Foellmer H G, Madri J A, Furthmayr H
Lab Invest. 1983 May;48(5):639-49.
Type IV collagen is one of the main constituents of basement membranes, yet it is unknown whether the structural framework at different sites is assembled from one unique type of molecule or whether different type IV collagen molecules exist. To study the composition, chemical identity, and organization of this protein in different organs we have prepared monoclonal antibodies to a type IV collagen preparation from human placenta. Swiss Webster mice were hyperimmunized, and splenic cells were fused with the three different myeloma cell lines SP2/0, NS1, and U1. Type IV collagen-specific hybrids were selected and cloned by limiting dilution and on hard agar. Monoclonal antibodies secreted by two clones were extensively characterized by ELISA-inhibition assay, immunoprecipitation, rotary shadowing, and immunofluorescence techniques. Unlike conventionally raised antibodies in rabbits, both monoclonal antibody reagents show species-specific binding exclusively to native type IV collagen from human placenta but not to a similar preparation from calf lung or to other types of collagen. After heat denaturation of the antigen binding was no longer observed. The M3F7 antibody-binding site is located within the triple helical domain of the type IV molecule, approximately 900 A removed from the amino terminal end as visualized by a metal shadow casting technique. The monoclonal antibody M3F7 precipitates material from pepsin-derived and radiolabeled type IV collagen, and analysis of the polypeptide chains in the immunoprecipitate by sodium dodecyl sulfate polyacrylamide gel electrophoresis suggests that two major fragments are contained in the precipitate, which yield polypeptides of about 100 and 50 kilodaltons. After rotary shadowing of antigen-antibody mixtures native collagen fragments of two different size classes that bind antibody are visualized. One fragment is approximately 1500 A in length, and the other measures about 2700 to 3000 A. The localization of the antigenic site on these fragments suggests that both are generated by pepsin cleavage at a site about 900 A removed from the amino terminal end. In immunofluorescence experiments the monoclonal antibodies stained all basement membranes in kidney, lung, placenta, or skin, suggesting that at least the type IV collagen molecule recognized by these monoclonal antibodies is shared by a variety of vascular and epithelial basement membranes.
IV型胶原是基底膜的主要成分之一，但不同部位的结构框架是由一种独特类型的分子组装而成，还是存在不同类型的IV型胶原分子，目前尚不清楚。为了研究这种蛋白质在不同器官中的组成、化学特性和组织方式，我们制备了针对人胎盘IV型胶原制剂的单克隆抗体。用瑞士韦伯斯特小鼠进行超免疫，然后将脾细胞与三种不同的骨髓瘤细胞系SP2/0、NS1和U1融合。通过有限稀释法和在硬琼脂上培养筛选并克隆出IV型胶原特异性杂交瘤。通过ELISA抑制试验、免疫沉淀、旋转投影和免疫荧光技术对两个克隆分泌的单克隆抗体进行了广泛的表征。与传统在兔子体内产生的抗体不同，这两种单克隆抗体试剂均表现出物种特异性结合，仅与人胎盘的天然IV型胶原结合，而不与小牛肺的类似制剂或其他类型的胶原结合。抗原热变性后，不再观察到抗体结合。通过金属投影技术观察到，M3F7抗体结合位点位于IV型分子的三螺旋结构域内，距离氨基末端约900埃。单克隆抗体M3F7从胃蛋白酶消化衍生的和放射性标记的IV型胶原中沉淀出物质，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分析免疫沉淀物中的多肽链，结果表明沉淀物中包含两个主要片段，分别产生约100和50千道尔顿的多肽。对抗原-抗体混合物进行旋转投影后，可以看到与抗体结合的两种不同大小类别的天然胶原片段。一个片段长度约为1500埃,另一个约为2700至3000埃。这些片段上抗原位点的定位表明，两者都是由胃蛋白酶在距离氨基末端约900埃的位点切割产生的。在免疫荧光实验中，单克隆抗体对肾脏、肺、胎盘或皮肤中的所有基底膜进行了染色，这表明至少这些单克隆抗体识别的IV型胶原分子存在于多种血管和上皮基底膜中。
