Összes szerző

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Hajdu Tímea

az alábbi absztraktok szerzői között szerepel:

Bihariné Batta Ágnes
Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements

Aug 29 - kedd

15:30 – 17:00

I. Poszterszekció

P05

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements

Ágnes Batta^1,2, Tímea Hajdu¹ and Péter Nagy¹

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

² Doctoral School of Molecular Medicine, University of Debrecen

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research. [1]

References

[1] Batta Á, Hajdu T, Nagy P, Cytometry Part A, 2023. DOI: 10.1002/cyto.a.24728

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.