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Updated: May 22, 2026
Multiplex Cytokine Profiling of Stimulated Mouse Splenocytes Using a Cytometric Bead-based Immunoassay Platform
Published on: November 9, 2017
Optimization and qualification of a multiplex bead array to assess cytokine and chemokine production by
Olivier D Defawe1, Youyi Fong, Evgenia Vasilyeva
1Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
Insights
A new multiplex bead array (MBA) assay effectively measures 12 cytokines and chemokines from peripheral blood mononuclear cells (PBMC), aiding vaccine immunogenicity studies. This assay complements flow cytometry for evaluating T-cell responses and immune correlates of protection.
Area of Science:
- Immunology
- Vaccinology
- Biotechnology
Background:
- T-cell responses are crucial for vaccine efficacy, but current flow cytometry methods have limitations in measuring multiple immune functions simultaneously.
- Assessing the magnitude and functional phenotype of vaccine-induced T-cell responses is critical for controlling pathogenic challenges.
Purpose of the Study:
- To optimize and qualify a multiplex bead array (MBA) assay for the quantitative and qualitative assessment of vaccine-induced immune responses.
- To evaluate the utility of MBA for measuring 12 cytokines and chemokines from stimulated peripheral blood mononuclear cells (PBMC).
Main Methods:
- Standardized Good Clinical Laboratory Practice (GCLP) procedures were used to optimize and qualify the MBA assay.
- Peripheral blood mononuclear cells (PBMC) were stimulated, and supernatants were analyzed using MBA to measure 12 cytokines and chemokines.
- Assay precision (intra-assay, inter-day, inter-technician) and linearity were assessed, and positivity criteria were developed.
Main Results:
- The optimized MBA assay demonstrated good precision (coefficients of variation <30%) and linearity for most analytes.
- Positivity criteria were established to confidently define a positive or negative immune response.
- The assay allows for quantitative and qualitative evaluation of vaccine-induced immunogenicity.
Conclusions:
- The qualified MBA assay is a valuable tool for assessing vaccine-induced immunogenicity and analyzing immune correlates of protection.
- This assay serves as an effective complement to existing methods, particularly for HIV vaccine clinical trials.
- The MBA assay enables comprehensive evaluation of T-cell functional phenotypes relevant to vaccine efficacy.
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.
