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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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ELISA results using individual T. b. rhodesiense infection and matched control sera.(A–F) Box plots (generated by Cleveland method) represent the 25th percentile to the 75th percentile boundaries in the box with the median line within the box, the whiskers indicate the 10th and 90th percentiles. The box plots represent the ELISA signals for each recombinant protein ELISA plate: (A) rISG64-1, (B) rISG64-2, (C) rISG64-3, (D) rISG65-1, (E) rISG65-2 and (F) rISG75) tested against individual sera diluted 1∶1000 from stage 1 T. b. rhodesiense infections (n = 5), stage 2 T. b. rhodesiense infections (n = 20) and matched uninfected controls (n = 20). (G) Heat maps of the same data for the individual sera versus the recombinant protein ELISA plates. (H) Receiver operating characteristics (ROC) plots of the same data. The output statistics for sensitivity and specificity are shown in (Table 2).
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pntd-0002087-g005: ELISA results using individual T. b. rhodesiense infection and matched control sera.(A–F) Box plots (generated by Cleveland method) represent the 25th percentile to the 75th percentile boundaries in the box with the median line within the box, the whiskers indicate the 10th and 90th percentiles. The box plots represent the ELISA signals for each recombinant protein ELISA plate: (A) rISG64-1, (B) rISG64-2, (C) rISG64-3, (D) rISG65-1, (E) rISG65-2 and (F) rISG75) tested against individual sera diluted 1∶1000 from stage 1 T. b. rhodesiense infections (n = 5), stage 2 T. b. rhodesiense infections (n = 20) and matched uninfected controls (n = 20). (G) Heat maps of the same data for the individual sera versus the recombinant protein ELISA plates. (H) Receiver operating characteristics (ROC) plots of the same data. The output statistics for sensitivity and specificity are shown in (Table 2).

Mentions: Recombinant protein ELISA plates that performed well in the pooled sera ELISAs were further screened against all of the individual sera. These antigens included three rISG64 proteins, two rISG65 proteins and one rISG75 protein. In this case, a total of 163 individual serum samples (145 from the WHO HAT specimen bank and 18 from the Institue of Tropical Medicine, Antwerp) were diluted and applied in triplicate to wells coated with single recombinant proteins. T. b. gambiense and T. b. rhodesiense patient sera ELISA results were analysed separately (Figure 4) and (Figure 5), respectively. The data are shown as box plots for each different recombinant antigen ELISA plate (Figures 4A and 5A) to provide a visualisation the range of antibody titres and the heat maps provide a different view of the same data (Figures 4B and 5B). Both views suggest that rISG65 proteins provide the highest detection sensitivity whereas the rISG64-1 may provide slightly greater specificity. The rISG75 protein did not perform as well as the rISG65 or rISG64 proteins by both criteria and, indeed, only the stage 2 sera had statistically significant levels of IgG to rISG75-1 compared to controls (Q = 4.616, P = <0.05). Dunn's post-hoc tests (not shown) demonstrated that, whereas there are significantly higher levels of anti-rISG64 and anti-rISG65 IgG antibodies in both stage 1 and stage 2 sera compared to uninfected controls, there is no statistically significant difference between the stage 1 and stage 2 groups. In other words, relative immunoreactivity to rISG64 or rISG65 antigens cannot be used to stage of the disease. Formal sensitivity (i.e., the proportion of correct positive results) and specificity (i.e., the proportion of correct negative results) parameters for each test were calculated by ROC curve analysis (Figure 4C and 5C) and are collated in (Table 2). The recombinant antigens that best discriminated between T. b. gambiense infected and control patients by ELISA were rISG65-1 and rISG64-1, which had areas under the ROC curve of 0.99 and 0.98 respectively (Figure 4C). The rISG65-1 ELISA antigen had sensitivity of 96.6% (with a 95% Confidence Interval (CI) of 88.3 to 99.6%) and specificity of 93.2% (95% CI of 83.5 to 98.1%), whereas sensitivity and specificity of rISG64-1 antigen was 93.2% (95% CI of 83.5 to 98.1%) and 94.9% (95% CI of 85.9 to 98.9%), respectively (Table 2).


