Comprehensive Guide For Luminex Multiplex Immunoassay Tips And Techniques

Comprehensive Guide For Luminex Multiplex Immunoassay Tips And Techniques

Comprehensive Guide For Luminex Multiplex Immunoassay Tips And Techniques

Multiplex assays allow simultaneous examination and quantification of multiple biological analytes in a single sample volume. Luminex has come up with several multiplex assays for all the disciplines of biological sciences, such as multiplex cytokine assays for the discovery and study of the immune system. Besides Luminex cytokine assays, Luminex analysis includes biomarkers, chemokines, metabolites, etc.

Simultaneous analysis of multiple analytes provides a broad range of benefits. It maximizes the limited amount of study sample, minimizes any experimental variability, optimizes productivity, and reduces time and resources. Moreover, if novel analytes or panels are needed, they develop custom Luminex assays, which are specifically tailored to meet all customer requirements.

What’s the technique behind Luminex multiplex immunoassay?

A Luminex multiplex immunoassay is a magnetic microsphere-based assay format that follows similar ELISA principles. Compared to traditional ELISA validation assays, researchers can quantify up to 100 analytes in lesser sample volumes. Luminex assays employ color-coded beads with discrete proportions of red or infrared fluorophores, each corresponding to a distinct bead region. Color-coded microspheres help quantify several biomarkers and cytokines in biological samples and provide a deeper understanding of the underlying disease processes.

Analyte-specific antibodies are conjugated to a unique bead region and then incubated with the study sample. After all unbound materials are washed away, a mixture of streptavidin-phycoerythrin reporter molecules and biotinylated detection antibodies are incubated with the study samples. The Luminex laser unit excites the microspheres to identify the bead region and its corresponding conjugated analyte. Another laser unit determines the signal generated by the reporter molecule. The generated signal is proportional to the concentrations of analyte bound.

Tips for Luminex multiplex immunoassays

Luminex samples generally need a dilution of a minimum of 2-fold. However, a higher volume of biomarkers may need additional sample dilution to bring the samples within the dynamic assay range. The extent to which a study matrix is validated depends on the type of Luminex assay. Not all sample types are validated in Luminex immunoassays. Generally, hemolyzed, lipemic, and icteric study samples are not suited for Luminex immunoassays.

Unless reagents are required to be kept cold, all reagents should be brought to room temperature before using them for experiments. Ideally, multichannel pipettes should be used to facilitate rapid experiments and consistent results. Avoid touching the bottom while pipetting the mixtures. Although changing pipette tips are not necessary between replicates, it is recommended to change them between standards or different samples to avoid contamination.

Whenever possible, use an automatic plate washer for consistent assay results. While washing, ensure that the buffer gets enough time of at least 30 seconds for soaking between washes. As a general rule, the incubation period should not deviate more than +/- 5 mins/hr of the incubation time. On the day of performing any assay, researchers should centrifuge all frozen and fresh assay samples before using them in the experiment.

The road ahead

Luminex multiplex assay has become the go-to technique in the analysis of multiple analytes in biological sciences. Nevertheless, future advancements in miniaturization and parallel processing are only going to skyrocket the use of multiplex assays.

The post Comprehensive Guide For Luminex Multiplex Immunoassay Tips And Techniques first appeared on Tekrati and is written by mariabrownema


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