A useful guide for analysis of immune markers by Fluorochrome (Luminex) technique

Abstract

The fluorochrome Luminex technique is a bead-based sandwich immunoassay that combines the enzyme-linked immuno sorbent assay (ELISA) with flow cytometry. The Luminex technique allows multiple cytokines to be measured simultaneously in small volumes and provides a convenient and sensitive tool for the detection of a large number of, e.g., extracellular secreted cytokines to characterize cytokine profiles. The technique is based on the so-called microspheres (beads) that serve as a solid phase for molecular detection. These individually dyed micro-beads have monoclonal antibodies directed against the cytokines and chemokines of interest and allow simultaneous detection of up to nearly 100 cytokines and chemokines in a dual-laser flow analyzer. Immune markers can be detected in serum and plasma samples as well as in cell culture supernatants from in vitro-stimulated peripheral blood mononuclear cells (PBMC). This chapter describes the Luminex technique for detection of multiple cytokines by magnetic bead sandwich immunoassay, with a special focus on some important pre-analytical factors, such as cell separation, cryopreservation, and PBMC thawing that may affect the detection outcome of immune markers. This method can also be easily adapted to measuring other biomarkers in biological samples.