Analysis of Antigen Binding Activity and Enzyme Inhibitory Activity of lgG Antibody to DNA Topoisomerase I from Patients with Collagen Diseases.

作者在日本独立发现的进展性系统性硬化症患者Ogino的抗og抗体抗原已被澄清为DNA拓扑异构酶I。为了更好地了解抗原抗体系统，纯化抗体阳性lgG并分析其抗原结合活性和对DNA拓扑异构酶I （topo I）的酶抑制活性。采用间接免疫荧光法筛选抗核抗体阳性血清。以兔胸腺提取物为抗原，采用双免疫扩散法在荧光抗核抗体阳性血清中筛选DNA拓扑异构酶I沉淀抗体。通过这些筛选试验，在261例PSS患者血清中共收集到72份含有抗topo I抗体的血清。采用免疫印迹法和酶免疫吸附法（ELISA）对17份含IgG抗体的血清进行topoⅰ级分离，并采用DE52柱层析分离IgG。以亲和纯化的兔胸腺提取物topo I为抗原，采用ELISA法同时测定IgG样品的抗原结合活性。通过测定IgG抑制牛胸腺DNA拓扑异构酶I酶对超螺旋DNA （pBR322质粒DNA）松弛的最小剂量，检测分离的IgG样品的酶抑制活性。IgG样品的抗原结合活性与酶抑制活性没有很好的相关性。有数据表明，DNA拓扑异构酶I的抗原表位是异质的，包括酶的活性中心。

The antigen of anti-Og antibody which had been found by the auther independently in Japan in a patient named Ogino with progressive systemic sclerosis has been clarified to be DNA topoisomerase I. To get a better understanding of the antigen antibody system, antibody positive lgG was purified and analysed for antigen binding activity and enzyme inhibitory activity to DNA topoisomerase I (topo I).Antinuclear antibody positive sera were first sereened by indirect immunofluorescent test. Then precipitating antibodies to DNA topoisomerase I were screened in fluorescent antinuclear antibody positive sera by double immunodiffusion test using rabbit thymus extracts as the antigen. Through these screening tests 72 sera containing anti-topo I antibody could be collected in 261 sefa from patients with PSS. By using immunoblotting technique and enzyme immunosorbent assay (ELISA), these sera were confirmed to contain IgG antibody to topo I. IgG fractions were separated by using DE52 column chromatography from 17 sera containing IgG antibodies. Antigen binding activities of the separated IgG samples were determined simultaneously by ELISA using affinity-purified topo I from rabbit thymus extract as the antigen. Enzyme inhibitory activity of the separated IgG samples were assayed by determining the minimal dosis of IgG to inhibit the relaxation of supercoiled DNA (pBR322 plasmid DNA) by calf thymus DNA topoisomerase I enzyme. The antigen binding activities of IgG samples did not correlate well to the enzyme inhibitory activities.There data indicate that the antigenic epitopes of DNA topoisomerase I are heterogeneous including the active center of the enzyme.