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

Lüdel, Frank; Bufe, Sandra; Bleymüller, Willem M.; de Jonge, Hugo; Iamele, Luisa; Niemann, Hartmut H.; Hellweg, Thomas

doi:10.3390/antib8040048

Open AccessArticle

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

by

Frank Lüdel

¹,

Sandra Bufe

^2,†,

Willem M. Bleymüller

²,

Hugo de Jonge

³,

Luisa Iamele

³,

Hartmut H. Niemann

²

and

Thomas Hellweg

^1,*

¹

Department of Chemistry, Physical and Biophysical Chemistry, Bielefeld University, 33615 Bielefeld, Germany

²

Department of Chemistry, Structural Biochemistry, Bielefeld University, 33615 Bielefeld, Germany

³

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; https://doi.org/10.3390/antib8040048

Submission 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)

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

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MDPI and ACS Style

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. https://doi.org/10.3390/antib8040048

AMA Style

Lüdel F, Bufe S, Bleymüller WM, de Jonge H, Iamele L, Niemann HH, Hellweg T. Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies. 2019; 8(4):48. https://doi.org/10.3390/antib8040048

Chicago/Turabian Style

Lüdel, Frank, Sandra Bufe, Willem M. Bleymüller, Hugo de Jonge, Luisa Iamele, Hartmut H. Niemann, and Thomas Hellweg. 2019. "Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering" Antibodies 8, no. 4: 48. https://doi.org/10.3390/antib8040048

APA Style

Lüdel, F., Bufe, S., Bleymüller, W. M., de Jonge, H., Iamele, L., Niemann, H. H., & Hellweg, T. (2019). Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies, 8(4), 48. https://doi.org/10.3390/antib8040048

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

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