Format

Send to

Choose Destination
Antibodies (Basel). 2019 Sep 23;8(4). pii: E48. doi: 10.3390/antib8040048.

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

Author information

1
Department of Chemistry, Physical and Biophysical Chemistry, Bielefeld University, 33615 Bielefeld, Germany. frank.luedel@mail.de.
2
Department of Chemistry, Structural Biochemistry, Bielefeld University, 33615 Bielefeld, Germany. bufe.sandra@mh-hannover.de.
3
Department of Chemistry, Structural Biochemistry, Bielefeld University, 33615 Bielefeld, Germany. willem_manuel.bleymueller@uni-bielefeld.de.
4
Department of Molecular Medicine, Division of Immunology and General Pathology, University of Pavia, 27100 Pavia, Italy. hugo.dejonge@unipv.it.
5
Department of Molecular Medicine, Division of Immunology and General Pathology, University of Pavia, 27100 Pavia, Italy. luisa.iamele@unipv.it.
6
Department of Chemistry, Structural Biochemistry, Bielefeld University, 33615 Bielefeld, Germany. hartmut.niemann@uni-bielefeld.de.
7
Department of Chemistry, Physical and Biophysical Chemistry, Bielefeld University, 33615 Bielefeld, Germany. thomas.hellweg@uni-bielefeld.de.

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 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; IgG; SAXS; SLS; Zimm-Plot; antibody contstruct

PMID:
31548495
DOI:
10.3390/antib8040048
Free full text

Supplemental Content

Full text links

Icon for Multidisciplinary Digital Publishing Institute (MDPI)
Loading ...
Support Center