1. Αποθετήριο Παραδοτέων Υποτρόφων Δράσεων ΕΣΠΑ Τριτοβάθμιας Εκπαίδευσης 2017-2023
2. Όλο το Αποθετήριο
3. Ερευνητικές ομάδες

Τύπος:	Άρθρο σε επιστημονικό περιοδικό
Τίτλος:	Development of a novel flow cytometry method for detecting pneumococcal-specific B cells
Συγγραφέας:	[EL] Τζοβάρα, Ειρήνη[EN] Tzovara, Irene [EL] Παπαδάτου, Ιωάννα[EN] Papadatou, Ioanna [EL] Τζανουδάκη, Μαριάννα[EN] Tzanoudaki, Marianna [EL] Σπούλου-Κλώνου, Βασιλική[EN] Spoulou, Vasiliki
Ημερομηνία:	09/05/2022
Περίληψη:	Antigen-specific B cell identification by flow cytometry is crucial for investigating their immunophenotype, subset distribution, and kinetics post-infection or immunization. Methods using biotinylated polysaccharide antigens have been described, but there is still room for improvement regarding sensitivity and applicability. The aim of this study was the development and validation of a multimer bead-based method for detecting pneumococcal polysaccharide serotypes (PS)-specific B cells following pneumococcal immunization. PS was chemically biotinylated and mounted on anti-biotin beads, and labeled with phycoerythrin (PE)-conjugated anti-biotin antibody to form a PS-multimer used for cell staining. Labeled beads were washed to remove excess fluorochrome and diminish non-specific labeling and background noise. Optimal ratios of PS-bead conjugate to PE and PS-multimer to cells were determined with titration assays. Comparison between the PS-multimer and a PS-PE monomer revealed enhanced detection of PS-specific cells and considerable signal amplification, attributed to the multimeric form of the detection probe and increased availability of antigen epitopes. To validate the specificity of the method, a competition assay using unbound PS was performed. Following pre-incubation with increasing PS concentrations, detection of PS-specific B cells with the PS-multimer was inhibited in a stepwise manner. Pre-incubation with excess PS completely blocked the fluorescent signal. This novel bead-based flow cytometry approach is a sensitive method demonstrating high specificity. It generated enhanced signals, provided clear-cut results, and was easily applicable, not requiring B cell pre-enrichment. It could be modified to adapt other antigens of interest, especially polysaccharides and proteins that could be used to probe antigen-specific B cell responses. The study of such responses may elucidate the underlying mechanisms involved in the establishment of long-term protection, provide evidence-based rationale for improving currently available vaccines and vaccination strategies, and pave the way for future vaccine development.
Γλώσσα:	Αγγλικά
Σελίδες:	8
DOI:	10.1002/cyto.a.24654
EISSN:	1552-4930
Θεματική κατηγορία:	[EL] Ανοσολογία[EN] Immunology
Λέξεις-κλειδιά:	13-valent pneumococcal conjugate vaccine; antigen-specific; antigen-specific B cells; antigen-specific multimer; Flow cytometry; memory B cells; pneumococcal vaccines; pneumococcal-specific B cells
Κάτοχος πνευματικών δικαιωμάτων:	© 2022 The Authors.Cytometry Part Apublished by Wiley Periodicals LLC on behalf of International Society for Advancement of Cytometry
Όροι και προϋποθέσεις δικαιωμάτων:	This is an open access article under the terms of theCreative Commons Attribution-NonCommercial-NoDerivsLicense, which permits use and distribution in anymedium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made
Ηλεκτρονική διεύθυνση του τεκμηρίου στον εκδότη:	https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.24654
Ηλεκτρονική διεύθυνση περιοδικού:	https://onlinelibrary.wiley.com/journal/15524930
Τίτλος πηγής δημοσίευσης:	Cytometry Part A – the Journal of Quantitative Cell Science
Τεύχος:	7
Τόμος:	101
Σελίδες τεκμηρίου (στην πηγή):	588-596
Σημειώσεις:	This work was financially supported by the European Social Fund – NSRF (National Strategic Reference Framework) as part of the Operational Program “Human Resources Development, Education and Lifelong Learning 2014–2020.”
Εμφανίζεται στις συλλογές:	Ερευνητικές ομάδες