Structural basis for the high specificity of a Trypanosoma congolense immunoassay targeting glycosomal aldolase

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.

An Unbiased Immunization Strategy Results in the Identification of Enolase as a Potential Marker for Nanobody-Based Detection of Trypanosoma evansi

“…Interestingly, both lowering and increasing the amount of capturing Nb11 appears to have a negative impact on the ELISA signal. This is reminiscent of the observations made for the Nb474-based homologous sandwich ELISA targeting TcoALD, which could be explained by "self-competition" or "washing out" effects [66]. Whether or not similar effects are at play for the Nb11-TevENO system remains to be investigated.…”

Development of a Nanobody-based lateral flow assay to detect active Trypanosoma congolense infections

“…For the Nb-based T . congolense immunoassay targeting Tco ALD 42 , we have recently shown that the molecular basis for specificity lies in the assay’s homologous sandwich design 76 . In the case of the Nb44/Nb42 assays, the reasons for their high T .…”

Use of camel single-domain antibodies for the diagnosis and treatment of zoonotic diseases

“…A VHH (Nb474) directed against T. congolese aldolase (TcoALD) has been developped for a sandwich immunoassay. This VHH is highly specific and did not recognize other trypanosomes such as T. brucei brucei, T. vivax and T. evansi [65,66].…”