Multiplex assays are crucial drivers for accelerating drug development timelines. Particularly, multiplex assays such as Luminex assays have helped pharmaceutical drug development complete projects rapidly. For example, multiplex cytokine assays have revolutionized how researchers analyze complex signaling pathways associated with disease and medical conditions.
Luminex Multiplex Bead assays employ microspheres to analyze multiple target analytes in a single assay sample. These bead-based techniques can quantify upto 100 analytes simultaneously in study samples. Such a robust high-throughput analysis is a sure path to excellence this coming year. Hence, the current article is a guide for multiplex and Luminex assays. We shall first understand the working and instrumentation of Luminex assays and then continue with tips for a robust Luminex assay.
The working of Luminex assays
As mentioned above, Luminex systems are bead-based assays that can quantify multiple analytes in complex biological matrices. Color-coded microspheres are dyed distinctly with varied proportions of infrared and red fluorophores. Each fluorophore represents a distinct bead region and gives a unique spectral signature. Detecting and quantifying multiple cytokines from a single sample can help scientists decipher crucial information about biological processes.
Researchers couple specific antibodies to a bead region and incubate the mixture with the study sample. Post washing, biotinylated detection antibodies and PE-reporter molecules are introduced into the reaction mixture. A Luminex instrument is then used to excite the beads. This system consists of two lasers; one excites the bead region while the other measures the PR-derived signal. This signal is proportional to the analyte present in the sample.
Luminex has several platforms for multiplexing biological samples. These platforms include FLEXMAP 3D, xMAP INTELLIFLEX, and Luminex 200 instruments. All three instruments are based on the principle of flow cytometry. The instrument injects the sample in flow through a sheath fluid, where a red laser identifies the bead region and another red laser quantifies the analyte. The instrument components include optics, lasers, high-speed digital processors, and advanced fluidics.
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Tips and tricks for a robust Luminex assay
Both the plasma and serum may provide different measurements. This difference is because plasma protein concentration is approximately 3-4% more than serum protein concentration. Besides, the presence of fibrinogen and other proteins makes serum and plasma qualitatively different. Hence, researchers must choose an ideal matrix where the measurements fall within the dynamic range and provide better accuracy and precision. A pro tip – don’t use the serum for hemolysis or gross lipemia.
Besides, plan your experiments. Planning will ensure all reagents are available till the end of the experiment. Ensure consistency among all experimental runs. Consistency can be achieved by following some basic rules.
Some of these rules are outlined below.
- Use low protein tubes
- Use multichannel pipettes
- Time all experimental runs
- Dilution tubes are better for serial dilution
- Employ the reverse pipetting technique
- Resuspend beads thoroughly
- Ideally, prepare reagents in excess
- Always run in replicates.
Notably, the Streptavidin-PE complex is light-sensitive. Hence, always protect the reagents from sunlight. Also, use foil paper to cover the plates while shaking.
