Kawasaki A, Shinkai Y, Kuwana Y, Furuya A, Iigo Y, Hanai N, Itoh S, Yagita H, Okumura K
Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan.
Int Immunol. 1990;2(7):677-84. doi: 10.1093/intimm/2.7.677.
Perforin is one of the important cytolytic factors in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. In this paper, we report rat mAbs against mouse perforin established by immunization with a recombinant mouse perforin fragment. These mAbs reacted with purified mouse perforin prepared from cytoplasmic granules of an NK-like cell line in ELISA and Western blot analysis. However, none of these mAbs blocked the hemolytic activity of mouse perforin or absorbed it when fixed in the solid phase. These results indicate that all of these mAbs react with denatured but not with native mouse perforin. By using a combination of the mAbs, we established a sandwich ELISA, for quantitating the cellular contents of perforin. These mAbs were also useful for immunohistochemical staining analysis, and perforin was detected in the cytoplasmic granules of CTL and NK cell lines. Perforin was also detected in a minor population of lymphocytes of the spleen, liver, and lymph node. In normal spleen cells of 5- to 8-week-old mice, 12-15% of asialo GM1+ cells and 7-21% of CD8+ T cells were perforin-positive, but CD4+ T cells, B cells, and macrophages were totally negative. These data clearly show that perforin is expressed in cells of a cytotoxic character in normal mice, in the same way as in primed mice.
穿孔素是细胞毒性T淋巴细胞（CTL）和自然杀伤（NK）细胞中的重要细胞溶解因子之一。在本文中，我们报道了通过用重组小鼠穿孔素片段免疫制备的抗小鼠穿孔素大鼠单克隆抗体。在ELISA和蛋白质印迹分析中，这些单克隆抗体与从NK样细胞系的细胞质颗粒中制备的纯化小鼠穿孔素发生反应。然而，这些单克隆抗体均未阻断小鼠穿孔素的溶血活性，也未在固定于固相时将其吸附。这些结果表明，所有这些单克隆抗体均与变性的而非天然的小鼠穿孔素发生反应。通过使用这些单克隆抗体的组合，我们建立了一种夹心ELISA，用于定量穿孔素的细胞含量。这些单克隆抗体也可用于免疫组织化学染色分析，并且在CTL和NK细胞系的细胞质颗粒中检测到了穿孔素。在脾脏、肝脏和淋巴结的少数淋巴细胞中也检测到了穿孔素。在5至8周龄小鼠的正常脾细胞中，12%至15%的去唾液酸GM1+细胞和7%至21%的CD8+ T细胞穿孔素呈阳性，但CD4+ T细胞、B细胞和巨噬细胞完全呈阴性。这些数据清楚地表明，穿孔素在正常小鼠的具有细胞毒性特征的细胞中表达，与致敏小鼠中的表达方式相同。