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)

ELISA results using individual T. b. rhodesiense infection and matched control sera.(A–F) Box plots (generated by Cleveland method) represent the 25th percentile to the 75th percentile boundaries in the box with the median line within the box, the whiskers indicate the 10th and 90th percentiles. The box plots represent the ELISA signals for each recombinant protein ELISA plate: (A) rISG64-1, (B) rISG64-2, (C) rISG64-3, (D) rISG65-1, (E) rISG65-2 and (F) rISG75) tested against individual sera diluted 1∶1000 from stage 1 T. b. rhodesiense infections (n = 5), stage 2 T. b. rhodesiense infections (n = 20) and matched uninfected controls (n = 20). (G) Heat maps of the same data for the individual sera versus the recombinant protein ELISA plates. (H) Receiver operating characteristics (ROC) plots of the same data. The output statistics for sensitivity and specificity are shown in (Table 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3584999&req=5

pntd-0002087-g005: ELISA results using individual T. b. rhodesiense infection and matched control sera.(A–F) Box plots (generated by Cleveland method) represent the 25th percentile to the 75th percentile boundaries in the box with the median line within the box, the whiskers indicate the 10th and 90th percentiles. The box plots represent the ELISA signals for each recombinant protein ELISA plate: (A) rISG64-1, (B) rISG64-2, (C) rISG64-3, (D) rISG65-1, (E) rISG65-2 and (F) rISG75) tested against individual sera diluted 1∶1000 from stage 1 T. b. rhodesiense infections (n = 5), stage 2 T. b. rhodesiense infections (n = 20) and matched uninfected controls (n = 20). (G) Heat maps of the same data for the individual sera versus the recombinant protein ELISA plates. (H) Receiver operating characteristics (ROC) plots of the same data. The output statistics for sensitivity and specificity are shown in (Table 2).
Mentions: Recombinant protein ELISA plates that performed well in the pooled sera ELISAs were further screened against all of the individual sera. These antigens included three rISG64 proteins, two rISG65 proteins and one rISG75 protein. In this case, a total of 163 individual serum samples (145 from the WHO HAT specimen bank and 18 from the Institue of Tropical Medicine, Antwerp) were diluted and applied in triplicate to wells coated with single recombinant proteins. T. b. gambiense and T. b. rhodesiense patient sera ELISA results were analysed separately (Figure 4) and (Figure 5), respectively. The data are shown as box plots for each different recombinant antigen ELISA plate (Figures 4A and 5A) to provide a visualisation the range of antibody titres and the heat maps provide a different view of the same data (Figures 4B and 5B). Both views suggest that rISG65 proteins provide the highest detection sensitivity whereas the rISG64-1 may provide slightly greater specificity. The rISG75 protein did not perform as well as the rISG65 or rISG64 proteins by both criteria and, indeed, only the stage 2 sera had statistically significant levels of IgG to rISG75-1 compared to controls (Q = 4.616, P = <0.05). Dunn's post-hoc tests (not shown) demonstrated that, whereas there are significantly higher levels of anti-rISG64 and anti-rISG65 IgG antibodies in both stage 1 and stage 2 sera compared to uninfected controls, there is no statistically significant difference between the stage 1 and stage 2 groups. In other words, relative immunoreactivity to rISG64 or rISG65 antigens cannot be used to stage of the disease. Formal sensitivity (i.e., the proportion of correct positive results) and specificity (i.e., the proportion of correct negative results) parameters for each test were calculated by ROC curve analysis (Figure 4C and 5C) and are collated in (Table 2). The recombinant antigens that best discriminated between T. b. gambiense infected and control patients by ELISA were rISG65-1 and rISG64-1, which had areas under the ROC curve of 0.99 and 0.98 respectively (Figure 4C). The rISG65-1 ELISA antigen had sensitivity of 96.6% (with a 95% Confidence Interval (CI) of 88.3 to 99.6%) and specificity of 93.2% (95% CI of 83.5 to 98.1%), whereas sensitivity and specificity of rISG64-1 antigen was 93.2% (95% CI of 83.5 to 98.1%) and 94.9% (95% CI of 85.9 to 98.9%), respectively (Table 2).

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