Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.

Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because...

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Main Authors: Emmanuel Obishakin, Benoit Stijlemans, Julien Santi-Rocca, Isabel Vandenberghe, Bart Devreese, Serge Muldermans, Philippe Bastin, Stefan Magez
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2014-01-01
Series:PLoS ONE
Online Access:https://doi.org/10.1371/journal.pone.0115893
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author Emmanuel Obishakin
Benoit Stijlemans
Julien Santi-Rocca
Isabel Vandenberghe
Bart Devreese
Serge Muldermans
Philippe Bastin
Stefan Magez
author_facet Emmanuel Obishakin
Benoit Stijlemans
Julien Santi-Rocca
Isabel Vandenberghe
Bart Devreese
Serge Muldermans
Philippe Bastin
Stefan Magez
author_sort Emmanuel Obishakin
collection DOAJ
description Trypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.
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spelling doaj.art-d57a371e1f524b66a1094c092c0c9d612022-12-21T19:14:43ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01912e11589310.1371/journal.pone.0115893Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.Emmanuel ObishakinBenoit StijlemansJulien Santi-RoccaIsabel VandenbergheBart DevreeseSerge MuldermansPhilippe BastinStefan MagezTrypanosomes are protozoan parasites that cause diseases in humans and livestock for which no vaccines are available. Disease eradication requires sensitive diagnostic tools and efficient treatment strategies. Immunodiagnostics based on antigen detection are preferable to antibody detection because the latter cannot differentiate between active infection and cure. Classical monoclonal antibodies are inaccessible to cryptic epitopes (based on their size-150 kDa), costly to produce and require cold chain maintenance, a condition that is difficult to achieve in trypanosomiasis endemic regions, which are mostly rural. Nanobodies are recombinant, heat-stable, small-sized (15 kDa), antigen-specific, single-domain, variable fragments derived from heavy chain-only antibodies in camelids. Because of numerous advantages over classical antibodies, we investigated the use of nanobodies for the targeting of trypanosome-specific antigens and diagnostic potential. An alpaca was immunized using lysates of Trypanosoma evansi. Using phage display and bio-panning techniques, a cross-reactive nanobody (Nb392) targeting all trypanosome species and isolates tested was selected. Imunoblotting, immunofluorescence microscopy, immunoprecipitation and mass spectrometry assays were combined to identify the target recognized. Nb392 targets paraflagellar rod protein (PFR1) of T. evansi, T. brucei, T. congolense and T. vivax. Two different RNAi mutants with defective PFR assembly (PFR2RNAi and KIF9BRNAi) were used to confirm its specificity. In conclusion, using a complex protein mixture for alpaca immunization, we generated a highly specific nanobody (Nb392) that targets a conserved trypanosome protein, i.e., PFR1 in the flagella of trypanosomes. Nb392 is an excellent marker for the PFR and can be useful in the diagnosis of trypanosomiasis. In addition, as demonstrated, Nb392 can be a useful research or PFR protein isolation tool.https://doi.org/10.1371/journal.pone.0115893
spellingShingle Emmanuel Obishakin
Benoit Stijlemans
Julien Santi-Rocca
Isabel Vandenberghe
Bart Devreese
Serge Muldermans
Philippe Bastin
Stefan Magez
Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
PLoS ONE
title Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
title_full Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
title_fullStr Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
title_full_unstemmed Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
title_short Generation of a nanobody targeting the paraflagellar rod protein of trypanosomes.
title_sort generation of a nanobody targeting the paraflagellar rod protein of trypanosomes
url https://doi.org/10.1371/journal.pone.0115893
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