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), A² (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 (10⁵ to 10⁶). 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.