Ishizaki M, Westerhausen-Larson A, Kino J, Hayashi T, Kao W W
Department of Ophthalmology, University of Cincinnati, Ohio 45267-0527.
Invest Ophthalmol Vis Sci. 1993 Aug;34(9):2680-9.
To examine the distribution of the collagen alpha 1(IV) chain and a novel collagen alpha(IV)-related chain in human ocular tissue.
Two monoclonal antibodies, JK199 and M3F7, against the alpha 1(IV) chain, and one monoclonal antibody, JK132, against a novel alpha(IV)-related chain were used in the avidin biotin peroxidase complex procedure of immunohistochemical studies. In situ hybridization and reverse transcription polymerase chain reaction were used to examine the presence of alpha 1(IV) messenger RNA in corneal epithelium.
Our data indicate that monoclonal antibodies JK199 and M3F7 react with most ocular basement membranes, but not with those of corneal epithelium. Similarly, monoclonal antibody JK132 reacts with most basement membranes of ocular tissues, with the exception of the inner limiting membrane of neural retina, Bruch's membrane, and corneal epithelial basement membrane. To examine if the epitopes recognized by the monoclonal antibodies were masked in corneal epithelium, the tissue sections were subjected to limited enzyme digestion, that is, pepsin, hyaluronidase, trypsin, and pronase E, or chemical treatments such as 0.1 N NaOH or 6 M urea. Proteinase treatment removed the JK132 epitope from all ocular basement membranes examined. Despite the pretreatment, corneal epithelial basement membrane was not stained by any of the monoclonal antibodies. However, the alpha 1(IV) messenger RNA was detected in corneal epithelium by in situ hybridization and reverse transcription polymerase chain reaction. Western immunoblotting indicates the presence of the alpha 1(IV) and the novel alpha(IV)-related chain in the basal lamella of corneal epithelium.
The epitopes recognized by JK199, M3F7, and JK132 are masked in basement membrane of corneal epithelium. Based on the tissue distribution and partial amino acid sequences of peptides recognized by JK132, the novel alpha(IV)-related chain differs from other known alpha(IV) chains.
研究胶原蛋白α1（IV）链和一种新型胶原蛋白α（IV）相关链在人眼组织中的分布。
两种抗α1（IV）链的单克隆抗体JK199和M3F7，以及一种抗新型α（IV）相关链的单克隆抗体JK132，用于免疫组织化学研究的抗生物素蛋白-生物素过氧化物酶复合物法。原位杂交和逆转录聚合酶链反应用于检测角膜上皮中α1（IV）信使核糖核酸的存在。
我们的数据表明，单克隆抗体JK199和M3F7与大多数眼基底膜发生反应，但不与角膜上皮的基底膜反应。同样，单克隆抗体JK132与眼组织的大多数基底膜发生反应，但神经视网膜内界膜、布鲁赫膜和角膜上皮基底膜除外。为了检查单克隆抗体识别的表位在角膜上皮中是否被掩盖，对组织切片进行了有限的酶消化，即胃蛋白酶、透明质酸酶、胰蛋白酶和链霉蛋白酶E，或化学处理，如0.1 N氢氧化钠或6 M尿素。蛋白酶处理从所有检测的眼基底膜中去除了JK132表位。尽管进行了预处理，角膜上皮基底膜未被任何单克隆抗体染色。然而，通过原位杂交和逆转录聚合酶链反应在角膜上皮中检测到了α1（IV）信使核糖核酸。蛋白质免疫印迹表明角膜上皮基底层中存在α1（IV）和新型α（IV）相关链。
JK199、M3F7和JK132识别的表位在角膜上皮基底膜中被掩盖。基于JK132识别的肽的组织分布和部分氨基酸序列，新型α（IV）相关链不同于其他已知的α（IV）链。
