Focal molography allows for affinity and concentration measurements of proteins in complex matrices with high accuracy
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Autor:in (Körperschaft)
Publikationsdatum
22.01.2025
Typ der Arbeit
Studiengang
Typ
01A - Beitrag in wissenschaftlicher Zeitschrift
Herausgeber:innen
Herausgeber:in (Körperschaft)
Betreuer:in
Übergeordnetes Werk
Biosensors
Themenheft
DOI der Originalpublikation
Link
Reihe / Serie
Reihennummer
Jahrgang / Band
15
Ausgabe / Nummer
2
Seiten / Dauer
66
Patentnummer
Verlag / Herausgebende Institution
MDPI
Verlagsort / Veranstaltungsort
Auflage
Version
Programmiersprache
Abtretungsempfänger:in
Praxispartner:in/Auftraggeber:in
Zusammenfassung
Characterizing biomolecular receptor–ligand interactions is critical for research and development. However, performing analyses in complex, biologically relevant matrices, such as serum, remains challenging due to non-specific binding that often impairs measurements. Here, we evaluated Focal Molography (FM) for determining KD and kinetic constants in comparison to gold-standard methods using single-domain heavy-chain antibodies in various systems. FM provided kinetic constants highly comparable to SPR and BLI in standard buffers containing blocking proteins, with KDs of soluble CD4 (sCD4) interactions within a 2.4-fold range across technologies. In buffers lacking blocking proteins, FM demonstrated greater robustness against non-specific binding and rebinding effects. In serum, FM exhibited stable baseline signals, unlike SPR and BLI, and yielded KDs of sCD4 interaction in 50% Bovine Serum within a 1.8-fold range of those obtained in standard buffers. For challenging molecules prone to non-specific binding (Granzyme B), FM successfully determined kinetic constants without external referencing. Finally, FM enabled direct analyte quantification in complex matrices. sCD4 quantification in cell culture media and 50% FBS showed recovery rates of 97.8–100.3% with an inter-assay CV below 1.3%. This study demonstrates the high potential of FM for kinetic affinity determination and biomarker quantification in complex matrices, enabling reliable measurements under biologically relevant conditions.
Schlagwörter
Focal molography, Surface plasmon resonance, Bio-layer interferometry, Biomolecular affinity, Kinetic affinity measurement, Antibody–antigen interaction, Receptor–ligand interaction, Complex matrices, Quantification of biomarkers, Non-specific binding
Fachgebiet (DDC)
Veranstaltung
Startdatum der Ausstellung
Enddatum der Ausstellung
Startdatum der Konferenz
Enddatum der Konferenz
Datum der letzten Prüfung
ISBN
ISSN
2079-6374
Sprache
Englisch
Während FHNW Zugehörigkeit erstellt
Ja
Zukunftsfelder FHNW
Publikationsstatus
Veröffentlicht
Begutachtung
Peer-Review der ganzen Publikation
Open Access-Status
Gold
Lizenz
Zitation
Dirscherl, L., Merz, L., Nieminen, R., Spies, P., Frutiger, A., Gatterdam, V., & Meinel, D. (2025). Focal molography allows for affinity and concentration measurements of proteins in complex matrices with high accuracy. Biosensors, 15(2), 66. https://doi.org/10.3390/bios15020066