Pinto Joar, Odongo Steven, Lee Felicity, Gaspariunaite Vaiva, Muyldermans Serge, Magez Stefan, Sterckx Yann G-J
Research Unit for Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Structural Biology Research Centre, VIB, Brussels, Belgium.
PLoS Negl Trop Dis. 2017 Sep 15;11(9):e0005932. doi: 10.1371/journal.pntd.0005932. eCollection 2017 Sep.
Animal 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 FINDINGS: The 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/SIGNIFICANCE: The 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.
动物非洲锥虫病（AAT）是一种被忽视的热带疾病，给撒哈拉以南非洲的畜牧业带来沉重负担。其病原体是锥虫寄生虫，其中刚果锥虫和活跃锥虫导致了大多数病例。最近，我们鉴定出一种纳米抗体（Nb474），并利用它开发了一种针对刚果锥虫果糖-1,6-二磷酸醛缩酶（TcoALD）的同源夹心酶联免疫吸附测定法。尽管锥虫醛缩酶之间具有高度的序列同一性，但基于Nb474的免疫测定法对刚果锥虫的检测具有高度特异性。本文给出的结果揭示了该测定法高度特异性背后的分子原理。
方法/主要发现：通过X射线晶体学确定了Nb474-TcoALD复合物的结构。结合分析型凝胶过滤，该结构表明单个TcoALD四聚体包含四个Nb474结合位点。通过与另外两种锥虫醛缩酶的晶体结构进行比较，确定TcoALD的Ala77和Leu106残基为特异性热点。通过酶联免疫吸附测定法和表面等离子体共振（SPR），我们证明这些残基的突变不会消除Nb474对TcoALD的识别，但会导致基于Nb474的同源夹心免疫测定法无法检测到。
结论/意义：结果表明，基于Nb474的免疫测定法的高度特异性不是由Nb474与TcoALD之间的初始识别事件决定的，而是由其同源夹心设计决定的。这（i）为该测定法的最佳设置提供了见解，（ii）对于现场应用可能具有重要意义，因为它可以解释某些刚果锥虫菌株潜在的检测逃逸情况，并且（iii）可能会引起开发类似测定法的人员的普遍兴趣。
