An evaluation of commercial fluorescent bead-based luminex cytokine assays

Abstract

The recent introduction of fluorescent bead-based technology, allowing the measurement of multiples analytes in a single 25-50 microl sample has revolutionized the study of cytokine responses. However, such multiplex approaches may compromise the ability of these assays to accurately measure actual cytokine levels. This study evaluates the performance of three commercially available multiplex cytokine fluorescent bead-based immunoassays (Bio-Rad's Cytokine 17-plex kit; LINCO Inc's 29-plex kit; and RnD System's Fluorokine-Multi Analyte Profiling (MAP) base kit A and B). The LINCO Inc kit was found to be the most sensitive assay for measuring concentrations of multiple recombinant cytokines in samples that had been spiked with serial dilutions of the standard provided by the manufacturer, followed respectively by the RnD Fluorokine-(MAP) and Bio-Rad 17-plex kits. A positive correlation was found in the levels of IFN-gamma measured in antigen stimulated whole blood culture supernatants by the LINCO Inc 29-plex, RnD Fluorokine-(MAP) and RnD system IFN-gamma Quantikine ELISA kits across a panel of controls and stimulated samples. Researchers should take the limitation of such multiplexed assays into account when planning experiments and the most appropriate use for these tests may currently be as screening tools for the selection of promising markers for analysis by more sensitive techniques.

Figure 1. Recoveries of the Bio-Rad 17-plex assay.

Un-stimulated whole blood culture supernatant samples from healthy donors were spiked at three (experiment 1) and five (experiment 2) different concentrations (2–8062 pg/ml) with the standard from the Bio-Rad 17-plex kit. Samples were assayed in duplicate and read at high and low RP1 targets on the Bio-plex system instrument. Recoveries were calculated for each of the cytokines in the panels for each of the spiked concentrations. The figure shows the recoveries obtained for each individual cytokine after two independent experiments.

Figure 2. Recoveries of the Linco 29-plex assay.

Un-stimulated whole blood culture supernatant samples from healthy donors were spiked at five (experiment 1) and two (experiment 2) different concentrations ranging from 10–5000 pg/ml with the standards from the LINCO 29-plex kit. Samples were assayed in duplicate and read at high and low RP1 targets on the Bio-plex system instrument. Recoveries were calculated for each of the cytokines in the panels for each of the spiked concentrations. The figure shows the recoveries obtained for each individual cytokine after two independent experiments.

Figure 3. Recoveries of RnD System's Fluorokine-MAP 13-plex base kits A and B.

(A) Un-stimulated whole blood culture supernatant samples from a healthy donor were spiked at different concentrations (14–19000 pg/ml) with the standards from RnD-System Fluorokine-MAP base kits A and B. Samples were assayed in duplicate, read at a low RP1 target on the Bio-plex system instrument and recoveries calculated for each of the cytokines in the panel. The figure shows the individual cytokine's best recovery obtained. (B) Serum samples from a healthy donor were spiked at concentrations (43.8–19000 pg/ml) with the standards from RnD-System's Fluorokine-MAP base kits A and B. Samples were assayed in duplicate, read at a low RP1 target on the Bio-plex system instrument and recoveries calculated for each of the cytokines in the panel. The figure shows the individual cytokine's recoveries obtained. The experiment was repeated for IFN-γ, ΤΝF-α and IL-4 only.