Burgoon M P, Owens G P, Carlson S, Maybach A L, Gilden D H
Department of Neurology, University of Colorado Health Sciences Center, Denver, CO 80262, USA.
J Immunol. 2001 Nov 15;167(10):6009-14. doi: 10.4049/jimmunol.167.10.6009.
The presence of increased IgG in the brains of humans with infectious and inflammatory CNS diseases of unknown etiology such as multiple sclerosis may be a clue to the cause of disease. For example, the intrathecally synthesized oligoclonal bands in diseases such as subacute sclerosing panencephalitis (SSPE) or cryptococcal meningitis have been shown to represent Ab directed against the causative agents, measles virus (MV), or Cryptococcus neoformans, respectively. Using SSPE as a model system, we developed a strategy to identify the antigenic targets of the intrathecal disease-relevant IgG in chronic human inflammatory and demyelinating diseases of the CNS. Libraries of cDNA Ags were displayed on the surface of T7Select bacteriophage and biopanned on IgG extracted from the brain of an SSPE patient, or on a monospecific recombinant Fab identified from SSPE brain. After three or six rounds of biopanning on either Ab, positive phage-displayed Ags reacting with IgG were enriched to 35-77% of all panned clones. Sequence analysis of the positive clones identified fragments of the nucleocapsid protein of MV, the cause of SSPE. The sensitivity of the system was determined by diluting the positive clones from this SSPE phage-displayed library at a ratio of 10(-6) into another phage-displayed library that did not contain any detectable MV Ags; after six rounds of panning, the positive clones comprised 34% of all phage and were also shown to be MV nucleocapsid specific. This strategy will be useful to identify potentially rare Ags in diseases of unknown cause.
在患有病因不明的感染性和炎症性中枢神经系统疾病（如多发性硬化症）的人类大脑中，IgG升高可能是疾病病因的一个线索。例如，在亚急性硬化性全脑炎（SSPE）或隐球菌性脑膜炎等疾病中，鞘内合成的寡克隆带已被证明分别代表针对病原体麻疹病毒（MV）或新型隐球菌的抗体。以SSPE为模型系统，我们开发了一种策略，用于识别慢性人类中枢神经系统炎症性和脱髓鞘疾病中鞘内疾病相关IgG的抗原靶点。cDNA抗原文库展示在T7Select噬菌体表面，并与从SSPE患者大脑中提取的IgG或从SSPE大脑中鉴定出的单特异性重组Fab进行生物淘选。在用任何一种抗体进行三轮或六轮生物淘选后，与IgG反应的阳性噬菌体展示抗原富集到所有淘选克隆的35 - 77%。对阳性克隆的序列分析鉴定出了SSPE病因MV的核衣壳蛋白片段。通过将来自该SSPE噬菌体展示文库的阳性克隆以10(-6)的比例稀释到另一个不包含任何可检测到的MV抗原的噬菌体展示文库中，来确定该系统的灵敏度；经过六轮淘选后，阳性克隆占所有噬菌体的34%，并且也被证明对MV核衣壳具有特异性。该策略将有助于识别病因不明疾病中潜在的罕见抗原。
