Kipriyanov S M, Dübel S, Breitling F, Kontermann R E, Little M
Recombinant Antibody Research Group (FSP 4/0445), German Cancer Research Center, Heidelberg.
Mol Immunol. 1994 Oct;31(14):1047-58. doi: 10.1016/0161-5890(94)90100-7.
A murine antibody single-chain Fv (scFv) fragment carrying five C-terminal histidine residues preceded by a cysteine residue and a marker peptide was expressed in Escherichia coli. Its variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody mAb215, which is specific for the largest subunit of RNA polymerase II of Drosophila melanogaster. ScFv' monomers, covalently linked (scFv')2 and non-covalent dimers, as well as aggregated antibody fragments, were isolated from an E. coli cell paste by immobilized metal affinity chromatography in 6 M urea followed by a renaturation procedure that does not use any sulfhydryl agents. In a final step, the components were separated by size exclusion chromatography. All the recombinant antibody fractions demonstrated high antigen-binding activity and specificity as shown by ELISA and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental monoclonal antibodies and four-fold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.
一种携带五个C末端组氨酸残基（前面有一个半胱氨酸残基和一个标记肽）的鼠源抗体单链Fv（scFv）片段在大肠杆菌中表达。其可变重链（VH）和轻链（VL）结构域源自小鼠单克隆抗体mAb215，该抗体对黑腹果蝇RNA聚合酶II的最大亚基具有特异性。通过固定金属亲和色谱法在6 M尿素中从大肠杆菌细胞裂解物中分离出scFv'单体、共价连接的（scFv'）2和非共价二聚体以及聚集的抗体片段，随后进行不使用任何巯基试剂的复性程序。在最后一步中，通过尺寸排阻色谱法分离各组分。如ELISA和Western印迹分析所示，所有重组抗体制剂均表现出高抗原结合活性和特异性。通过竞争性免疫测定进行的亲和力测量表明，共价连接的（scFv'）2的结合常数与亲本单克隆抗体的结合常数非常接近，比scFv'单体高四倍。通过游离巯基特异性生物素化的scFv衍生物在ELISA和Western印迹分析中识别相应抗原，从而证明了使用化学修饰的scFv抗体进行免疫检测的可能性。
