EliCell: a gel-phase dual antibody capture and detection assay to measure cytokine release from eosinophils

Eosinophils contain many preformed cytokines and chemokines, which are stor ed in specific granules along with cationic granule proteins. Mobilization and release of these granule contents can be selective and mediated by vesi cular transport. We have developed a sensitive method to detect and quantit ate eosinophil vesicular transport-mediated release of specific eosinophil proteins. Our EliCell assay is based on microscopic observations of individ ual viable eosinophils embedded in an agarose matrix that contains: immobil ized antibody to the protein of interest. Following stimulation of eosinoph ils, released protein is bound by the capture antibody at its site of relea se and is detected by a fluorochrome-conjugated detection antibody. We have validated this assay by evaluating interferon-gamma -induced release of RA NTES from eosinophils. Extracellularly released RANTES was visualized as fo cal immunoflourescent staining and was quantitated by scoring the numbers o f eosinophils releasing RANTES and by measuring the fluorescent intensity o ver individual eosinophils. In comparison with ELISA assays of RANTES relea sed into supernatant fluids by interferon-gamma -stimulated eosinophils, El iCell assays were more sensitive enabling detection of RANTES release at ea rlier times and at lower levels of interferon-gamma stimulation. The EliCel l assay provides a sensitive method to study the regulated release of eosin ophil-derived cytokines, chemokines and other granule proteins. (C) 2000 El sevier Science B.V. All rights reserved.