Cellular Technology Ltd, 20521 Chagrin Blvd., Shaker Heights, OH, 44122 USA.
Cells. 2015 Jan 29;4(1):71-83. doi: 10.3390/cells4010071.
Comprehensive immune monitoring requires that frequencies of T cells, producing different cytokines, are measured to establish the magnitude of Th1, Th2, and Th17 components of cell-mediated immunity. Antigen titration provides additional information about the affinity of T cell response. In tumor immunity, it is also advisable to account for determinant spreading by testing multiple epitopes. Efforts for comprehensive immune monitoring would require substantial numbers of PBMC to run the above tests systematically, which in most test cases is limiting. Immune monitoring with ELISPOT assays have been performed, thus far, in a 96-well format. In this study we show that one can increase cell utilization by performing the assay in 384-well plates whose membrane surface area is one third that of 96-well plates. Systematic testing of PBMC for antigen-specific T cell response in the two formats demonstrated that the 384-well assay corresponds to a one-in-three miniaturization of the 96-well assay. The lowest number of cells that can be used in the 384-well format, while allowing for sufficient contact with APC, is 33,000 PBMC/well. Therefore, with one million PBMC typically obtained from 1 mL of blood, a 30 well T cell ELISPOT assay can be performed in a 384-well format.
全面的免疫监测需要测量产生不同细胞因子的 T 细胞的频率，以确定细胞介导免疫的 Th1、Th2 和 Th17 成分的幅度。抗原滴定提供了关于 T 细胞反应亲和力的额外信息。在肿瘤免疫中，通过测试多个表位来考虑决定簇的扩展也是明智的。全面的免疫监测需要大量的 PBMC 来系统地运行上述测试，而在大多数测试情况下，这是有限制的。迄今为止，ELISPOT 测定已在 96 孔板格式中进行了免疫监测。在这项研究中，我们表明，通过在 384 孔板中进行测定，可以增加细胞的利用率，384 孔板的膜表面积是 96 孔板的三分之一。在两种格式下对 PBMC 进行抗原特异性 T 细胞反应的系统测试表明，384 孔测定相当于 96 孔测定的三分之一缩小。在允许与 APC 充分接触的情况下，384 孔板中可以使用的最少细胞数为 33,000 PBMC/孔。因此，通常从 1 毫升血液中获得 100 万个 PBMC，可以在 384 孔板中进行 30 孔 T 细胞 ELISPOT 测定。
