Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by vaccine-specific cells.
J Immunol Methods
; 382(1-2): 117-28, 2012 Aug 31.
Article
em En
| MEDLINE
| ID: mdl-22626638
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Imunoensaio
/
Linfócitos T
/
Citocinas
/
Vacinas contra a AIDS
/
Quimiocinas
Tipo de estudo:
Qualitative_research
Limite:
Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos