Kanamaru Y, Naziruddin B, Graves D C, Reyes de la Rocha S, Sachdev G P
College of Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
J Immunol Methods. 1991 Jun 3;139(2):167-79. doi: 10.1016/0022-1759(91)90186-j.
The purpose of this investigation was to demonstrate the presence of different species (subpopulations) in the purified human tracheobronchial mucin (HTM-1). Mucin was highly purified from sputum specimens collected from a cystic fibrosis (CF) patient using a protocol involving sequential chromatography on Bio-Gel A-5m and hydroxylapatite columns. SDS-composite gel electrophoresis followed by periodic acid-Schiff's reagent staining was unable to detect mucin species. However, using enzyme-linked immunoelectrotransfer blot (EITB) method and polyclonal antibodies raised against HTM-1, at least four different migrating mucin species were detected. Further immunological characterization of these mucin species was carried out using a library of 16 monoclonal antibodies (MAbs) developed against the purified mucin. Nine MAbs belonged to the IgM class, two MAbs were IgG1, one IgG2a and remaining four were of the IgG3 subclass. Periodate oxidation of the mucin antigen was used to establish the nature of the mucin epitopes recognized by the MAbs. 11 MAbs recognized carbohydrate epitopes in the mucin molecule that were sensitive to periodate, while five MAbs reacted with periodate resistant carbohydrate epitopes or the protein portion of the mucin molecule. Enzyme-linked immunoelectrotransfer blot analysis of the MAbs against HTM-1 showed the presence of at least three distinct mucin species. Chromatography of the mucin on immunoaffinity columns (MAbs H(13.3), M(33.3) and CCK 061 conjugated to CNBr-activated Sepharose 4B), followed by ELISA and EITB analyses, established the mucin species recognized by the antibodies. These experiments further indicated that both unique and shared epitopes were present in the mucin species. These monoclonal antibodies may provide a promising approach to differentiate the secretory products of the tracheobronchial tree.
本研究的目的是证明纯化的人气管支气管粘蛋白（HTM-1）中存在不同的种类（亚群）。使用涉及在Bio-Gel A-5m和羟基磷灰石柱上进行连续色谱的方案，从一名囊性纤维化（CF）患者收集的痰标本中高度纯化粘蛋白。十二烷基硫酸钠复合凝胶电泳后用高碘酸希夫试剂染色无法检测到粘蛋白种类。然而，使用酶联免疫电转移印迹（EITB）方法和针对HTM-1产生的多克隆抗体，检测到至少四种不同迁移的粘蛋白种类。使用针对纯化粘蛋白产生的16种单克隆抗体（MAb）文库对这些粘蛋白种类进行了进一步的免疫学表征。9种MAb属于IgM类，2种MAb是IgG1，1种IgG2a，其余4种属于IgG3亚类。粘蛋白抗原的高碘酸盐氧化用于确定MAb识别的粘蛋白表位的性质。11种MAb识别粘蛋白分子中对高碘酸盐敏感的碳水化合物表位，而5种MAb与抗高碘酸盐的碳水化合物表位或粘蛋白分子的蛋白质部分反应。针对HTM-1的MAb的酶联免疫电转移印迹分析显示至少存在三种不同的粘蛋白种类。将粘蛋白在免疫亲和柱（与溴化氰活化的琼脂糖4B偶联的MAb H(13.3)、M(33.3)和CCK 061）上进行色谱分析，随后进行ELISA和EITB分析，确定了抗体识别的粘蛋白种类。这些实验进一步表明，粘蛋白种类中存在独特和共享的表位。这些单克隆抗体可能为区分气管支气管树的分泌产物提供一种有前景的方法。
