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Ogino进行性系统性硬化症患者中独立发现的抗Og抗体的抗原为DNA拓扑异构酶I。为进一步了解抗原抗体系统，对抗体阳性IgG进行了纯化、抗原结合活性和对DNA拓扑异构酶I（topoisomerase I，topo I）的酶抑制活性分析，并首次用间接免疫荧光试验筛选抗核抗体阳性血清。以兔胸腺提取物为抗原，用免疫双扩散法从荧光抗核抗体阳性血清中筛选出抗DNA拓扑异构酶Ⅰ沉淀抗体。通过这些筛选试验，从261例PSS患者的sefa中收集到72份含有抗Topo I抗体的血清。用免疫印迹技术和酶联免疫吸附试验（ELISA）证实这些血清中含有抗Topo I的IgG抗体。用DE 52柱层析法从17份含IgG抗体的血清中分离IgG组分。用亲和纯化的兔胸腺提取物topo I作为抗原，通过ELISA同时测定分离的IgG样品的抗原结合活性。通过测定IgG抑制小牛胸腺DNA拓扑异构酶I松弛超螺旋DNA（pBR 322质粒DNA）的最小剂量来测定分离的IgG样品的酶抑制活性。IgG的抗原结合活性与酶抑制活性之间没有很好的相关性，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.