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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

Performance of the prototype lateral flow device in a blinded study with eighty randomised serum samples.(A) Visual scores of test line density from rISG65-1 prototype lateral flow devices (scored in increments of 1 from 0 to 5, with very faint test line shadows represented as 0.5) are plotted against the subsequently decoded patient status (stage 1 T. b. gambiense infections (n = 8), stage 2 T. b. gambiense infections (n = 32) and matched uninfected controls (n = 40). (B) The same test strips were removed from the devices and scanned by CAMAG densitometer. The data are plotted directly below the results for the visual scores for the same samples. The R2 of a scatter plot was 0.96, showing very good correlation between visual score and CAMAG reading. (C–E) Box plots of the results for the same serum samples analysed by (C) rISG65-1 ELISA, (D) rISG65-1 lateral flow prototype with visual scoring and (E) rISG65-1 lateral flow prototype with CAMAG scanner scoring.
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pntd-0002087-g007: Performance of the prototype lateral flow device in a blinded study with eighty randomised serum samples.(A) Visual scores of test line density from rISG65-1 prototype lateral flow devices (scored in increments of 1 from 0 to 5, with very faint test line shadows represented as 0.5) are plotted against the subsequently decoded patient status (stage 1 T. b. gambiense infections (n = 8), stage 2 T. b. gambiense infections (n = 32) and matched uninfected controls (n = 40). (B) The same test strips were removed from the devices and scanned by CAMAG densitometer. The data are plotted directly below the results for the visual scores for the same samples. The R2 of a scatter plot was 0.96, showing very good correlation between visual score and CAMAG reading. (C–E) Box plots of the results for the same serum samples analysed by (C) rISG65-1 ELISA, (D) rISG65-1 lateral flow prototype with visual scoring and (E) rISG65-1 lateral flow prototype with CAMAG scanner scoring.

Mentions: Eighty randomised and coded WHO ‘test’ T. b. gambiense sera, comprising forty infected and forty non-infected sera were applied to the lateral flow prototypes. Each serum sample (5 µl) was diluted with 15 µl of PBS and applied to a lateral flow device sample pad. Within about 30 s, 80 µl of chase buffer was added and the test was left for 30 min, at which point a visual score was recorded. The sample pads were removed to prevent back flow and the visual scores were decoded (Figure 7). Sensitivity and specificity were calculated by ROC curve analysis, and for visual scores a cut off of 2.5 gave 100% sensitivity (95% CI of 91.1 to 100) and 87.5% specificity (95% CI of 73.2 to 95.8%). An analysis of the test lines was also carried out using a densitometer, where an arbitrary cut off at 265.6 RU gave 100% sensitivity (95% CI of 91.2 to 100%) and 92.5% specificity (95% of CI 79.6 to 98.4%) (Table 3) indicating there is potential for separation between infection and non-infected individual scores. Principally the end user will interpret the results visually therefore further optimisation of the test line will be necessary to reduce false positive results due to non-specific binding. A checklist, Supporting Information (Table S2), and flow diagram, Supporting Information (Figure S2), are provided according to the STAndards for the Reporting of Diagnostic accuracy studies (STARD) guidelines.


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)

Performance of the prototype lateral flow device in a blinded study with eighty randomised serum samples.(A) Visual scores of test line density from rISG65-1 prototype lateral flow devices (scored in increments of 1 from 0 to 5, with very faint test line shadows represented as 0.5) are plotted against the subsequently decoded patient status (stage 1 T. b. gambiense infections (n = 8), stage 2 T. b. gambiense infections (n = 32) and matched uninfected controls (n = 40). (B) The same test strips were removed from the devices and scanned by CAMAG densitometer. The data are plotted directly below the results for the visual scores for the same samples. The R2 of a scatter plot was 0.96, showing very good correlation between visual score and CAMAG reading. (C–E) Box plots of the results for the same serum samples analysed by (C) rISG65-1 ELISA, (D) rISG65-1 lateral flow prototype with visual scoring and (E) rISG65-1 lateral flow prototype with CAMAG scanner scoring.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3584999&req=5

pntd-0002087-g007: Performance of the prototype lateral flow device in a blinded study with eighty randomised serum samples.(A) Visual scores of test line density from rISG65-1 prototype lateral flow devices (scored in increments of 1 from 0 to 5, with very faint test line shadows represented as 0.5) are plotted against the subsequently decoded patient status (stage 1 T. b. gambiense infections (n = 8), stage 2 T. b. gambiense infections (n = 32) and matched uninfected controls (n = 40). (B) The same test strips were removed from the devices and scanned by CAMAG densitometer. The data are plotted directly below the results for the visual scores for the same samples. The R2 of a scatter plot was 0.96, showing very good correlation between visual score and CAMAG reading. (C–E) Box plots of the results for the same serum samples analysed by (C) rISG65-1 ELISA, (D) rISG65-1 lateral flow prototype with visual scoring and (E) rISG65-1 lateral flow prototype with CAMAG scanner scoring.
Mentions: Eighty randomised and coded WHO ‘test’ T. b. gambiense sera, comprising forty infected and forty non-infected sera were applied to the lateral flow prototypes. Each serum sample (5 µl) was diluted with 15 µl of PBS and applied to a lateral flow device sample pad. Within about 30 s, 80 µl of chase buffer was added and the test was left for 30 min, at which point a visual score was recorded. The sample pads were removed to prevent back flow and the visual scores were decoded (Figure 7). Sensitivity and specificity were calculated by ROC curve analysis, and for visual scores a cut off of 2.5 gave 100% sensitivity (95% CI of 91.1 to 100) and 87.5% specificity (95% CI of 73.2 to 95.8%). An analysis of the test lines was also carried out using a densitometer, where an arbitrary cut off at 265.6 RU gave 100% sensitivity (95% CI of 91.2 to 100%) and 92.5% specificity (95% of CI 79.6 to 98.4%) (Table 3) indicating there is potential for separation between infection and non-infected individual scores. Principally the end user will interpret the results visually therefore further optimisation of the test line will be necessary to reduce false positive results due to non-specific binding. A checklist, Supporting Information (Table S2), and flow diagram, Supporting Information (Figure S2), are provided according to the STAndards for the Reporting of Diagnostic accuracy studies (STARD) guidelines.

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