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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

Prototype lateral flow device for detecting antibodies to rISG65-1 protein.Representative results using serum samples from a matched uninfected patient (left) and a stage 2 T. b. gambiense infected patient (right). The visual scores for these test lines were 0 and 5, respectively, and the CAMAG densitometry measurements were 24.2 and 597.4, respectively. The inset shows the principle of detection, with patient antibody to ISG65 forming a bridge between rISG56-1 immobilised on the nitrocellulose strip and the colloidal gold-coupled rISG65-1 picked up from the sample pad.
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pntd-0002087-g006: Prototype lateral flow device for detecting antibodies to rISG65-1 protein.Representative results using serum samples from a matched uninfected patient (left) and a stage 2 T. b. gambiense infected patient (right). The visual scores for these test lines were 0 and 5, respectively, and the CAMAG densitometry measurements were 24.2 and 597.4, respectively. The inset shows the principle of detection, with patient antibody to ISG65 forming a bridge between rISG56-1 immobilised on the nitrocellulose strip and the colloidal gold-coupled rISG65-1 picked up from the sample pad.

Mentions: Based on the ROC curve analyses of the performances of the ELISA plates, we selected rISG65-1 (ROC curve area 0.99 for T. b.gambiense sera) for development of a lateral flow prototype. Purified rISG65-1 was supplied to BBInternational (Dundee, www.bbigold.com) a company that specialises in lateral flow technology. The lateral flow approach that was utilised is illustrated in (Figure 6). Thus, rISG65-1 was both immobilised in a band on a nitrocellulose membrane and coupled to colloidal gold that was then localised in the conjugate pad. When the sera and chase buffer are applied to the sample pad, the rISG65-colloidal gold conjugate is resuspended. The absorbent pad at the top of the lateral flow device draws the liquid across the nitrocellulose membrane. During this time, any anti-rISG65 antibody in the serum binds to the rISG65-gold conjugate and when the antibodies reach the rISG65 test band, one Fab arm of the IgG binds to the immobilised rISG65 while the other Fab domain bridges to the rISG65-gold-conjugate. Accumulation of this specific antibody sandwich generates a visible test line. The control line is an internal positive control for the lateral flow test and does not relate to the infection status of the patient but indicates successful test flow. The final reading of this test should be as follows; the appearance of only a control line (upper band) indicates non-infected sera, whereas, the appearance of two lines, a control and test line (upper & lower bands) indicates infected sera, examples are shown in (Figure 6). Absence of a control line (upper band) indicates an invalid test, irrespective of the appearance of the test line and the test should be repeated.


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)

Prototype lateral flow device for detecting antibodies to rISG65-1 protein.Representative results using serum samples from a matched uninfected patient (left) and a stage 2 T. b. gambiense infected patient (right). The visual scores for these test lines were 0 and 5, respectively, and the CAMAG densitometry measurements were 24.2 and 597.4, respectively. The inset shows the principle of detection, with patient antibody to ISG65 forming a bridge between rISG56-1 immobilised on the nitrocellulose strip and the colloidal gold-coupled rISG65-1 picked up from the sample pad.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0002087-g006: Prototype lateral flow device for detecting antibodies to rISG65-1 protein.Representative results using serum samples from a matched uninfected patient (left) and a stage 2 T. b. gambiense infected patient (right). The visual scores for these test lines were 0 and 5, respectively, and the CAMAG densitometry measurements were 24.2 and 597.4, respectively. The inset shows the principle of detection, with patient antibody to ISG65 forming a bridge between rISG56-1 immobilised on the nitrocellulose strip and the colloidal gold-coupled rISG65-1 picked up from the sample pad.
Mentions: Based on the ROC curve analyses of the performances of the ELISA plates, we selected rISG65-1 (ROC curve area 0.99 for T. b.gambiense sera) for development of a lateral flow prototype. Purified rISG65-1 was supplied to BBInternational (Dundee, www.bbigold.com) a company that specialises in lateral flow technology. The lateral flow approach that was utilised is illustrated in (Figure 6). Thus, rISG65-1 was both immobilised in a band on a nitrocellulose membrane and coupled to colloidal gold that was then localised in the conjugate pad. When the sera and chase buffer are applied to the sample pad, the rISG65-colloidal gold conjugate is resuspended. The absorbent pad at the top of the lateral flow device draws the liquid across the nitrocellulose membrane. During this time, any anti-rISG65 antibody in the serum binds to the rISG65-gold conjugate and when the antibodies reach the rISG65 test band, one Fab arm of the IgG binds to the immobilised rISG65 while the other Fab domain bridges to the rISG65-gold-conjugate. Accumulation of this specific antibody sandwich generates a visible test line. The control line is an internal positive control for the lateral flow test and does not relate to the infection status of the patient but indicates successful test flow. The final reading of this test should be as follows; the appearance of only a control line (upper band) indicates non-infected sera, whereas, the appearance of two lines, a control and test line (upper & lower bands) indicates infected sera, examples are shown in (Figure 6). Absence of a control line (upper band) indicates an invalid test, irrespective of the appearance of the test line and the test should be repeated.

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