Optimization and qualification of an 8-color intracellular cytokine staining assay for quantifying T cell responses in rhesus macaques for pre-clinical vaccine studies.
J Immunol Methods
; 386(1-2): 10-21, 2012 Dec 14.
Article
em En
| MEDLINE
| ID: mdl-22955212
ABSTRACT
Vaccination and SIV challenge of macaque species is the best animal model for evaluating candidate HIV vaccines in pre-clinical studies. As such, robust assays optimized for use in nonhuman primates are necessary for reliable ex vivo measurement of immune responses and identification of potential immune correlates of protection. We optimized and qualified an 8-color intracellular cytokine staining assay for the measurement of IFNγ, IL-2, and TNF from viable CD4 and CD8 T cells from cryopreserved rhesus macaque PBMC stimulated with peptides. After optimization, five laboratories tested assay performance using the same reagents and PBMC samples; similar results were obtained despite the use of flow cytometers with different configurations. The 8-color assay was then subjected to a pre-qualification study to quantify specificity and precision. These data were used to set positivity thresholds and to design the qualification protocol. Upon completion of the qualification study, the assay was shown to be highly reproducible with low inter-aliquot, inter-day, and inter-operator variability according to the qualification criteria with an overall variability of 20-40% for each outcome measurement. Thus, the 8-color ICS assay was formally qualified according to the ICH guidelines Q2 (R1) for specificity and precision indicating that it is considered a standardized/robust assay acceptable for use in pre-clinical trial immunogenicity testing.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Coloração e Rotulagem
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Linfócitos T
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Citocinas
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Síndrome de Imunodeficiência Adquirida dos Símios
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Vírus da Imunodeficiência Símia
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Vacinas contra a AIDS
Tipo de estudo:
Diagnostic_studies
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Evaluation_studies
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Guideline
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Prognostic_studies
Limite:
Animals
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Humans
Idioma:
En
Revista:
J Immunol Methods
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
Estados Unidos