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Antibody microarray analysis of inflammatory mediator release by human leukemia T cells and human non‐small cell lung cancer cells
Author(s) -
Morgan Aric,
Garcia Bradley,
Hargrave Aubrey,
Hommema Eric,
Kilmer Greg,
Narahari Janaki,
Webb Brian,
Wiese Rick
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a632-b
Subject(s) - jurkat cells , antibody microarray , cytokine , microbiology and biotechnology , antibody , cell culture , chemokine , tumor necrosis factor alpha , biotinylation , chemistry , leukemia inhibitory factor , t cell , cancer research , immunology , biology , inflammation , interleukin 6 , immune system , genetics
In order to expedite the analysis of samples for multiple cytokines/chemokines, we have developed slide‐based ExcelArray™ antibody sandwich microarrays. Each slide consists of 16 subarrays (wells) 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 DyLight™ 649 dye for quantitation. The Jurkat cell line was used as a model for human T cell leukemia and the A549 cell line was used as model for human lung cancer in our experiments. In order to evoke a cytokine/chemokine response, cells were stimulated with Tumor Necrosis Factor alpha (TNFa), Phorbol‐12‐myristate‐13‐acetate (PMA, TPA) and Phytohemagglutinin (PHA). Cell supernatants derived from both untreated and stimulated cells were analyzed on 41 unique analytes. Stimulated cells showed an increase in the expression level of many of the test analytes, including IL‐8, TNF‐a, and MIP‐1a, 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.