REVERSE ELISPOT ASSAY FOR CLONAL ANALYSIS OF CYTOKINE PRODUCTION .2. ENUMERATION OF INTERLEUKIN-1-SECRETING CELLS BY AMPLIFIED (AVIDIN-BIOTIN ANTIPEROXIDASE) ASSAY

Detection of cytokine-producing cells can be accomplished by reverse modifications of the ELISPOT assay using cytokine-specific unconjugated and enzyme-labelled antibodies as solid phase capture system and detecting reagents, respectively. However, in certain situations where the secreted cytokine is produced in minute amounts such as in the case of interleukin-1 (IL-1), the sensitivity of the indicator immunoenzyme system employed may be insufficient to permit detection of the corresponding secreting cells. We have developed a novel immunoenzyme amplification procedure that involves the use of a biotinylated secondary anti-enzyme antibody reagent to enhance the signal provided by the primary enzyme-labelled antibody conjugate. Following addition of enzyme-conjugated avidin, ELISPOT assay wells are developed with a suitable chromogen substrate yielding spots located at the former position of cells secreting the analyte under study. As a model system, the detection of IL-1-beta-secreting cells by human peripheral blood monocytes is described.