Kobayashi K, Kobayashi H, Ueda M, Honda Y
Department of Ophthalmology, Faculty of Medicine, Kyoto University, Japan.
Brain Res. 1996 Nov 18;740(1-2):57-65. doi: 10.1016/s0006-8993(96)00852-9.
Astrocytes exhibit a diverse morphology and numerous functions in the central nervous system as well as in the retina. In order to obtain markers for the analysis of astrocytes, we prepared monoclonal antibodies that recognized antigens specific to astrocytes. Monoclonal antibody (mAb), designated KK1, reacted with the processes of astrocytes in the nerve fiber layer and the ganglion cell layer in the human retina as detected by indirect immunofluorescence. Normal Müller cells, whose processes are localized vertically in retina, were not labeled by KK1 mAb. In mouse brain, KK1 mAb reacted specifically with astrocytes in the white matter, but not with those in the gray matter. Studies employing a high-resolution confocal laser scanning microscope and double-labeling with KK1 mAb and commercially available anti-glial fibrillary acidic protein (GFAP) mAb (GA5) revealed that KK1 mAb visualized the processes that were not recognized by anti-GFAP mAb (GA5) in both human retina and mouse brain. In cultured mouse astrocytes, KK1 mAb reacted only with anti-GFAP mAb (GA5)-positive cells, but a small percentage of anti-GFAP mAb (GA5)-positive cells were labeled with KK1 mAb. In addition, the subcellular distribution of the KK1 antigen in cultured astrocytes apparently differed from that of GFAP labeled by anti-GFAP mAb (GA5). The antigen that was purified from the normal mouse brain by KK1 mAb-conjugated beads reacted with anti-GFAP mAb(GA5) in immunoblotting. No reactivity of KK1 mAb was observed in immunohistochemical analysis in GFAP -/- mutant mouse brain. These results demonstrate that KK1 mAb specifically recognized an epitope of GFAP that did not react with other anti-GFAP mAb (GA5). Retinal astrocytes and a subtype of astrocytes in the white matter of mouse brain shared the epitope that was recognized by KK1 mAb. KK1 mAb might be a powerful tool to investigate a subtype of astrocytes.
星形胶质细胞在中枢神经系统以及视网膜中呈现出多样的形态和众多功能。为了获得用于分析星形胶质细胞的标志物，我们制备了能识别星形胶质细胞特异性抗原的单克隆抗体。名为KK1的单克隆抗体，通过间接免疫荧光检测发现，它与人视网膜神经纤维层和神经节细胞层中的星形胶质细胞突起发生反应。正常的穆勒细胞，其突起垂直分布于视网膜中，未被KK1单克隆抗体标记。在小鼠脑中，KK1单克隆抗体与白质中的星形胶质细胞特异性反应，但不与灰质中的星形胶质细胞反应。使用高分辨率共聚焦激光扫描显微镜以及KK1单克隆抗体与市售抗胶质纤维酸性蛋白（GFAP）单克隆抗体（GA5）进行双重标记的研究表明，在人视网膜和小鼠脑中，KK1单克隆抗体能显示出抗GFAP单克隆抗体（GA5）未识别的突起。在培养的小鼠星形胶质细胞中，KK1单克隆抗体仅与抗GFAP单克隆抗体（GA5）阳性细胞反应，但一小部分抗GFAP单克隆抗体（GA5）阳性细胞被KK1单克隆抗体标记。此外，培养的星形胶质细胞中KK1抗原的亚细胞分布明显不同于抗GFAP单克隆抗体（GA5）标记的GFAP的分布。通过与KK1单克隆抗体偶联的珠子从正常小鼠脑中纯化的抗原，在免疫印迹中与抗GFAP单克隆抗体（GA5）反应。在GFAP -/- 突变小鼠脑的免疫组织化学分析中未观察到KK1单克隆抗体的反应性。这些结果表明，KK1单克隆抗体特异性识别GFAP的一个表位，该表位不与其他抗GFAP单克隆抗体（GA5）反应。视网膜星形胶质细胞和小鼠脑白质中的一种星形胶质细胞亚型共享被KK1单克隆抗体识别的表位。KK1单克隆抗体可能是研究星形胶质细胞亚型的有力工具。
