ZellNet Consulting, Inc., 555 North Avenue, Suite 25-S, Fort Lee, NJ 07024, USA.
Autoimmun Diagnostika GmbH, Ebinger Strasse 4, D-72479 Strassberg, Germany.
Cells. 2014 Nov 27;3(4):1102-15. doi: 10.3390/cells3041102.
ELISpot is one of the most commonly used immune monitoring assays, which allows the functional assessment of the immune system at the single cell level. With its outstanding sensitivity and ease of performance, the assay has recently advanced from the mere single function cell analysis to multifunctional analysis by implementing detection reagents that are labeled with fluorophores (FluoroSpot), allowing the detection of secretion patterns of two or more analytes in a single well. However, the automated evaluation of such assays presents various challenges for image analysis. Here we dissect the technical and methodological requirements for a reliable analysis of FluoroSpot assays, introduce important quality control measures and provide advice for proper interpretation of results obtained by automated imaging systems.
ELISpot 是最常用的免疫监测分析方法之一,它可以在单细胞水平上对免疫系统进行功能评估。由于其出色的灵敏度和易于操作,该检测方法最近已从单纯的单细胞分析发展为多功能分析,通过使用荧光标记的检测试剂(FluoroSpot),可以在单个孔中检测两种或更多分析物的分泌模式。然而,此类检测方法的自动评估对图像分析提出了各种挑战。在这里,我们将剖析可靠分析 FluoroSpot 检测方法的技术和方法学要求,介绍重要的质量控制措施,并为自动成像系统获得的结果提供适当的解释建议。