Zuber Bartek, Levitsky Victor, Jönsson Gun, Paulie Staffan, Samarina Arina, Grundström Susanna, Metkar Sunil, Norell Håkan, Callender Glenda G, Froelich Christopher, Ahlborg Niklas
Mabtech AB, Box 1233, SE-131 28 Nacka, Sweden.
J Immunol Methods. 2005 Jul;302(1-2):13-25. doi: 10.1016/j.jim.2005.04.015.
The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.
穿孔素（PFN）蛋白对于细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞清除靶细胞至关重要。由于缺乏灵敏的方法，对释放PFN的细胞的研究受到了阻碍。因此，我们制备了PFN反应性单克隆抗体（mAb），并开发了捕获酶联免疫吸附测定（ELISA）和酶联免疫斑点测定（ELISpot）。产生了三种mAb，并显示它们与PFN的独特决定簇发生反应。通过流式细胞术和免疫组织化学评估，所有mAb均识别出人外周血单个核细胞（PBMC）中的细胞内PFN。利用其中两种mAb进行捕获，第三种mAb进行检测，开发了功能性PFN捕获ELISA和ELISpot测定。在检测YT淋巴瘤细胞系释放的PFN时，ELISpot显示出比ELISA更高的检测灵敏度。使用ELISpot对CTL克隆释放的PFN进行评估，结果与平行的（51）Cr释放细胞毒性测定一致。此外，在用常见病毒的特定CTL表位进行体外刺激后，PBMC释放的PFN可以通过ELISpot和ELISA进行定量。这些新型免疫测定对于进一步研究颗粒介导的细胞凋亡的机制将具有重要价值。此外，捕获免疫测定可为研究感染性疾病和肿瘤疾病中的CTL反应以及疫苗开发提供工具。
