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

Lüdel F, Bufe S, Bleymüller WM, de Jonge H de, Iamele L, Niemann H, Hellweg T (2019)
Antibodies 8(4): 48.

Zeitschriftenaufsatz | Veröffentlicht| Englisch
 
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Autor/in
Lüdel, FrankUniBi ; Bufe, Sandra; Bleymüller, Willem M.; de Jonge, Hugo de; Iamele, Luisa; Niemann, HartmutUniBi; Hellweg, ThomasUniBi
Abstract / Bemerkung
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 kD , 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. 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.
Stichworte
DLS; SAXS; SLS; Zimm-Plot; IgG; antibody contstruct
Erscheinungsjahr
2019
Zeitschriftentitel
Antibodies
Band
8
Ausgabe
4
Art.-Nr.
48
eISSN
2073-4468
Finanzierungs-Informationen
Article Processing Charge funded by the Deutsche Forschungsgemeinschaft and the Open Access Publication Fund of Bielefeld University.
Page URI
https://pub.uni-bielefeld.de/record/2937628

Zitieren

Lüdel F, Bufe S, Bleymüller WM, et al. Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies. 2019;8(4): 48.
Lüdel, F., Bufe, S., Bleymüller, W. M., de Jonge, H. de, Iamele, L., Niemann, H., & Hellweg, T. (2019). Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies, 8(4), 48. doi:10.3390/antib8040048
Lüdel, F., Bufe, S., Bleymüller, W. M., de Jonge, H. de, Iamele, L., Niemann, H., and Hellweg, T. (2019). Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies 8:48.
Lüdel, F., et al., 2019. Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies, 8(4): 48.
F. Lüdel, et al., “Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering”, Antibodies, vol. 8, 2019, : 48.
Lüdel, F., Bufe, S., Bleymüller, W.M., de Jonge, H. de, Iamele, L., Niemann, H., Hellweg, T.: Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering. Antibodies. 8, : 48 (2019).
Lüdel, Frank, Bufe, Sandra, Bleymüller, Willem M., de Jonge, Hugo de, Iamele, Luisa, Niemann, Hartmut, and Hellweg, Thomas. “Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering”. Antibodies 8.4 (2019): 48.
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2019-09-27T08:56:36Z
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