Perforin, a pore-forming protein detectable by monoclonal antibodies, is a functional marker for killer cells

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.