Linear gene fusions of antibody fragments with streptavidin can be linked to biotin labelled secondary molecules to form bispecific reagents.
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.
Pearce
L A
LA
CSIRO, Division of Biomolecular Engineering, Parkville, Australia.
Oddie
G W
GW
Coia
G
G
Kortt
A A
AA
Hudson
P J
PJ
Lilley
G G
GG
D000818:Animals; D000906:Antibodies; D018033:Antibodies, Bispecific / Q000235:genetics / Q000378:metabolism; D019677:Artificial Gene Fusion / Q000379:methods; D015374:Biosensing Techniques; D001710:Biotin / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009980:Influenza A virus / Q000201:enzymology; D051379:Mice; D009439:Neuraminidase / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D000906:Antibodies; D018033:Antibodies, Bispecific; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D001710:Biotin; D019809:Streptavidin; D009439:Neuraminidase