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Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay

The quantification of frequency of IFN-γ–producing T cells responding to donor alloantigen using the IFN-γ enzyme linked immunosorbent spot (ELISPOT) holds potential for pretransplant and posttransplant immunological risk stratification. The effectiveness of this assay, and the ability to compare re...

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Autores principales: Carroll, Robert, Troelnikov, Alexander, Chong, Anita S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Lippincott Williams & Wilkins 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096438/
https://www.ncbi.nlm.nih.gov/pubmed/27826604
http://dx.doi.org/10.1097/TXD.0000000000000621
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author Carroll, Robert
Troelnikov, Alexander
Chong, Anita S.
author_facet Carroll, Robert
Troelnikov, Alexander
Chong, Anita S.
author_sort Carroll, Robert
collection PubMed
description The quantification of frequency of IFN-γ–producing T cells responding to donor alloantigen using the IFN-γ enzyme linked immunosorbent spot (ELISPOT) holds potential for pretransplant and posttransplant immunological risk stratification. The effectiveness of this assay, and the ability to compare results generated by different studies, is dependent on the utilization of a standardized operating procedure (SOP). Key factors in assay standardization include the identification of primary and secondary antibody pairs, and the reading of the ELISPOT plate with a standardized automated algorithm. Here, we describe in detail, an SOP that should provide low coefficient of variation results. For multicenter trials, it is recommended that groups perform the ELISPOT assays locally but use a centralized ELISPOT reading facility, as this has been shown to be beneficial in reducing coefficient of variation between laboratories even when the SOP is strictly adhered to.
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spelling pubmed-50964382016-11-08 Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay Carroll, Robert Troelnikov, Alexander Chong, Anita S. Transplant Direct Laboratory Methods The quantification of frequency of IFN-γ–producing T cells responding to donor alloantigen using the IFN-γ enzyme linked immunosorbent spot (ELISPOT) holds potential for pretransplant and posttransplant immunological risk stratification. The effectiveness of this assay, and the ability to compare results generated by different studies, is dependent on the utilization of a standardized operating procedure (SOP). Key factors in assay standardization include the identification of primary and secondary antibody pairs, and the reading of the ELISPOT plate with a standardized automated algorithm. Here, we describe in detail, an SOP that should provide low coefficient of variation results. For multicenter trials, it is recommended that groups perform the ELISPOT assays locally but use a centralized ELISPOT reading facility, as this has been shown to be beneficial in reducing coefficient of variation between laboratories even when the SOP is strictly adhered to. Lippincott Williams & Wilkins 2016-10-20 /pmc/articles/PMC5096438/ /pubmed/27826604 http://dx.doi.org/10.1097/TXD.0000000000000621 Text en Copyright © 2016 The Authors. Transplantation Direct. Published by Wolters Kluwer Health, Inc. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND) (http://creativecommons.org/licenses/by-nc-nd/4.0/) , where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
spellingShingle Laboratory Methods
Carroll, Robert
Troelnikov, Alexander
Chong, Anita S.
Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title_full Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title_fullStr Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title_full_unstemmed Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title_short Virtual Global Transplant Laboratory Standard Operating Protocol for Donor Alloantigen-specific Interferon-gamma ELISPOT Assay
title_sort virtual global transplant laboratory standard operating protocol for donor alloantigen-specific interferon-gamma elispot assay
topic Laboratory Methods
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096438/
https://www.ncbi.nlm.nih.gov/pubmed/27826604
http://dx.doi.org/10.1097/TXD.0000000000000621
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