sepharose (lane 1) and non-infection (control) IgG-Sepharose (lane 2). The gel lanes were excised in 18 slices per lane, as indicated between lanes 1 and 2, and analysed by LC-MS/MS after reduction, S-alkylation and tryptic digestion. The positions of molecular weight markers are indicated on the left.
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pntd-0002087-g002: Immuno-affinity chromatography and identification of potential diagnostic antigens.(A) Schematic representation of the preparation of IgG-Sepharose from T. b. gambiense infection and non-infection (control) sera, the immune-affinity capture of trypanosome antigens from a whole detergent lysate and their subsequent elution and concentration by ethanol precipitation. (B) Colloidal Comassie blue stained SDS-PAGE gel of the proteins eluted from infection IgG-Sepharose (lane 1) and non-infection (control) IgG-Sepharose (lane 2). The gel lanes were excised in 18 slices per lane, as indicated between lanes 1 and 2, and analysed by LC-MS/MS after reduction, S-alkylation and tryptic digestion. The positions of molecular weight markers are indicated on the left.

Mentions: We took a non-biased proteomics approach to identify proteins that adsorb selectively to pooled infection IgG, and not to pooled control IgG (Figure 2A). Each serum pool contained four individual sera of patients clinically defined as having an infection with T. b. gambiense or as being uninfected. IgG fractions were purified from the pooled sera by affinity chromatography on protein G and then immobilised to cyanogen bromide-activated Sepharose beads. Equal amounts of infection and control IgG-Sepharose were incubated with equal amounts of T. b. brucei detergent cell lysate. Proteins that bound to the IgG columns were eluted by low pH, precipitated with cold ethanol, dissolved in SDS-sample buffer, reduced, separated by SDS-PAGE and stained with colloidal Coomassie blue (Figure 2B). More protein was seen in the eluate from the infection IgG column, consistent with infection-specific anti-trypanosome immune responses. Equivalent sections were cut out from the infection and control lanes, as indicated (Figure 2B). The excised gel pieces underwent in-gel S-alkylation and tryptic digestion, and the tryptic peptides were analysed by LC-MS/MS. Mascot software matched the peptide spectra to proteins in the T. b. brucei predicted protein database and scored the quality of the identifications. Lists of the proteins retained by infection IgG-Sepharose and control IgG-Sepharose were compared in each gel section. Twenty-four proteins with a MASCOT protein score above 50 were found uniquely in the infection IgG eluate and these are described in (Table 1). Several of the infection-specific proteins were defined as ‘hypothetical’, but other hits included known cell surface proteins, such as: Invariant Surface Glycoprotein (ISG) 75, ISG65, ISG64, Gene Related to Expression Site Associated Gene (GRESAG) 4, and the transferrin receptor subunits ESAG 6 and 7. As a starting point, the proteins with high MASCOT scores were prioritised. The rationale for this selection was that, by using an excess of trypanosome lysate in the affinity purification step, the amount of an eluted antigen should reflect, to a first approximation, the relative amount of antigen-specific immobilised IgG. The latter should, in turn, correspond to the immune response to that antigen in infected patients. Using this criterion, the protein antigens selected for study were ISG75, ESAG7, GRESAG4, ISG65, ISG64 and ESAG6 (Table 1). Next, we looked into the likely ease of protein expression of these antigens in E. coli. At this stage, we de-selected ESAG6 and ESAG7 because they form a heterodimer (adding the complication of dual expression) and because successful (but low level) protein expression has only been reported in a eukaryotic baculovirus expression system [47]. On the other hand, E. coli recombinant expression of domains of ISG75, ISG65 and ISG64 had either been reported in the literature [48] or were known to the authors (Mark Carrington, unpublished data). Consequently, we selected all three ISGs for protein expression trials. Finally, we performed expression trials on the predicted extracellular domain of GRESAG4, for which there was no literature precedent.


