Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.

TitleOptimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.
Publication TypeJournal Article
Year of Publication2012
AuthorsDefawe OD, Fong Y, Vasilyeva E, Pickett M, Carter DK, Gabriel E, Rerks-Ngarm S, Nitayaphan S, Frahm N, McElrath JM, De Rosa SC
JournalJ Immunol Methods
Volume382
Issue1-2
Pagination117-28
Date Published2012 Aug 31
ISSN1872-7905
Abstract

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.

DOI10.1016/j.jim.2012.05.011
Alternate JournalJ. Immunol. Methods
PubMed ID22626638