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High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease

Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficul...

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Autores principales: Fiema, Bryan, Harris, Andrew C., Gomez, Aurelie, Pongtornpipat, Praechompoo, Lamiman, Kelly, Vander Lugt, Mark T., Paczesny, Sophie
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MyJove Corporation 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499072/
https://www.ncbi.nlm.nih.gov/pubmed/23149907
http://dx.doi.org/10.3791/4247
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author Fiema, Bryan
Harris, Andrew C.
Gomez, Aurelie
Pongtornpipat, Praechompoo
Lamiman, Kelly
Vander Lugt, Mark T.
Paczesny, Sophie
author_facet Fiema, Bryan
Harris, Andrew C.
Gomez, Aurelie
Pongtornpipat, Praechompoo
Lamiman, Kelly
Vander Lugt, Mark T.
Paczesny, Sophie
author_sort Fiema, Bryan
collection PubMed
description Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2Rα(5), TNFR1(6), HGF(7), IL-8(8), elafin(2), and REG3α(3) (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials. This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents.
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spelling pubmed-34990722012-11-19 High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease Fiema, Bryan Harris, Andrew C. Gomez, Aurelie Pongtornpipat, Praechompoo Lamiman, Kelly Vander Lugt, Mark T. Paczesny, Sophie J Vis Exp Medicine Unbiased discovery proteomics strategies have the potential to identify large numbers of novel biomarkers that can improve diagnostic and prognostic testing in a clinical setting and may help guide therapeutic interventions. When large numbers of candidate proteins are identified, it may be difficult to validate candidate biomarkers in a timely and efficient fashion from patient plasma samples that are event-driven, of finite volume and irreplaceable, such as at the onset of acute graft-versus-host disease (GVHD), a potentially life-threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Here we describe the process of performing commercially available ELISAs for six validated GVHD proteins: IL-2Rα(5), TNFR1(6), HGF(7), IL-8(8), elafin(2), and REG3α(3) (also known as PAP1) in a sequential fashion to minimize freeze-thaw cycles, thawed plasma time and plasma usage. For this procedure we perform the ELISAs in sequential order as determined by sample dilution factor as established in our laboratory using manufacturer ELISA kits and protocols with minor adjustments to facilitate optimal sequential ELISA performance. The resulting plasma biomarker concentrations can then be compiled and analyzed for significant findings within a patient cohort. While these biomarkers are currently for research purposes only, their incorporation into clinical care is currently being investigated in clinical trials. This technique can be applied to perform ELISAs for multiple proteins/cytokines of interest on the same sample(s) provided the samples do not need to be mixed with other reagents. If ELISA kits do not come with pre-coated plates, 96-well half-well plates or 384-well plates can be used to further minimize use of samples/reagents. MyJove Corporation 2012-10-31 /pmc/articles/PMC3499072/ /pubmed/23149907 http://dx.doi.org/10.3791/4247 Text en Copyright © 2012, Journal of Visualized Experiments http://creativecommons.org/licenses/by-nc-nd/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/
spellingShingle Medicine
Fiema, Bryan
Harris, Andrew C.
Gomez, Aurelie
Pongtornpipat, Praechompoo
Lamiman, Kelly
Vander Lugt, Mark T.
Paczesny, Sophie
High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title_full High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title_fullStr High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title_full_unstemmed High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title_short High Throughput Sequential ELISA for Validation of Biomarkers of Acute Graft-Versus-Host Disease
title_sort high throughput sequential elisa for validation of biomarkers of acute graft-versus-host disease
topic Medicine
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3499072/
https://www.ncbi.nlm.nih.gov/pubmed/23149907
http://dx.doi.org/10.3791/4247
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