SundarRaj N, Barbacci-Tobin E, Howe W E, Robertson S M, Limetti G
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pennsylvania 15213.
Invest Ophthalmol Vis Sci. 1987 Oct;28(10):1678-86.
Macular corneal dystrophy is an inherited corneal disease characterized by corneal opacities resulting from intra- and extracellular deposits within the corneal stroma. Several monoclonal antibodies developed against antigens of corneal fibroblasts were screened for their reactivity with these abnormal deposits in corneas with macular dystrophy using an indirect peroxidase-conjugated immunostaining technique. One of these monoclonal antibodies (designated 8F1-3) reacted very strongly with these abnormal deposits. Although the antigen recognized by this monoclonal antibody was present in the normal corneal stromal and endothelial cells, its concentration in the cells in the corneas with macular dystrophy appeared to be considerably higher, based on the intensity of the immunostaining reaction. Corneal fibroblasts grown in tissue culture were employed for further characterization of the antigen. After fixing with paraformaldehyde and permeabilizing with Triton X-100, immunofluorescent staining of the corneal fibroblasts using these monoclonal antibodies revealed a filamentous pattern of staining which resembled that seen for vimentin filaments. On treatment of corneal fibroblasts with colchicine, the filaments recognized by this antibody were withdrawn from their cytoplasmic array to form a perinuclear cap as also observed for vimentin-containing intermediate filaments. Immunoelectron microscopic studies using colloidal gold-conjugated antimouse IgG indicated that this monoclonal antibody recognized an antigen associated with intermediate-type filament. However, antivimentin antibody did not react with the abnormal deposits in the corneas with macular dystrophy, indicating that the antigen identified in the present study, although associated with intermediate filaments, was not vimentin. Analyses of cytoskeletal antigens by the immunoblotting technique further revealed that this monoclonal antibody recognized two polypeptides with Mr48,000 and 45,000, while antivimentin antibody reacted with 58,000 Mr polypeptide (vimentin).
黄斑角膜营养不良是一种遗传性角膜疾病，其特征是角膜基质内细胞内和细胞外沉积物导致角膜混浊。使用间接过氧化物酶结合免疫染色技术，筛选了几种针对角膜成纤维细胞抗原产生的单克隆抗体，以检测它们与黄斑营养不良角膜中这些异常沉积物的反应性。其中一种单克隆抗体（命名为8F1-3）与这些异常沉积物反应非常强烈。尽管该单克隆抗体识别的抗原存在于正常角膜基质和内皮细胞中，但根据免疫染色反应的强度，其在黄斑营养不良角膜细胞中的浓度似乎要高得多。在组织培养中生长的角膜成纤维细胞用于该抗原的进一步鉴定。用多聚甲醛固定并用Triton X-100通透处理后，使用这些单克隆抗体对角膜成纤维细胞进行免疫荧光染色，显示出丝状染色模式，类似于波形蛋白丝的染色模式。用秋水仙碱处理角膜成纤维细胞后，该抗体识别的细丝从其细胞质排列中缩回，形成核周帽，这与含波形蛋白的中间丝的情况相同。使用胶体金结合抗小鼠IgG的免疫电子显微镜研究表明，该单克隆抗体识别一种与中间丝相关的抗原。然而，抗波形蛋白抗体与黄斑营养不良角膜中的异常沉积物不反应，这表明本研究中鉴定的抗原虽然与中间丝相关，但不是波形蛋白。通过免疫印迹技术对细胞骨架抗原的分析进一步表明，该单克隆抗体识别两种分子量分别为48,000和45,000的多肽，而抗波形蛋白抗体与分子量为58,000的多肽（波形蛋白）反应。
