Revolutionizing multiplexed protein profiling: dual-reporter capabilities of Luminex xMAP^® technology

Luminex® xMAP® Technology is a flow cytometry-based multiplexing platform that utilizes colored magnetic microspheres (beads) coupled with target-specific molecules such as antigens, antibodies, receptors, enzyme substrates, as well as oligonucleotides. This technology facilitates the simultaneous detection of up to 500 analytes in a single reaction. The xMAP® Technology platform offers a wide range of preconfigured or custom applications, delivering results with sensitivity and reliability comparable to conventional singleplex assays like ELISA, western blot, and qPCR, but with greater efficiency and throughput. Consequently, it has become an invaluable tool in fields such as proteomics, genomics, clinical diagnostics, and drug discovery.

Here we introduce the xMAP INTELLIFLEX® System, featuring innovative dual-reporter capabilities, as a novel tool for multiplexed protein profiling applications. This system enables the detection of two parameters per analyte on the same bead in a single well. The dual-reporter functionality has been successfully implemented in antibody isotyping assays to monitor immune responses to specific infections or vaccinations, facilitating the detection of two immunoglobulin (Ig) isotypes for multiple antigens simultaneously. Examples include the concurrent detection of IgG/IgM, IgA/IgM, and IgG/IgA responses to various pathogens, such as multiple Borrelia species (Häring et al. 2023), SARS-Cov-2 (Fhied et al 2022), Epstein-Barr Virus (Schrieber et al. 2023) and Neisseria gonorrhoeae (Stover et al. 2024). In addition to monitoring antibody titers, dual reporter assays can also determine antibody neutralization potential, as demonstrated by the ability of the ACE2 receptor to block antibody binding to S and RBD (Cameron et al. 2022).

The dual-reporter capabilities extend to various assay formats where simultaneous measurements of analyte pairs are critical. Examples include analyzing post-translational modifications, such as phosphorylation in protein signaling pathways, and distinguishing between free and bound drug forms in pharmacokinetic/pharmacodynamic (PK/PD) studies. Additionally, dual-reporter assays can effectively characterize bi-specific/fusion proteins, used for profiling of extracellular vesicles, and bioparticles, like viral particles (Sahi et al. 2024), and detect mutant proteins in cell and tissue lysates. Moreover, xMAP® dual-reporter capabilities facilitate protein-protein interaction studies, allowing determination of interaction affinities or execution of competition experiments. Notably, this latter application has been proven sensitive in assessing vaccine-induced antibody blockade of PD-1/PD-L1 interactions (Overholster et al. 2023).

In conclusion, Luminex xMAP® Technology, combined with intelligent workflow automation, revolutionizes protein identification and characterization. It elevates proteomics research in your laboratory by maximizing the number of results while minimizing sample input.