Pearce L A, Oddie G W, Coia G, Kortt A A, Hudson P J, Lilley G G
CSIRO, Division of Biomolecular Engineering, Parkville, Australia.
Biochem Mol Biol Int. 1997 Sep;42(6):1179-88. doi: 10.1080/15216549700203651.
Monomeric single chain antibody (scFv) fragments lack both the avidity of the bivalent IgG, or (Fab')2 fragment, and the effector functions conferred by the Fc domain. For certain diagnostic or therapeutic applications it may be desirable to link these molecules to other proteins, antibodies, enzymes or peptide ligands, and chemical or recombinant methods have been developed to produce many of these crosslinked reagents. One approach has been to link an antibody fragment to streptavidin which can bind a second biotinylated molecule to create a higher affinity, bifunctional or bispecific molecule. To demonstrate the applicability of this technology, an anti-neuraminidase NC10 scFv-streptavidin fusion was expressed in E. coli and the product was refolded and purified to homogeneity from 6 M guanidine hydrochloride. Analysis in a BIAcore biosensor showed that the NC10 scFv moiety reacted with immobilised neuraminidase and that the core streptavidin moiety was able to bind biotinylated anti-ferritin Fab' to produce a new model bispecific reagent which bound ferritin. Conceptually, this design principle can be applied to the creation of useful diagnostic and possibly therapeutic molecules.
单体单链抗体（scFv）片段既缺乏二价IgG或（Fab'）2片段的亲和力，也缺乏Fc结构域赋予的效应器功能。对于某些诊断或治疗应用，可能需要将这些分子与其他蛋白质、抗体、酶或肽配体连接起来，并且已经开发出化学或重组方法来生产许多此类交联试剂。一种方法是将抗体片段与链霉亲和素连接，链霉亲和素可以结合第二个生物素化分子，以产生更高亲和力的双功能或双特异性分子。为了证明该技术的适用性，在大肠杆菌中表达了抗神经氨酸酶NC10 scFv-链霉亲和素融合蛋白，并将产物从6 M盐酸胍中重折叠并纯化至同质。在BIAcore生物传感器中的分析表明，NC10 scFv部分与固定化的神经氨酸酶反应，并且核心链霉亲和素部分能够结合生物素化的抗铁蛋白Fab'，以产生一种结合铁蛋白的新型双特异性试剂。从概念上讲，这种设计原则可应用于创建有用的诊断分子以及可能的治疗分子。
