Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay.
Analysis of allelic cross-reactivity of monoclonal IgG antibodies by a multiplexed reverse FluoroSpot assay.
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DOI：
10.7554/elife.79245
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发表时间：
2022-07-15
期刊：
影响因子：
7.7
通讯作者：
Quintana, Maria del Pilar
中科院分区：
文献类型：
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作者：
Hoffmann-Veltung, Henriette;Anabire, Nsoh Godwin;Ofori, Michael Fokuo;Janhmatz, Peter;Ahlborg, Niklas;Hviid, Lars;Quintana, Maria del Pilar
抗体交叉反应问题在免疫学中具有核心重要性，尤其是在对恶性疟原虫的保护性免疫方面，这种疟疾的关键抗原显示出实质性的等位基因变异(多态)。然而，血清学分析往往不能区分真正的交叉反应(一种抗体识别多个抗原变体)和明显的交叉反应(存在多个变体特异性抗体)，因为它需要在单一B细胞/单抗水平上进行分析。ELISPOT是一种能够实现这一点的检测方法，最近开发的一种ELISpot(荧光点)的多重变体有助于同时评估B细胞/抗体对几种不同抗原的反应性。在这项研究中，我们提出了对该方法的进一步改进，使直接分析单抗水平与等位基因变异的交叉反应成为可能。使用VAR2CSA型PfEMP1-一种在胎盘疟疾发病机制中臭名昭著的多态抗原-作为模型，我们证明了该方法的稳健性及其在分析自然暴露个体中VAR2CSA特异性单抗的真正交叉反应中的适用性。该分析方法适用于其他多态抗原的分析，使其成为研究疟疾和许多其他疾病免疫的有力工具。
The issue of antibody cross-reactivity is of central importance in immunology, and not least in protective immunity to Plasmodium falciparum malaria, where key antigens show substantial allelic variation (polymorphism). However, serological analysis often does not allow the distinction between true cross-reactivity (one antibody recognizing multiple antigen variants) and apparent cross-reactivity (presence of multiple variant-specific antibodies), as it requires analysis at the single B-cell/monoclonal antibody level. ELISpot is an assay that enables that, and a recently developed multiplexed variant of ELISpot (FluoroSpot) facilitates simultaneous assessment of B-cell/antibody reactivity to several different antigens. In this study, we present a further enhancement of this assay that makes direct analysis of monoclonal antibody-level cross-reactivity with allelic variants feasible. Using VAR2CSA-type PfEMP1—a notoriously polymorphic antigen involved in the pathogenesis of placental malaria—as a model, we demonstrate the robustness of the assay and its applicability to analysis of true cross-reactivity of monoclonal VAR2CSA-specific antibodies in naturally exposed individuals. The assay is adaptable to the analysis of other polymorphic antigens, rendering it a powerful tool in studies of immunity to malaria and many other diseases.