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Updated: Jul 11, 2026
Humanized Mediator Release Assay as a Read-Out for Allergen Potency
Published on: June 29, 2021
Antibody microarray analysis of inflammatory mediator release by human leukemia T-cells and human non small cell lung
Bradley H Garcia1, Aubrey Hargrave, Aric Morgan
1Thermo Fisher Scientific, 3747 N. Meridian Road, Rockford, IL 61105, USA.
Insights
We developed antibody microarrays for rapid analysis of cytokines and chemokines, crucial for immune response. This method efficiently profiles cellular inflammatory mediator release from cell culture supernatants.
Area of Science:
- Immunology
- Cell Biology
- Biotechnology
Background:
- Cytokines and chemokines regulate inflammation and immune responses.
- Traditional methods like ELISA and Western blot are time-consuming for multiplex analysis.
- Efficient profiling of cellular inflammatory mediators is essential for understanding disease mechanisms.
Purpose of the Study:
- To develop and validate a slide-based antibody microarray for expedited multiplex analysis of cytokines and chemokines.
- To assess the utility of antibody microarrays for profiling cellular inflammatory mediator release.
- To quantify multiple analytes from cell culture supernatants using a novel microarray platform.
Main Methods:
- Developed Thermo Scientific ExcelArray Antibody Sandwich Microarrays with 16 subarrays per slide, each containing 12 antibodies in triplicate.
- Utilized a sandwich ELISA-based architecture with biotinylated detection antibodies and DyLight 649 Dye for quantitation.
- Analyzed cell supernatants from Jurkat and A549 cell lines stimulated with TNF-alpha, PMA, and PHA using four specialized arrays (Inflammation I, II, Angiogenesis, Chemotaxis).
Main Results:
- Achieved assay sensitivity below 10 pg/mL.
- Quantified 41 unique analytes from stimulated and unstimulated cell supernatants.
- Observed significant increases in IL-8, TNF-alpha, and MIP-1alpha expression in stimulated cells compared to controls.
Conclusions:
- Antibody microarray analysis provides a rapid and efficient method for multiplex cytokine and chemokine profiling.
- The developed platform is effective for analyzing cellular inflammatory mediator release from cell culture supernatants.
- This technology facilitates comprehensive analysis of immune responses and inflammatory processes.
Abstract:
Cytokines and chemokines are responsible for regulating inflammation and the immune response. Cytokine and chemokine release is typically measured by quantitative enzyme-linked immunosorbant assay (ELISA) or Western blot analysis. To expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide-based Thermo Scientific ExcelArray Antibody Sandwich Microarrays. Each slide consists of 16 subarrays (wells), each printed with 12 specific antibodies in triplicate and positive and negative control elements. This 16-well format allows for the analysis of 10 test samples using a six-point standard curve. The array architecture is based on the "sandwich" ELISA, in which an analyte protein is sandwiched between an immobilized capture antibody and a biotinylated detection antibody, using streptavidin-linked Thermo Scientific DyLight 649 Dye for quantitation. The observed sensitivity of this assay was <10 pg/mL. In our experiments, the Jurkat cell line was used as a model for human T-cell leukemia, and the A549 cell line was used as a model for human non-small cell lung cancer. To evoke a cytokine/chemokine response, cells were stimulated with tumor necrosis factor alpha (TNFalpha), phorbol-12-myristate-13-acetate (PMA, TPA), and phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on four different arrays (Inflammation I, Inflammation II, Angiogenesis, and Chemotaxis), enabling the quantitation of 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL-8, TNF-alpha, and MIP-1alpha, compared to the non-treated controls. Our experiments clearly demonstrate the utility of antibody microarray analysis of cell-culture supernatants for the profiling of cellular inflammatory mediator release.
