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Bio-Plex Cytokine Immunoassays and ELISA: Comparison of Two Methodologies in Testing Samples From Asthmatic and Healthy Children, Rev A

oplate well (Vignali 2000).

The goal of our study was to compare the Bio-Plex cytokine assays with cytokine ELISA developed in-house for testing cell culture samples from asthmatic and healthy children.


Methods
Cell Isolation and Cultures
Human peripheral blood mononuclear cells (PBMC) were isolated from fresh blood donated by atopic asthmatic children and non-atopic healthy children between the ages of 13 and 15 (Bttcher et al. in press). The cells (1 x 106/ml) were cultured in AIM-V serum-free medium (Life Technologies AB, Sweden) with 20 mM mercaptoethanol (Sigma-Aldrich, Stockholm, Sweden). The cells were stimulated with timothy grass, cat, and dog allergen extracts from ALK (Horsholm, Denmark) at 10,000 standardized units/ml each. The cells were cultured at 37C with 5% CO2 for 144 hr. The supernatants were collected after centrifugation and stored at 70C. Samples were tested simultaneously for cytokines IL-5, IL-10, IL-13, and IFN-γ using both a Bio-Plex human cytokine 4-plex assay and an ELISA made from commercially available antibodies.

Multiplex Assay
A Bio-Plex human cytokine assay for simultaneous quantitation of IL-5, IL-10, IL-13, and IFN-γ was run according to the recommended procedure. In brief, the premixed standards were reconstituted in 0.5 ml of culture medium, generating a stock concentration of 50,000 pg/ml for each cytokine. The standard stock was serially diluted in the same culture medium to generate 8 points for the standard curve. The assay was performed in a 96-well filtration plate supplied with the assay kit. Premixed beads (50 l) coated with target capture antibodi
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