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

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.背景：动物非洲锥虫病（Animal African trypanosomosis, AAT）是一种被忽视的热带病，给撒哈拉以南非洲的畜牧业带来沉重负担。其病原体为锥虫属寄生虫，其中刚果锥虫（T. congolense）与维氏锥虫（T. vivax）为绝大多数病例的致病原。本研究团队近期鉴定出一种纳米抗体（Nanobody, Nb474），并基于此开发了针对刚果锥虫果糖-1,6-二磷酸醛缩酶（TcoALD）的同源夹心酶联免疫吸附试验（ELISA）。尽管锥虫体醛缩酶的序列同源性较高，但基于Nb474的免疫检测方法对刚果锥虫的检测具有极高特异性。本文所呈现的研究结果揭示了该检测方法高特异性的分子机制。 方法学与主要发现：研究通过X射线晶体学解析了Nb474-TcoALD复合物的晶体结构。结合分析型凝胶过滤实验结果，该结构显示单个TcoALD四聚体可结合四个Nb474分子。通过与另外两种锥虫体醛缩酶的晶体结构进行比对，研究确定了TcoALD的Ala77和Leu106残基为特异性识别的关键位点。随后通过酶联免疫吸附试验与表面等离子体共振（Surface Plasmon Resonance, SPR）实验证实，对这两个残基进行突变并不会阻断Nb474对TcoALD的初始识别，但会导致基于Nb474的同源夹心免疫检测方法无法完成有效检测。 结论与意义：研究结果表明，基于Nb474的免疫检测方法的高特异性并非源于Nb474与TcoALD之间的初始识别事件，而是源于其同源夹心的实验设计。这一发现（i）为检测方法的优化构建提供了理论依据；（ii）具备重要的现场应用价值，可解释部分刚果锥虫菌株逃脱该检测的潜在机制；（iii）可为同类检测方法的开发提供通用参考。