Luminex assays are magnetic bead-based immunoassays employing similar principles to sandwich ELISA assays. Luminex assays are multiplex assays that can simultaneously analyze up to 100 analytes with less sample volume compared to traditional ELISAs. They employ color-coded beads dyed with different amounts of red and infrared fluorophores. As Luminex ELISAs can quantify multiple analytes, Luminex multiplex cytokine assays and Luminex protein assays have become crucial in understanding biological and disease processes.
Although Luminex assays are similar to sandwich ELISAs, they have unique requirements. In the current article, we share a complete guide to setting up an accurate Luminex ELISA assay for several clinical drug development studies.
Working and Principle of Luminex ELISA assay
First, researchers mix the study sample with color-coded beads that are coated with capture antibodies specific to the analyte of interest. Once the antibodies bind to the analytes, biotinylated detection antibodies are added to form an antibody-antigen sandwich. Later phycoerythrin-conjugated streptavidin reporter molecules are added that bind to the detection antibodies.
Magnetic beads are read on dual-laser flow-based systems such as FlexMap analyzer and Luminex 200. One laser in the dual-laser system classifies and determines the analyte, whereas the second laser deduces the magnitude of the PE-streptavidin signal. This generated signal is directly proportional to the amount of bound analyte. Other than flow-based analyzers, researchers can also use Luminex MAGPIX analyzers. This analyzer uses a magnet to capture and hold the beads while illuminating them through two different LEDs. One LED detects the analyte, while the other determines the magnitude of the PE-streptavidin signal.
Sample preparation is crucial for Luminex ELISA assays. Depending on each drug development pathway, there may be different samples for analysis. However, Luminex ELISAs need a minimum 2-fold dilution. Besides, highly abundant biomarkers may need additional dilutions. Although Luminex supports several biological samples, they are not suitable for analyzing icteric, lipemic, and hemolyzed samples. Below we share sample preparation for a range of biological matrices.
- Plasma: Use heparin or EDTA as an anticoagulant while collecting plasma. It is necessary to centrifuge the plasma within 30 mins of collection, for 15 mins at 1000*g.
- Cell culture supernates: Centrifuge the samples to remove particulates.
- Tissue Lysate: Rinse the tissue, cut it into smaller pieces and homogenize it in PBS. Add cell lysis buffer to lyse at room temperature. Finally, centrifuge to remove debris.
- Serum: Allow the sample to clot at room temperature. Centrifuge for 15 mins at 1000*g and remove the serum.
- Saliva: Centrifuge the saliva for 5 mins at 10,000*g and collect the aqueous section for analysis.
- Urine: Collect the first-morning urine (mid-stream) into a sterile container. Remove particulate matter by centrifuging for 4 mins at 16,000*g.
- Human milk: Analyze the sample immediately or store them below -20℃.
- Platelet-poor plasma: Use heparin or EDTA to collect plasma and centrifuge at 1000*g for 15 mins within 30 mins of collection. Additional centrifugation for 10 mins at 10000*g is recommended to remove the platelets.
Hence, Luminex assays are ideal for several biological matrices. However, it is necessary to note that Luminex assays have not been validated against all study samples, and researchers should verify them before using any specific Luminex kit.