Weber Malte, Weiss Etienne, Engel Alfred M
Department of Rare Reagents, Roche Diagnostics GmbH, Nonnenwald 2, Penzberg 82372, Germany.
J Immunol Methods. 2003 Jul;278(1-2):249-59. doi: 10.1016/s0022-1759(03)00228-x.
Scl-70 is the major antigen recognised by autoantibodies in the sera of patients with systemic sclerosis (SSc). The autoantibodies that specifically react with Scl-70 are highly characteristic of the disease and represent valuable markers for the diagnosis of SSc. We describe a novel strategy for cloning autoantibody fragments starting with a small blood sample from an SSc patient. B cells isolated from the collected peripheral blood mononuclear cells (PBMCs) were cultured in vitro using the EL4-B5 system. Anti-Scl-70 IgG-producing cells were pooled for RNA preparation followed by the generation of phagemid libraries of approximately 10(7) independent single-chain Fvs (scFvs). The screening of these libraries by phage display allowed us to isolate four anti-Scl-70 scFvs following three rounds of biopanning. About 10 times more starting blood material was needed to generate scFv libraries of similar size from PBMCs of an SSc patient and only two anti-Scl-70 scFvs were isolated after three rounds of phage selection. Together, this work shows that functional autoantibody fragments can be advantageously cloned after in vitro expansion of B cells. The isolated anti-Scl-70 autoantibody fragments represent useful tools for calibrating SSc diagnostic assays.
Scl-70是系统性硬化症（SSc）患者血清中自身抗体识别的主要抗原。与Scl-70特异性反应的自身抗体是该疾病的高度特征性表现，也是诊断SSc的重要标志物。我们描述了一种从SSc患者的少量血样开始克隆自身抗体片段的新策略。从收集的外周血单核细胞（PBMC）中分离出的B细胞，使用EL4-B5系统进行体外培养。将产生抗Scl-70 IgG的细胞汇集起来用于制备RNA，随后构建约10⁷个独立单链抗体片段（scFv）的噬菌粒文库。通过噬菌体展示对这些文库进行筛选，经过三轮生物淘选，我们分离出了4个抗Scl-70 scFv。从SSc患者的PBMC中产生类似大小的scFv文库大约需要多10倍的起始血液材料，并且在三轮噬菌体筛选后仅分离出2个抗Scl-70 scFv。总之，这项工作表明，B细胞体外扩增后可以有利地克隆功能性自身抗体片段。分离出的抗Scl-70自身抗体片段是校准SSc诊断检测的有用工具。
