Johansson C, Butkowski R, Wieslander J
Department of Nephrology, University Hospital, Lund, Sweden.
Connect Tissue Res. 1991;25(3-4):229-41. doi: 10.3109/03008209109029159.
Monoclonal antibodies were produced against NC1, the globular noncollagenous domain of collagen IV, isolated from bovine glomerular basement membrane. Cells from eight positive wells were cloned and the resulting monoclonal antibodies were studied in detail by immunofluorescence on human kidney sections, by Western blot and by ELISA against denatured subunits from NC1 hexamers and against native NC1 hexamers from different tissues. The monoclonal antibodies could be divided into two groups. Firstly, those monoclonal antibodies that, in ELISA and Western blot, reacted with peptides related to the alpha 1 chain of collagen IV and stained all basement membranes in the kidney. Secondly, a monoclonal antibody that, in ELISA and Western blot, reacted with peptides related to the Goodpasture antigen, the alpha 3 chain of collagen IV. When this antibody was applied to human kidney sections it stained the glomerular basement membrane very intensively. Bowman's capsule and some tubular basement membrane were also stained, although to a lesser extent. This staining pattern is the same as that observed with sera from patients with Goodpasture's syndrome. An attempt was made to separate different subtypes of the NC1 hexamer. A monoclonal antibody from the first group was used to make an affinity chromatography column. Glomerular basement membrane digested with collagenase was separated on this column and the collected fractions were analyzed by ELISA and SDS-PAGE. The result from this study support the idea that glomerular basement membrane is composed of at least two different subtypes of type IV collagen.
制备了针对从牛肾小球基底膜分离出的IV型胶原球状非胶原结构域（NC1）的单克隆抗体。对八个阳性孔中的细胞进行克隆，并通过对人肾切片进行免疫荧光、蛋白质印迹以及针对NC1六聚体的变性亚基和来自不同组织的天然NC1六聚体进行酶联免疫吸附测定（ELISA），对所得单克隆抗体进行了详细研究。单克隆抗体可分为两组。首先，那些在ELISA和蛋白质印迹中与IV型胶原α1链相关肽发生反应并能使肾脏中所有基底膜染色的单克隆抗体。其次，一种在ELISA和蛋白质印迹中与Goodpasture抗原（IV型胶原α3链）相关肽发生反应的单克隆抗体。当将该抗体应用于人肾切片时，它能非常强烈地染色肾小球基底膜。鲍曼囊和一些肾小管基底膜也被染色，尽管程度较轻。这种染色模式与Goodpasture综合征患者血清所观察到的相同。尝试分离NC1六聚体的不同亚型。使用第一组中的一种单克隆抗体制备亲和层析柱。用胶原酶消化的肾小球基底膜在该柱上进行分离，收集的组分通过ELISA和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）进行分析。该研究结果支持肾小球基底膜由至少两种不同亚型的IV型胶原组成这一观点。
