Ross C N, Turner N, Savage P, Cashman S J, Spooner R A, Pusey C D
Department of Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, United Kingdom.
Lab Invest. 1996 Jun;74(6):1051-9.
Goodpasture's disease is defined by the presence of autoantibodies to the glomerular basement membrane and characterized clinically by rapidly progressive glomerulonephritis and pulmonary hemorrhage. P1, a murine monoclonal antibody to the Goodpasture antigen (the noncollagenous domain of the alpha 3 chain of type IV collagen, alpha 3(IV)NC1), has been a valuable reagent in investigating the pathogenesis of this disorder. The purpose of this study was to generate and characterize a recombinant form of P1 as a single-chain Fv (scFv). First strand cDNA was made from RNA extracted from the P1 hybridoma cell line, and DNA encoding the antibody light and heavy chain variable domains was amplified by polymerase chain reaction, using universal oligonucleotides. The purified products were ligated sequentially into an expression plasmid separated by a sequence encoding a 15 amino acid flexible oligopeptide linker. The resulting scFv was expressed in E. coli. Functional scFv, designated HBR-3, was obtained by denaturing and refolding the expressed product. HBR-3 was shown by ELISA, immunoblotting, and immunohistologic techniques, to have the same specificity for alpha 3(IV)NC1 as P1 and autoantibodies from patients with Goodpasture's disease. HBR-3 and P1 were shown to have similar affinity for their mutual ligand. On sections of normal human kidney, the scFv bound only to glomerular basement membrane and distal tubular basement membrane. It did not bind to the glomerular basement membrane of patients with Alport's syndrome, in whom the Goodpasture antigen is often not expressed in an antigenic form. We have, therefore, generated a scFv which reproduces the specific binding properties of the parent monoclonal antibody, P1. The potential of HBR-3 as a diagnostic reagent in Alport's syndrome has been demonstrated. The development of this recombinant molecule should permit new approaches to the investigation of Goodpasture's disease.
肺出血肾炎综合征（Goodpasture's disease）是由抗肾小球基底膜自身抗体的存在所定义的，临床上以快速进展性肾小球肾炎和肺出血为特征。P1是一种针对肺出血肾炎综合征抗原（IV型胶原α3链的非胶原结构域，α3(IV)NC1）的鼠单克隆抗体，在研究该疾病的发病机制中一直是一种有价值的试剂。本研究的目的是制备并鉴定一种重组形式的P1单链抗体片段（scFv）。从P1杂交瘤细胞系提取的RNA合成第一链cDNA，使用通用寡核苷酸通过聚合酶链反应扩增编码抗体轻链和重链可变区的DNA。纯化产物依次连接到一个表达质粒中，中间由一个编码15个氨基酸的柔性寡肽接头序列隔开。所得的scFv在大肠杆菌中表达。通过对表达产物进行变性和复性获得了具有功能的scFv，命名为HBR-3。通过酶联免疫吸附测定（ELISA）、免疫印迹和免疫组织学技术表明，HBR-3对α3(IV)NC1的特异性与P1以及肺出血肾炎综合征患者的自身抗体相同。HBR-3和P1对其共同配体表现出相似的亲和力。在正常人肾脏切片上，scFv仅与肾小球基底膜和远端肾小管基底膜结合。它不与Alport综合征患者的肾小球基底膜结合，在Alport综合征患者中，肺出血肾炎综合征抗原通常不以抗原形式表达。因此，我们制备了一种scFv，它再现了亲本单克隆抗体P1的特异性结合特性。已证明HBR-3作为Alport综合征诊断试剂的潜力。这种重组分子应能为肺出血肾炎综合征的研究带来新的方法。
