Multiplexed fluorescent bead-based immunoassays for quantitation of human cytokines in serum and culture supernatants

Abstract

Background: An increasing volume of data suggests a relationship between cytokine levels in human body fluids and disease pathogenesis. Traditionally, many individual assays would be performed to measure the large number of known cytokines and determine their associations with disease. A new technique for the simultaneous measurement of multiple cytokines in cell culture supernatants by fluorescent microsphere-based flow cytometry was adapted to human sera. Methods: Multiplexed sandwich immunoassays for eight cytokines were developed by coupling cytokine-specific capture antibodies to beads with different emission spectra. The binding of biotinylated detection antibodies bound with a streptavidin-conjugated fluorochrome was analyzed. Recovery of "spiked" cytokines, sensitivity, and variability of the assays were evaluated. In addition, the results of the bead assays were compared with the results of commercial enzyme-linked immunosorbent assays (ELISAs) that used the same antibody pairs. Results: Correlations of the bead assays and the ELISAs were 0.974 (n = 18) for supernatant samples and 0.859 (n = 28) for serum samples. High, false-positive values observed with some sera, assumed to be produced by heterophilic antibodies, were reduced by preincubation with a cocktail of animal sera. Conclusions: Fluorescent bead-based immunoassays can be used to quantitate multiple cytokines in human sera and contribute to an understanding of the role of cytokines in disease processes. This methodogy is applicable to many combinations of purified analytes and high-affinity antibodies.