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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

ELISA results with pooled human sera.(A) Pooled human sera representing stage 1 T. b. gambiense infections (pool of 10 sera), stage 2 T. b. gambiense infections (pool of 40 sera) and matched uninfected controls (pool of 50 sera) were diluted 1∶1000 and used in triplicate on ELISA plates coated with the rISG75, rISG65-1, rISG65-2, rISG64-1, rISG64-2, rISG64-3 and rGRESAG4a recombinant proteins described in (Figure 2). The mean ELISA signals ± SEM are plotted against the recombinant protein used in the ELISA. (B) As panel A but using pooled human sera representing stage 1 T. b. rhodesiense infections (pool of 5 sera), stage 2 T. b. rhodesiense infections (pool of 20 sera) and matched uninfected controls (pool of 25 sera).
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pntd-0002087-g003: ELISA results with pooled human sera.(A) Pooled human sera representing stage 1 T. b. gambiense infections (pool of 10 sera), stage 2 T. b. gambiense infections (pool of 40 sera) and matched uninfected controls (pool of 50 sera) were diluted 1∶1000 and used in triplicate on ELISA plates coated with the rISG75, rISG65-1, rISG65-2, rISG64-1, rISG64-2, rISG64-3 and rGRESAG4a recombinant proteins described in (Figure 2). The mean ELISA signals ± SEM are plotted against the recombinant protein used in the ELISA. (B) As panel A but using pooled human sera representing stage 1 T. b. rhodesiense infections (pool of 5 sera), stage 2 T. b. rhodesiense infections (pool of 20 sera) and matched uninfected controls (pool of 25 sera).

Mentions: The selected purified recombinant trypanosome proteins, see Supporting Information (Figure S1), were used to prepare ELISA plates, as described in Experimental Procedures, and these were screened against various pooled human sera. These pools were derived from the 145 individual serum samples provided by the WHO Human African Trypanosomiasis specimen bank. The pooled sera were for stage 1 T. b. gambiense patients (n = 10), stage 2 T. b. gambiense patients (n = 40) and matched uninfected patients (n = 50); and from stage 1 T. b. rhodesiense patients (n = 5), stage 2 T. b. rhodesiense patients (n = 20) and matched uninfected patients (n = 20). The results indicated that both stage 1 and stage 2 T. b. gambiense infection sera have significant antibody titres against all of the rISG64 and rISG65 proteins, compared to pooled non-infection sera (Figure 3A), whereas infection sera titres against rISG75 and GRESAG4a were much closer to those for the control sera. The best performing recombinant protein was ISG65-1, which had the highest infection to control signal. For the T. b. rhodesiense pooled sera, the signals were generally significantly lower, with the stage 2 pooled sera giving a significantly higher signal than the stage 1 pooled sera. There was one exception to this; the T. b. rhodesiense stage 1 pool had the highest antibody titre against rISG75 (Figure 3B). However, as will be described later, the rISG75 result was due to a very high antibody titre in a single individual. From these results, all the rISG proteins were taken forward and screened against the individual sera but GRESAG4a (rG4a) was abandoned at this stage because it had poor infection versus non-infection discrimination.


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 with pooled human sera.(A) Pooled human sera representing stage 1 T. b. gambiense infections (pool of 10 sera), stage 2 T. b. gambiense infections (pool of 40 sera) and matched uninfected controls (pool of 50 sera) were diluted 1∶1000 and used in triplicate on ELISA plates coated with the rISG75, rISG65-1, rISG65-2, rISG64-1, rISG64-2, rISG64-3 and rGRESAG4a recombinant proteins described in (Figure 2). The mean ELISA signals ± SEM are plotted against the recombinant protein used in the ELISA. (B) As panel A but using pooled human sera representing stage 1 T. b. rhodesiense infections (pool of 5 sera), stage 2 T. b. rhodesiense infections (pool of 20 sera) and matched uninfected controls (pool of 25 sera).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3584999&req=5

pntd-0002087-g003: ELISA results with pooled human sera.(A) Pooled human sera representing stage 1 T. b. gambiense infections (pool of 10 sera), stage 2 T. b. gambiense infections (pool of 40 sera) and matched uninfected controls (pool of 50 sera) were diluted 1∶1000 and used in triplicate on ELISA plates coated with the rISG75, rISG65-1, rISG65-2, rISG64-1, rISG64-2, rISG64-3 and rGRESAG4a recombinant proteins described in (Figure 2). The mean ELISA signals ± SEM are plotted against the recombinant protein used in the ELISA. (B) As panel A but using pooled human sera representing stage 1 T. b. rhodesiense infections (pool of 5 sera), stage 2 T. b. rhodesiense infections (pool of 20 sera) and matched uninfected controls (pool of 25 sera).
Mentions: The selected purified recombinant trypanosome proteins, see Supporting Information (Figure S1), were used to prepare ELISA plates, as described in Experimental Procedures, and these were screened against various pooled human sera. These pools were derived from the 145 individual serum samples provided by the WHO Human African Trypanosomiasis specimen bank. The pooled sera were for stage 1 T. b. gambiense patients (n = 10), stage 2 T. b. gambiense patients (n = 40) and matched uninfected patients (n = 50); and from stage 1 T. b. rhodesiense patients (n = 5), stage 2 T. b. rhodesiense patients (n = 20) and matched uninfected patients (n = 20). The results indicated that both stage 1 and stage 2 T. b. gambiense infection sera have significant antibody titres against all of the rISG64 and rISG65 proteins, compared to pooled non-infection sera (Figure 3A), whereas infection sera titres against rISG75 and GRESAG4a were much closer to those for the control sera. The best performing recombinant protein was ISG65-1, which had the highest infection to control signal. For the T. b. rhodesiense pooled sera, the signals were generally significantly lower, with the stage 2 pooled sera giving a significantly higher signal than the stage 1 pooled sera. There was one exception to this; the T. b. rhodesiense stage 1 pool had the highest antibody titre against rISG75 (Figure 3B). However, as will be described later, the rISG75 result was due to a very high antibody titre in a single individual. From these results, all the rISG proteins were taken forward and screened against the individual sera but GRESAG4a (rG4a) was abandoned at this stage because it had poor infection versus non-infection discrimination.

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