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Keywords = antibody contstruct

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19 pages, 3153 KB  
Article
Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering
by Frank Lüdel, Sandra Bufe, Willem M. Bleymüller, Hugo de Jonge, Luisa Iamele, Hartmut H. Niemann and Thomas Hellweg
Antibodies 2019, 8(4), 48; https://doi.org/10.3390/antib8040048 - 23 Sep 2019
Cited by 9 | Viewed by 12730
Abstract
Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone [...] Read more.
Depending on the linker length between the V H and the V L domain, single-chain Fv (scFv) antibody fragments form monomers, dimers (diabodies) or higher oligomers. We aimed at generating a diabody of the anti-MET antibody 3H3 to use it as crystallization chaperone to promote crystallization of the MET ectodomain through the introduction of a pre-formed twofold axis of symmetry. Size exclusion chromatography, however, suggested the protein to be monomeric. Hence, we used scattering techniques applied to solutions to further investigate its oligomerization state. The small angle X-ray scattering (SAXS) curve measured for our protein nicely fits to the scattering curve calculated from the known crystal structure of a diabody. In addition, concentration-dependent photon correlation spectroscopy (PCS) measurements revealed a hydrodynamic radius of 3.4 nm at infinite dilution and a negative interaction parameter k D , indicating attractive interactions that are beneficial for crystallization. Both SAXS and PCS measurements clearly suggest our antibody fragment to be a diabody in solution. Chemical cross-linking with glutaraldehyde and cell motility assays confirmed this conclusion. Full article
(This article belongs to the Special Issue Higher Order Structure Characterization of Therapeutic Antibodies)
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