Kyriatsoulis A, Manns M, Roth U, Gerken G, Lohse A, Wollensak S, Reske K, Meyer zum Büschenfelde K H
I. Medizinische Klinik und Poliklinik, Johannes Gutenberg-Universität Mainz, F.R.G.
J Immunol Methods. 1988 Apr 22;109(1):113-21. doi: 10.1016/0022-1759(88)90448-6.
We present a strategy to characterize specific antigen/autoantibody systems using polyclonal sera from patients as a probe and crude antigenic preparations such as mitoplasts. Sera from patients with primary biliary cirrhosis (PBC) are characterized by at least one of two specific subtypes of antimitochondrial antibodies (AMA), anti-p48 or anti-p62. Immunoblotting of such sera with mitoplast preparations derived from human, rat and rabbit livers revealed three proteins of approximately 27, 48 and 68 kDa as target antigens. On the basis of the molecular weight of these antigens we were able to purify them by elution from preparative SDS gels. Immunization of NZB mice with the high molecular weight component (the 68 kDa antigen from human liver mitoplasts) elicited a monoclonal antibody. The 68 kDa protein was then isolated by affinity chromatography and may well represent the prime target antigen of anti-p62 antimitochondrial antibodies. This experimental approach could be applied to protein target antigens of other autoantibodies.
我们提出了一种策略，以患者的多克隆血清为探针，使用诸如线粒体膜等粗制抗原制剂来表征特定的抗原/自身抗体系统。原发性胆汁性肝硬化（PBC）患者的血清具有两种特定亚型抗线粒体抗体（AMA）中的至少一种，即抗-p48或抗-p62。用源自人、大鼠和兔肝脏的线粒体膜制剂对这些血清进行免疫印迹分析，发现三种分子量约为27、48和68 kDa的蛋白质为靶抗原。基于这些抗原的分子量，我们能够通过从制备型SDS凝胶上洗脱来纯化它们。用高分子量成分（来自人肝脏线粒体膜的68 kDa抗原）免疫NZB小鼠，产生了一种单克隆抗体。然后通过亲和层析分离出68 kDa的蛋白质，它很可能代表抗-p62抗线粒体抗体的主要靶抗原。这种实验方法可应用于其他自身抗体的蛋白质靶抗原。
