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Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium
Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA)....
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493492/ https://www.ncbi.nlm.nih.gov/pubmed/36159843 http://dx.doi.org/10.3389/fimmu.2022.984642 |
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author | Waerlop, Gwenn Leroux-Roels, Geert Lambe, Teresa Bellamy, Duncan Medaglini, Donata Pettini, Elena Cox, Rebecca Jane Trieu, Mai-Chi Davies, Richard Bredholt, Geir Montomoli, Emanuele Gianchecchi, Elena Clement, Frédéric |
author_facet | Waerlop, Gwenn Leroux-Roels, Geert Lambe, Teresa Bellamy, Duncan Medaglini, Donata Pettini, Elena Cox, Rebecca Jane Trieu, Mai-Chi Davies, Richard Bredholt, Geir Montomoli, Emanuele Gianchecchi, Elena Clement, Frédéric |
author_sort | Waerlop, Gwenn |
collection | PubMed |
description | Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection. |
format | Online Article Text |
id | pubmed-9493492 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-94934922022-09-23 Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium Waerlop, Gwenn Leroux-Roels, Geert Lambe, Teresa Bellamy, Duncan Medaglini, Donata Pettini, Elena Cox, Rebecca Jane Trieu, Mai-Chi Davies, Richard Bredholt, Geir Montomoli, Emanuele Gianchecchi, Elena Clement, Frédéric Front Immunol Immunology Influenza continues to be the most important cause of viral respiratory disease, despite the availability of vaccines. Today’s evaluation of influenza vaccines mainly focuses on the quantitative and functional analyses of antibodies to the surface proteins haemagglutinin (HA) and neuraminidase (NA). However, there is an increasing interest in measuring cellular immune responses targeting not only mutation-prone surface HA and NA but also conserved internal proteins as these are less explored yet potential correlates of protection. To date, laboratories that monitor cellular immune responses use a variety of in-house procedures. This generates diverging results, complicates interlaboratory comparisons, and hampers influenza vaccine evaluation. The European FLUCOP project aims to develop and standardize assays for the assessment of influenza vaccine correlates of protection. This report describes the harmonization and qualification of the influenza-specific interferon-gamma (IFN-γ) Enzyme-Linked ImmunoSpot (ELISpot) assay. Initially, two pilot studies were conducted to identify sources of variability during sample analysis and spot enumeration in order to develop a harmonized Standard Operating Procedure (SOP). Subsequently, an assay qualification study was performed to investigate the linearity, intermediate precision (reproducibility), repeatability, specificity, Lower and Upper Limits of Quantification (LLOQ-ULOQ), Limit of Detection (LOD) and the stability of signal over time. We were able to demonstrate that the FLUCOP harmonized IFN-γ ELISpot assay procedure can accurately enumerate IFN-γ secreting cells in the analytical range of 34.4 Spot Forming Units (SFU) per million cells up to the technical limit of the used reader and in the linear range from 120 000 to 360 000 cells per well, in plates stored up to 6 weeks after development. This IFN-γ ELISpot procedure will hopefully become a useful and reliable tool to investigate influenza-specific cellular immune responses induced by natural infection or vaccination and can be an additional instrument in the search for novel correlates of protection. Frontiers Media S.A. 2022-09-08 /pmc/articles/PMC9493492/ /pubmed/36159843 http://dx.doi.org/10.3389/fimmu.2022.984642 Text en Copyright © 2022 Waerlop, Leroux-Roels, Lambe, Bellamy, Medaglini, Pettini, Cox, Trieu, Davies, Bredholt, Montomoli, Gianchecchi and Clement https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Immunology Waerlop, Gwenn Leroux-Roels, Geert Lambe, Teresa Bellamy, Duncan Medaglini, Donata Pettini, Elena Cox, Rebecca Jane Trieu, Mai-Chi Davies, Richard Bredholt, Geir Montomoli, Emanuele Gianchecchi, Elena Clement, Frédéric Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title | Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title_full | Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title_fullStr | Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title_full_unstemmed | Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title_short | Harmonization and qualification of an IFN-γ Enzyme-Linked ImmunoSpot assay (ELISPOT) to measure influenza-specific cell-mediated immunity within the FLUCOP consortium |
title_sort | harmonization and qualification of an ifn-γ enzyme-linked immunospot assay (elispot) to measure influenza-specific cell-mediated immunity within the flucop consortium |
topic | Immunology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9493492/ https://www.ncbi.nlm.nih.gov/pubmed/36159843 http://dx.doi.org/10.3389/fimmu.2022.984642 |
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