An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection
An antigen-specific, four-color, B-cell FluoroSpot assay utilizing tagged antigens for detection
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DOI：
10.1016/j.jim.2016.02.020
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发表时间：
2016-06-01
影响因子：
2.2
通讯作者：
Ahlborg, Niklas
中科院分区：
文献类型：
--
作者：
Jahnmatz, Peter;Bengtsson, Theresa;Ahlborg, Niklas
FluoroSpot测定法是利用荧光检测的ELISpot的变体，迄今为止主要用于评估T细胞，其中同时检测几种细胞因子允许对功能不同的T细胞进行更定性的分析。当分析B细胞时，测量多种分析物的潜力也呈现出几个优点。我们的目标是开发一种适用于各种抗原研究的B细胞FluoroSpot检测。该测定利用固定在96孔滤膜板中的抗IgG抗体。在细胞培养过程中，抗体分泌细胞（ASC）分泌的IgG抗体在这些细胞中的每一个的附近被捕获，并且使用用于检测的抗原来定义单个ASC的特异性。用生物素或肽标签标记抗原，使得能够用荧光团缀合的链霉亲和素或标签特异性抗体进行二次检测。同时利用多达四种不同的标签系统和荧光团的测定，使用杂交瘤和免疫脾细胞作为ASC进行评价。开发了测定变体，其可以：i）鉴定具有不同抗原特异性的多种ASC; ii）检测与不同但相关的抗原显示交叉反应性的ASC;以及iii）定义抗原特异性，并且通过包括抗IgG亚类检测试剂，同时确定ASC分泌的抗体的IgG亚类。如本文所示，使用基于标签的检测系统的B细胞FluoroSpot测定提供了一种通用且强大的工具来研究单个细胞的抗体应答，该工具可以容易地适应于各种抗原特异性ASC的研究。(C)2016作者由爱思唯尔公司出版
The FluoroSpot assay, a variant of ELISpot utilizing fluorescent detection, has so far been used primarily for assessment of T cells, where simultaneous detection of several cytokines has allowed a more qualitative analysis of functionally distinct T cells. The potential to measure multiple analytes also presents several advantages when analyzing B cells. Our aim was to develop a B-cell FluoroSpot assay adaptable to studies of a variety of antigens. The assay utilizes anti-IgG antibodies immobilized in 96-well filter membrane plates. During cell culture, IgG antibodies secreted by antibody-secreting cells (ASCs) are captured in the vicinity of each of these cells and the specificity of single ASCs is defined using antigens for detection. The antigens were labeled with biotin or peptide tags enabling secondary detection with fluorophore-conjugated streptavidin or tag-specific antibodies. The assay, utilizing up to four different tag systems and fluorophores simultaneously, was evaluated using hybridomas and immunized splenocytes as ASCs. Assay variants were developed that could: i) identify multiple ASCs with different antigen specificities; ii) detect ASCs showing cross-reactivity with different but related antigens; and iii) define the antigen-specificity and, by including anti-IgG subclass detection reagents, simultaneously determine the IgG subclass of antibodies secreted by ASCs. As demonstrated here, the B-cell FluoroSpot assay using tag-based detection systems provides a versatile and powerful tool to investigate antibody responses by individual cells that can be readily adapted to studies of a variety of antigen-specific ASCs. (C) 2016 The Authors. Published by Elsevier B.V.