Construction and expression of anti-RBC diabody

ScFv is univalent small Ab molecule comprising a VH domain and a VL domain connected by a polypeptide linker. In order to construct bivalent molecules, we modified the anti-human RBC ScFv expressing vector by shortening the linker from 17 amino acid residues [SR (GGGGS)3] to 6 amino acid residues (SRGGGS) to force the pairing of VH and VL between two different molecules to form bivalent antibody fragment (diabody). The bivalency of E. coli expressed diabody was proved by its ability to agglutinate human RBC. The dimerization of diabody was also demonstrated by gel filtration (size-exclution) chromatography.