Összes szerző

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Bécsi Bálint

az alábbi absztraktok szerzői között szerepel:

Nagy Péter
The effect of fluorescence labeling on the function and dynamical properties of antibodies

Aug 30 - szerda

14:18 – 14:36

Sejtanalitika biofizikai megközelítéssel

E33

The effect of fluorescence labeling on the function and dynamical properties of antibodies

Tímea Hajdu¹, Gábor Mocsár¹, István Rebenku¹, Ágnes Batta¹, Bálint Bécsi², Ferenc Erdődi² and Peter Nagy¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen

² Department of Medical Chemistry, Faculty of Medicine, University of Debrecen

Fluorescent antibodies have been the cornerstone of cell biological investigations in the last couple of decades due to their relatively straightforward application. While fluorescence labeling has been shown to deteriorate the affinity of antibodies and the fluorescence properties of the dyes, an effect that may significantly affect the reliability of quantitative biophysical measurements [1], the background and further implications of these findings have not been explored. Here we show that fluorescence labeling of antibodies not only deteriorates their epitope binding capability, but functions linked to other IgG domains, and the extent of these effects reveals remarkably similar dependence on the degree of labeling. The melting temperature of the Fab and Fc domains of unlabeled and fluorescently-tagged antibodies were identical according to differential scanning fluorometry implying that the overall stability of antibody domains is not affected by fluorescence labeling. According to time-dependent measurements, the decay rate of fluorescence anisotropy increased by the degree of labeling suggesting that the wagging motion of antibody domains in accelerated by the presence of the fluorophores. This conclusion is corroborated by FRET measurements between the Fc domain and the IgG-bound epitope in which the steady-state energy transfer efficiency was higher in antibodies with a high degree of labeling implying that the Fc and epitope-binding domains approach each other more closely in highly labeled antibodies on average over time. The investigations suggest that the effect of fluorescence labeling on all antibody functions may be due to altered antibody dynamics.

Acknowledgment

The project was supported by research grants from the National Research, Development and Innovation Office (K138075, ANN133421).

References

[1] Szabó Á, Szatmári T, Ujlaky-Nagy L, Rádi I, Vereb G, Szöllősi J, Nagy P (2018) Biophys J, 114: 668-700.