Cancer Sciences Division, Southampton University Hospitals, Southampton, UK.
Cancer Immunol Immunother. 2010 Apr;59(4):619-27. doi: 10.1007/s00262-009-0814-4. Epub 2010 Jan 6.
The Cancer Immunotherapy Immunoguiding Program has conducted an IFN-gamma ELISPOT proficiency panel to examine the influence of serum supplementation of test media on assay performance. Sixteen European laboratories analyzed the same PBMC samples using different locally established protocols. Participants generated two simultaneous data sets-one using medium supplemented with serum and one without serum. Performances of the two test conditions were compared by quantifying: (1) the number of viable cells, (2) background spot formation induced in the medium only control and (3) the ability to detect antigen-specific T cell responses. The study demonstrated that the number of viable cells recovered and the overall background spot production were not significantly different between the two conditions. Furthermore, overall laboratory performance was equivalent for the two test conditions; 11 out of 16 laboratories reported equal or greater detection rates using serum-free medium, while 5 laboratories reported decreased detections rates under serum-free conditions. These results show that good performance of the IFN-gamma ELISPOT assay can be achieved under serum-free conditions. Optimization of the protocol for serum-free conditions should result in excellent detection rates and eliminate the requirement of serum batch and stability testing, allowing further harmonization of the assay.
癌症免疫治疗免疫指导计划进行了 IFN-γ ELISPOT 能力验证面板，以检查测试介质中血清补充对检测性能的影响。16 家欧洲实验室使用不同的本地建立的方案分析了相同的 PBMC 样本。参与者生成了两个同时的数据组 - 一个使用补充有血清的培养基，另一个不使用血清。通过定量以下方面来比较两种测试条件的性能：（1）活细胞数量，（2）仅培养基对照诱导的背景斑点形成，以及（3）检测抗原特异性 T 细胞反应的能力。研究表明，两种条件之间活细胞的回收数量和总体背景斑点产生没有显著差异。此外，两种测试条件的整体实验室性能相当；16 个实验室中有 11 个报告使用无血清培养基时具有相等或更高的检测率，而 5 个实验室报告在无血清条件下检测率降低。这些结果表明，IFN-γ ELISPOT 测定可以在无血清条件下获得良好的性能。无血清条件下的方案优化应导致出色的检测率，并消除对血清批次和稳定性测试的需求，从而进一步实现测定的协调。
