Abstract
BackgroundAnimal African trypanosomosis (AAT) is a neglected tropical disease which imposes a heavy burden on the livestock industry in Sub-Saharan Africa. Its causative agents are Trypanosoma parasites, with T. congolense and T. vivax being responsible for the majority of the cases. Recently, we identified a Nanobody (Nb474) that was employed to develop a homologous sandwich ELISA targeting T. congolense fructose-1,6-bisphosphate aldolase (TcoALD). Despite the high sequence identity between trypanosomatid aldolases, the Nb474-based immunoassay is highly specific for T. congolense detection. The results presented in this paper yield insights into the molecular principles underlying the assay’s high specificity.Methodology/Principal findingsThe structure of the Nb474-TcoALD complex was determined via X-ray crystallography. Together with analytical gel filtration, the structure reveals that a single TcoALD tetramer contains four binding sites for Nb474. Through a comparison with the crystal structures of two other trypanosomatid aldolases, TcoALD residues Ala77 and Leu106 were identified as hot spots for specificity. Via ELISA and surface plasmon resonance (SPR), we demonstrate that mutation of these residues does not abolish TcoALD recognition by Nb474, but does lead to a lack of detection in the Nb474-based homologous sandwich immunoassay.Conclusions/SignificanceThe results show that the high specificity of the Nb474-based immunoassay is not determined by the initial recognition event between Nb474 and TcoALD, but rather by its homologous sandwich design. This (i) provides insights into the optimal set-up of the assay, (ii) may be of great significance for field applications as it could explain the potential detection escape of certain T. congolense strains, and (iii) may be of general interest to those developing similar assays.
Highlights
The Trypanosoma genus represents a diverse group of extracellular hemoflagellated parasites of which some members can infect and cause disease in humans and livestock
Animal African Trypanosomosis leads to considerable economic losses and renders sustainable livestock industry in Sub-Saharan Africa very difficult
We recently described the use of an assay to detect T. congolense infections in both experimentally and naturally infected animals
Summary
The Trypanosoma genus represents a diverse group of extracellular hemoflagellated parasites of which some members can infect and cause disease in humans and livestock. The practice of indiscriminate administration of anti-trypanosomal drugs to both healthy and diseased animals has led to the emergence of drug-resistant parasite strains [2,3] For this reason, there are ongoing efforts by the research community to develop both DNA- and proteinbased tests to improve diagnosis of AAT in the field. The antibody-based tests, which rely on the detection of circulating parasite-induced host antibodies, have two Structure of Nb474 and Trypanosoma congolense glycosomal aldolase main disadvantages: (i) a low specificity due to antibody cross-reactivity [10] and (ii) the inability of differentiating between past and ongoing infections as a consequence of long lasting circulating antibodies after parasite clearance [11,12,13]. The results presented in this paper yield insights into the molecular principles underlying the assay’s high specificity
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