Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot® Software Based on Center of Mass Distance Algorithm.
Multiplexing T- and B-Cell FLUOROSPOT Assays: Experimental Validation of the Multi-Color ImmunoSpot® Software Based on Center of Mass Distance Algorithm.
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多重 T 细胞和 B 细胞 FLUOROSPOT 检测：基于质心距离算法的多色ImmunoSpot® 软件的实验验证。
作者：
A. Karulin;Z. Megyesi;R. Caspell;Jodi Hanson;P. Lehmann
在过去的十年中，ELISPOT已成为免疫学研究、免疫监测和疫苗开发中高度实施的主流检测方法。独特的单细胞分辨率以及高通量潜力将ELISPOT与流式细胞术，ELISA，微阵列和基于珠的多重检测分开。明确识别单个T细胞和B细胞是否共表达某些分析物（包括产生多功能细胞因子的T细胞）的必要性刺激了多色ELISPOT检测的发展。这些检测的成功也受到样品/细胞可用性有限以及试剂和劳动力资源限制的推动。目前市面上很少有可用于多色FLUOROSPOT的测试套件和仪器。除了对竞争系统的商业描述之外，人们对它们在实验环境中检测分泌多种分析物的单个细胞与随机覆盖的斑点的准确性知之甚少。在这里，我们提出了三色和四色T细胞和b细胞FLUOROSPOT数据分析的理论和实验验证研究。我们使用的ImmunoSpot®Fluoro-X™分析系统包括一个自动图像采集单元，可生成无光谱重叠的单个彩色图像，以及基于不同颜色斑点中心之间的最大允许距离或质心距离（COMD）的多色斑点计数软件。采用四色b细胞FLUOROSPOT检测IgM、IgA、IgG1、IgG3；和三/四色t细胞FLUOROSPOT检测IL-2、IFN-γ、TNF-α和GzB，在一系列稀释实验中，我们证明了Fluoro-X™多色斑点计数算法的有效性和准确性。基于泊松空间分布的统计预测，加上混乱的图像计数，允许对真实的多色斑点计数进行客观校正，以排除随机叠加的斑点。
Over the past decade, ELISPOT has become a highly implemented mainstream assay in immunological research, immune monitoring, and vaccine development. Unique single cell resolution along with high throughput potential sets ELISPOT apart from flow cytometry, ELISA, microarray- and bead-based multiplex assays. The necessity to unambiguously identify individual T and B cells that do, or do not co-express certain analytes, including polyfunctional cytokine producing T cells has stimulated the development of multi-color ELISPOT assays. The success of these assays has also been driven by limited sample/cell availability and resource constraints with reagents and labor. There are few commercially available test kits and instruments available at present for multi-color FLUOROSPOT. Beyond commercial descriptions of competing systems, little is known about their accuracy in experimental settings detecting individual cells that secrete multiple analytes vs. random overlays of spots. Here, we present a theoretical and experimental validation study for three and four color T- and B-cell FLUOROSPOT data analysis. The ImmunoSpot® Fluoro-X™ analysis system we used includes an automatic image acquisition unit that generates individual color images free of spectral overlaps and multi-color spot counting software based on the maximal allowed distance between centers of spots of different colors or Center of Mass Distance (COMD). Using four color B-cell FLUOROSPOT for IgM, IgA, IgG1, IgG3; and three/four color T-cell FLUOROSPOT for IL-2, IFN-γ, TNF-α, and GzB, in serial dilution experiments, we demonstrate the validity and accuracy of Fluoro-X™ multi-color spot counting algorithms. Statistical predictions based on the Poisson spatial distribution, coupled with scrambled image counting, permit objective correction of true multi-color spot counts to exclude randomly overlaid spots.
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DOI：
10.1016/j.tube.2010.12.005
发表时间：
2011-05
期刊：
Tuberculosis (Edinburgh, Scotland)
影响因子：
--
作者：
El Fenniri L;Toossi Z;Aung H;El Iraki G;Bourkkadi J;Benamor J;Laskri A;Berrada N;Benjouad A;Mayanja-Kizza H;Betts MR;El Aouad R;Canaday DH
通讯作者：
Canaday DH
影响因子：
20.3
作者：
Betts, Michael R.;Nason, Martha C.;Koup, Richard A.
通讯作者：
Koup, Richard A.
影响因子：
6.4
作者：
Day, Cheryl L.;Mkhwanazi, Nompumelelo;Walker, Bruce D.
通讯作者：
Walker, Bruce D.
影响因子：
5.5
作者：
Hutnick, Natalie A.;Carnathan, Diane;Demers, Korey;Makedonas, George;Ertl, Hildegund C. J.;Betts, Michael R.
通讯作者：
Betts, Michael R.
影响因子：
1.5
作者：
Kuerten, Stefanie;Nowacki, Tobias M.;Tary-Lehmann, Magdalena
通讯作者：
Tary-Lehmann, Magdalena