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)

Immuno-affinity chromatography and identification of potential diagnostic antigens.(A) Schematic representation of the preparation of IgG-Sepharose from T. b. gambiense infection and non-infection (control) sera, the immune-affinity capture of trypanosome antigens from a whole detergent lysate and their subsequent elution and concentration by ethanol precipitation. (B) Colloidal Comassie blue stained SDS-PAGE gel of the proteins eluted from infection IgG-Sepharose (lane 1) and non-infection (control) IgG-Sepharose (lane 2). The gel lanes were excised in 18 slices per lane, as indicated between lanes 1 and 2, and analysed by LC-MS/MS after reduction, S-alkylation and tryptic digestion. The positions of molecular weight markers are indicated on the left.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002087-g002: Immuno-affinity chromatography and identification of potential diagnostic antigens.(A) Schematic representation of the preparation of IgG-Sepharose from T. b. gambiense infection and non-infection (control) sera, the immune-affinity capture of trypanosome antigens from a whole detergent lysate and their subsequent elution and concentration by ethanol precipitation. (B) Colloidal Comassie blue stained SDS-PAGE gel of the proteins eluted from infection IgG-Sepharose (lane 1) and non-infection (control) IgG-Sepharose (lane 2). The gel lanes were excised in 18 slices per lane, as indicated between lanes 1 and 2, and analysed by LC-MS/MS after reduction, S-alkylation and tryptic digestion. The positions of molecular weight markers are indicated on the left.
Mentions: We took a non-biased proteomics approach to identify proteins that adsorb selectively to pooled infection IgG, and not to pooled control IgG (Figure 2A). Each serum pool contained four individual sera of patients clinically defined as having an infection with T. b. gambiense or as being uninfected. IgG fractions were purified from the pooled sera by affinity chromatography on protein G and then immobilised to cyanogen bromide-activated Sepharose beads. Equal amounts of infection and control IgG-Sepharose were incubated with equal amounts of T. b. brucei detergent cell lysate. Proteins that bound to the IgG columns were eluted by low pH, precipitated with cold ethanol, dissolved in SDS-sample buffer, reduced, separated by SDS-PAGE and stained with colloidal Coomassie blue (Figure 2B). More protein was seen in the eluate from the infection IgG column, consistent with infection-specific anti-trypanosome immune responses. Equivalent sections were cut out from the infection and control lanes, as indicated (Figure 2B). The excised gel pieces underwent in-gel S-alkylation and tryptic digestion, and the tryptic peptides were analysed by LC-MS/MS. Mascot software matched the peptide spectra to proteins in the T. b. brucei predicted protein database and scored the quality of the identifications. Lists of the proteins retained by infection IgG-Sepharose and control IgG-Sepharose were compared in each gel section. Twenty-four proteins with a MASCOT protein score above 50 were found uniquely in the infection IgG eluate and these are described in (Table 1). Several of the infection-specific proteins were defined as ‘hypothetical’, but other hits included known cell surface proteins, such as: Invariant Surface Glycoprotein (ISG) 75, ISG65, ISG64, Gene Related to Expression Site Associated Gene (GRESAG) 4, and the transferrin receptor subunits ESAG 6 and 7. As a starting point, the proteins with high MASCOT scores were prioritised. The rationale for this selection was that, by using an excess of trypanosome lysate in the affinity purification step, the amount of an eluted antigen should reflect, to a first approximation, the relative amount of antigen-specific immobilised IgG. The latter should, in turn, correspond to the immune response to that antigen in infected patients. Using this criterion, the protein antigens selected for study were ISG75, ESAG7, GRESAG4, ISG65, ISG64 and ESAG6 (Table 1). Next, we looked into the likely ease of protein expression of these antigens in E. coli. At this stage, we de-selected ESAG6 and ESAG7 because they form a heterodimer (adding the complication of dual expression) and because successful (but low level) protein expression has only been reported in a eukaryotic baculovirus expression system [47]. On the other hand, E. coli recombinant expression of domains of ISG75, ISG65 and ISG64 had either been reported in the literature [48] or were known to the authors (Mark Carrington, unpublished data). Consequently, we selected all three ISGs for protein expression trials. Finally, we performed expression trials on the predicted extracellular domain of GRESAG4, for which there was no literature precedent.

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