Comparison of multiplex immunoassay platforms

Abstract

Background: Candidate biomarkers discovered with high-throughput proteomic techniques (along with many biomarkers reported in the literature) must be rigorously validated. The simultaneous quantitative assessment of multiple potential biomarkers across large cohorts presents a major challenge to the field. Multiplex immunoassays represent a promising solution, with the potential to provide quantitative data via parallel analyses. These assays also require substantially less sample and reagents than the traditional ELISA (which is further limited by its ability to measure only a single antigen). We have measured the reproducibility, reliability, robustness, accuracy, and throughput of commercially available multiplex immunoassays to ascertain their suitability for serum biomarker analysis and validation.

Methods: Assay platforms MULTI-ARRAY (Meso Scale Discovery), Bio-Plex (Bio-Rad Laboratories), A(2) (Beckman Coulter), FAST Quant (Whatman Schleicher & Schuell BioScience), and FlowCytomix (Bender MedSystems) were selected as representative examples of technologies currently used for high-throughput immunoanalysis. All assays were performed according to protocols specified by the manufacturers and with the reagents (diluents, calibrators, blocking reagents, and detecting-antibody mixtures) included with their kits.

Results: The quantifiable interval determined for each assay and antigen was based on precision (CV < 25%) and percentage recovery (measured concentration within 20% of the actual concentration). The MULTI-ARRAY and Bio-Plex assays had the best performance with the lowest limits of detection, and the MULTI-ARRAY system had the most linear signal output over the widest concentration range (10(5) to 10(6)). Cytokine concentrations in unspiked and cytokine-spiked serum samples from healthy individuals were further investigated with the MULTI-ARRAY and Bio-Plex assays.

Conclusions: The MULTI-ARRAY and Bio-Plex multiplex immunoassay systems are the most suitable for biomarker analysis or quantification.

Fig. 1. Calibration curve performance and comparison of MULTI-ARRAY (blue), Bio-Plex (green), A² (light blue), and FAST Quant (red) multiplex assays

The following cytokine kits were compared: Human Pro-Inflammatory 9-Plex MULTI-ARRAY kit [granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon γ (IFN-γ), IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12p70, tumor necrosis factor-α (TNF-α)] from Meso Scale Discovery; Bio-Plex Human Cytokine 27-Plex Panel [IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12p70, IL-13, IL-15, IL-17, eotaxin, basic fibroblast growth factor, granulocyte colony-stimulating factor, GM-CSF, IFN-γ, IFN-γ–induced protein 10 (IP-10), monocyte chemoattractant protein 1 (MCP-1/MCAF), macrophage inflammatory protein 1α (MIP-1α), MIP-1β, platelet-derived growth factor BB (PDGF-BB), RANTES (regulated upon activation, normal T cell expressed and secreted), TNF-α, VEGF (vascular endothelial growth factor)] from Bio-Rad Laboratories; A² human 13-cytokine assay kit (GM-CSF, IFN-γ, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12p70, IL-13, TNF-α) from Beckman Coulter; FAST Quant Human II Kit 10-plex assay (IL-1β, IL-10, IL-2, IL-12p70, IL-4, GM-CSF, IL-6, RANTES, IL-8, MCP-1) from Whatman Schleicher & Schuell BioScience. (A), Log plots of raw IL-6 data (fluorescence signal intensity vs IL-6 concentration). (B), Corresponding plot of CV vs IL-6 concentration. The CV was calculated from 2 spots (2 wells in MULTI-ARRAY), 8 spots (2 pads in FAST Quant), 12 spots (3 wells in A²), and 2 wells (in Bio-Plex). (C), Plot of percentage calibrator recovery vs IL-6 concentration. A 4-parameter algorithm and a 1/y² weighting function were used to fit (with Prism software) each data set from the MULTI-ARRAY, Bio-Plex, A², and FAST Quant systems to a sigmoidal dose–response curve with a variable slope. Curve back-fitting is done by calculating the observed concentration of each calibrator and then comparing it with the original concentration (expected concentration) used in each analyte assay. For these tests, actual and expected concentrations were calculated, and the percentage recovery of calibrator was calculated as: [(observed concentration)/(expected concentration)] × 100.