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Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Department of Chemistry, Physical and Biophysical Chemistry, Bielefeld University, 33615 Bielefeld, Germany
Department of Chemistry, Structural Biochemistry, Bielefeld University, 33615 Bielefeld, Germany
Department of Molecular Medicine, Division of Immunology and General Pathology, University of Pavia, 27100 Pavia, Italy
Author to whom correspondence should be addressed.
Current address: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, 30625 Hannover, Germany.
Antibodies 2019, 8(4), 48;
Received: 18 April 2019 / Revised: 10 September 2019 / Accepted: 13 September 2019 / Published: 23 September 2019
(This article belongs to the Special Issue Higher Order Structure Characterization of Therapeutic Antibodies)
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.
Keywords: DLS; SAXS; SLS; Zimm-Plot; IgG; antibody contstruct DLS; SAXS; SLS; Zimm-Plot; IgG; antibody contstruct
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Lüdel, F.; Bufe, S.; Bleymüller, W.M.; de Jonge, H.; Iamele, L.; Niemann, H.H.; Hellweg, T. Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies 2019, 8, 48.

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