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

TitleGeneration of a nanobody targeting the paraflagellar rod protein of trypanosomes.
Publication TypeJournal Article
Year of Publication2014
AuthorsObishakin, E., B. Stijlemans, J. Santi-Rocca, I. Vandenberghe, B. Devreese, S. Muldermans, P. Bastin, and S. Magez
JournalPLoS One
Volume9
Issue12
Paginatione115893
Date Published2014
ISSN1932-6203
KeywordsAmino Acid Sequence, Animals, Antigens, Protozoan, Camelids, New World, Flagella, Immunoglobulin Heavy Chains, Molecular Sequence Data, Protozoan Proteins, Recombinant Proteins, RNA Interference, RNA, Small Interfering, Sequence Alignment, Single-Chain Antibodies, Trypanocidal Agents, Trypanosoma, Trypanosomiasis
Abstract

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.

DOI10.1371/journal.pone.0115893
Alternate JournalPLoS ONE
PubMed ID25551637
PubMed Central IDPMC4281110
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