PhD Scientific Days 2025
Budapest, 7-9 July 2025
Poster Session II. - G: Pharmaceutical Sciences and Health Technologies
Optimization and Comparison of Conventional Filtration and SPA WGA Beads-Based Binding Assays Utilizing Radiolabeled Ligand for the α7 nACh Receptor
Előadó neve
Dr. Lóska Dóra
Neptun code
ZAAQQ5
Előadó munkahelye
Department of Pharmacodynamics
Előadó telefonszáma
+36202957541
Előadó e-mail címe
loska.dorka@gmail.com
Az előadás címe
Optimization and Comparison of Conventional Filtration and SPA WGA Beads-Based Binding Assays Utilizing Radiolabeled Ligand for the α7 nACh Receptor
Szerző(k) neve és munkahelye
Dóra Lóska1, Ferenc Horti2, András Boros3, Tamás Tábi1, Balázs Lendvai3
1: Department of Pharmacodynamics
2: Gedeon Richter Plc.
3: Gedeon Richter Plc., Semmelweis University Richter Department
Bemutatás módja
Poszter
Szekció
Poster Session II. - G: Pharmaceutical Sciences and Health Technologies
Language of the presentation
Hungarian
Preferred session
Pharmaceutical Sciences and Health Technologies
Összefoglaló szövege
Introduction: The α7 nicotinic acetylcholine receptor (nAChR) plays a key role in several neurological processes including cognition, making it a valuable target for pharmacological research. The binding properties of positive allosteric modulators (PAMs) are still not fully understood, however receptor binding assays remained to be established for their characterization.
Aims: Our aim was to optimize the α7 nACh receptor binding assay using both conventional and scintillation proximity assay (SPA) methods, and to compare the two approaches.
Method: We prepared membrane homogenate from a Flp-In 293 cells stably expressing recombinant nicotinic α7 nACh subunits forming functional homopentameric ion channels. For binding experiments the membrane was incubated with a series of concentrations of the orthosteric antagonist radioligand [3H]ASEM (0,03125-4 nM) for 120 minutes at 25 °C. Non-specific binding (NSB) was determined in the presence of 10 µM methyllycaconitine. After incubation, the samples were filtered over UniFilter® GF/C (Revvity), or we added 1 mg/ml SPA beads. Radioactivity was measured using a microplate counter.
Results: Our results show that the dissociation constant (Kd) value was nearly identical with both methods, but the value for the number of binding sites (Bmax) was significantly lower with the SPA method. Increasing concentrations of the radioligand resulted in typical saturation binding curves, but we observed a much higher background of NSB in the case of the SPA method.
Conclusion: The SPA method proved to be a faster and more practical alternative to the conventional method, offering a simplified workflow and reduced handling steps. However, the lower Bmax values and higher NSB observed with SPA suggest that this assay is less sensitive. While the traditional method remains more accurate for quantitative binding measurements, the SPA technique holds promise for high-throughput applications, provided further optimization is performed to improve its sensitivity. In addition to the investigation of α7 nACh receptor allosteric modulators, particularly PAMs, which are of significant interest for therapeutic development, we plan to extend our studies to primary rat brain tissue. This will enable us to better understand the modulatory effects of PAMs in more physiologically relevant settings.
Funding: Supported by Richter Gedeon Plc, SE 250+
University
Semmelweis University
Supervisor
Tamás Tábi
Publication of my abstract
I give consent to the publication of my abstract on the website of the congress.
phd.section.field
in doctoral studies before complex exam (PhD)
Kind
Szabad
Status
elfogadva
Accepted presentation method
poszter
Előadás fájl jóváhagyás
nem rendelkezett róla
Előadó
8244
Start
18:54
End
19:00