Document Detail


Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.
MedLine Citation:
PMID:  22626638     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The magnitude and functional phenotype (e.g. proliferation, immune stimulation) of vaccine-induced T-cell responses are likely to be critical in defining responses that can control pathogenic challenge. Current multi-parameter flow cytometric techniques may not be sufficient to measure all of these different functions, since characterizing T-cell responses by flow cytometry is presently limited to concurrent measurement of at most 10 cytokines/chemokines. Here, we describe extensive studies conducted using standardized GCLP procedures to optimize and qualitatively/quantitatively qualify a multiplex bead array (MBA) performed on supernatant collected from stimulated peripheral blood mononuclear cells (PBMC) to assess 12 cytokines and chemokines of interest. Our optimized MBA shows good precision (intra-assay, inter-day, inter-technician; coefficients of variation <30%) and linearity for most of the analytes studied. We also developed positivity criteria that allow us to define a response as positive or negative with a high degree of confidence. In conclusion, we provide a detailed description of the qualification of an MBA, which permits quantitative and qualitative evaluation of vaccine-induced immunogenicity and analysis of immune correlates of protection. This assay provides an excellent complement to the existing repertoire of assays for assessing immunogenicity in HIV vaccine clinical trials.
Authors:
Olivier D Defawe; Youyi Fong; Evgenia Vasilyeva; Melissa Pickett; Donald K Carter; Erin Gabriel; Supachai Rerks-Ngarm; Sorachai Nitayaphan; Nicole Frahm; M Juliana McElrath; Stephen C De Rosa
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2012-05-22
Journal Detail:
Title:  Journal of immunological methods     Volume:  382     ISSN:  1872-7905     ISO Abbreviation:  J. Immunol. Methods     Publication Date:  2012 Aug 
Date Detail:
Created Date:  2012-07-09     Completed Date:  2012-10-15     Revised Date:  2013-09-03    
Medline Journal Info:
Nlm Unique ID:  1305440     Medline TA:  J Immunol Methods     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  117-28     Citation Subset:  IM    
Copyright Information:
Copyright © 2012 Elsevier B.V. All rights reserved.
Affiliation:
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
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MeSH Terms
Descriptor/Qualifier:
AIDS Vaccines / immunology*
Chemokines / biosynthesis*,  immunology
Cytokines / biosynthesis*,  immunology
Flow Cytometry
HIV Antigens / immunology
Humans
Immunoassay / methods*
Leukocytes, Mononuclear / immunology
T-Lymphocytes / immunology*,  metabolism,  secretion*
Grant Support
ID/Acronym/Agency:
(P30 AI027757/AI/NIAID NIH HHS; P30 AI027757/AI/NIAID NIH HHS; U01 AI069481/AI/NIAID NIH HHS; UM1 AI068618/AI/NIAID NIH HHS; UM1 AI068618/AI/NIAID NIH HHS; UM1 AI068635/AI/NIAID NIH HHS; UM1 AI068635/AI/NIAID NIH HHS; UM1 AI069481/AI/NIAID NIH HHS
Chemical
Reg. No./Substance:
0/AIDS Vaccines; 0/Chemokines; 0/Cytokines; 0/HIV Antigens
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