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

es were transferred to each well of the filter plate (5,000 beads per well per cytokine) and washed twice with Bio-Plex wash buffer. Premixed standards or samples (50 l) were added to each well containing washed beads. The samples were used directly without further dilution. The plate was shaken for 30 sec and then incubated at room temperature for 30 min with low-speed shaking (300 rpm). After incubation and washing, premixed detection antibodies (50 l, final concentration of 2 g/ml) were added to each well. The incubation was terminated after shaking for 10 min at room temperature. After 3 washes, the beads were resuspended in 125 l of Bio-Plex assay buffer. Beads were read on the Bio-Plex suspension array system, and the data were analyzed using Bio-Plex Manager software (v 3.0) with 5PL curve fitting.

ELISA
ELISAs were performed as described in Jenmalm et al. (2001). Human cytokines IL-10 and IL-13 were analyzed using PeliPair reagent set antibodies (CLB, Amsterdam, the Netherlands). IL-5 was analyzed using an antibody pair from Pharmingen (San Diego, CA): capture antibody TRFK5 and detection antibody JES1-5. IFN-γ was measured using an antibody pair from R&D Systems (Abingdon, UK): capture antibody 2571.811 and a polyclonal detection antibody (goat anti-human). The detection antibodies were biotinylated and streptavidin-horseradish peroxidase was the enzyme label reagent.


Results and Discussion
Correlation between the Bio-Plex assay and ELISA was evaluated by measuring 22 samples for four cytokine targets (IL-5, IL-10, IL-13, and IFN-γ; see table). The concentrations of the majority of samples measured by ELISA compared well with concentration
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