Spiller O B, Hanna S M, Morgan B P
Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, UK.
Immunology. 1999 Jul;97(3):374-84. doi: 10.1046/j.1365-2567.1999.00776.x.
In humans, decay-accelerating factor (DAF) is a widely distributed, cell-bound inhibitor of the complement activation enzymes and plays a key role in regulating complement activation, preventing the generation of anaphylotoxins and opsonins, and protecting against complement-mediated lysis. Rodent analogues of DAF have recently been identified, providing a new avenue for the analysis of function. Rat DAF was cloned in our laboratory. Here we describe the generation of monoclonal antibodies (mAbs) against rat DAF, using transfected cells as immunogen, and their use in the analysis of the distribution of DAF in the rat by flow cytometry, Western blot analysis and immunohistochemistry. One of the mAbs was found to block the complement inhibitory function of rat DAF, offering the prospect of neutralization of DAF function in vivo. The antibodies have also been used for purification of DAF from rat erythrocytes by affinity chromatography. Rat DAF purified in this manner was similar in molecular mass to human DAF. The purified protein incorporated into lipid membranes, confirming the presence of a glycolipid anchor, and incorporated protein strongly inhibited the rat C3 convertase. Rat DAF was strongly expressed on endothelia throughout the animal and was also present in most tissues and organs. DAF expression was weak or absent in the brain and on circulating and spleen-resident T cells. Strong DAF expression observed in the kidney was restricted to the glomerulus and Bowman's capsule. DAF expression in the testis was found only in association with the later stages of spermatogenesis.
在人类中，衰变加速因子（DAF）是一种广泛分布的、与细胞结合的补体激活酶抑制剂，在调节补体激活、防止过敏毒素和调理素的产生以及保护机体免受补体介导的细胞溶解方面发挥着关键作用。最近已鉴定出DAF的啮齿动物类似物，为功能分析提供了新途径。大鼠DAF是在我们实验室克隆的。在此，我们描述了以转染细胞为免疫原产生抗大鼠DAF的单克隆抗体（mAb），以及它们在通过流式细胞术、蛋白质印迹分析和免疫组织化学分析大鼠DAF分布中的应用。发现其中一种mAb可阻断大鼠DAF的补体抑制功能，为体内中和DAF功能提供了前景。这些抗体还被用于通过亲和层析从大鼠红细胞中纯化DAF。以这种方式纯化的大鼠DAF在分子量上与人DAF相似。纯化的蛋白质整合到脂质膜中，证实存在糖脂锚定，并且整合的蛋白质强烈抑制大鼠C3转化酶。大鼠DAF在整个动物体内的内皮细胞上强烈表达，也存在于大多数组织和器官中。在脑以及循环和脾脏驻留T细胞上，DAF表达较弱或不存在。在肾脏中观察到的强烈DAF表达仅限于肾小球和鲍曼囊。在睾丸中，仅在精子发生后期发现DAF表达。
