Cytokines are small soluble proteins or peptides that play a crucial role in modulating the immune response. They are produced by immune cells, including B cells, T cells, mast cells, fibroblasts, stromal cells, and macrophages. Cytokines regulate the maturation of the immune cell population, modulate the humoral and cellular immune response, and are vital mediators of several cell signaling pathways. Hence, understanding the working and mechanism of cytokines is crucial to discovering insights into the working of disease and pathological processes.
Luminex multiplex assays have emerged as robust tools for analyzing cytokines in complex biological matrices. Because of its multiplexing capacities, they can measure and quantify multiple analytes in single sample volumes. This platform can monitor and compare cytokines within and between experiments and provide a comprehensive picture of disease mechanisms in individual patients. Hence, the current article highlights the advantage and limitations of Luminex multiplex assays to detect cytokines in biological samples.
Luminex-based assays have several advantages over traditional elisa immunoassay. The primary benefit of Luminex multiplex assay is its ability to analyze multiple analytes in a single assay volume. Conceptually, each Luminex microsphere is an ELISA on a bead. This multiplexing ability means Luminex assays can analyze up to 100 analytes in a single well. Each microsphere is an individual test. Besides, the small surface area and three-dimensional structure facilitate faster reaction kinetics, and assays are completed in a short period compared to conventional ELISAs.
Luminex ELISA multiplex assays have the flexibility to attach customized probes to the beads. This feature helps Luminex assays to adapt to specific mutations observed in particular populations, making it an ideal screening tool. Besides, Luminex assays require only 50 μl of sample volume per well, significantly reducing the sample volume required for analysis. Thus, valuable biological samples are conserved while large volumes of data are generated.
Luminex assays offer high-throughput and highly-dense analyses to yield reliable, reproducible, and accurate results. . Although PCR and ELISA can provide high-throughput analysis, they cannot analyze more than one analyte at a time. On the other hand, microarrays screen several tests at a time but lack the reproducibility required for high-throughput analysis. Thus, Luminex assays have several advantages in drug discovery and drug development projects. However, there are some limitations to the utility of Luminex assays. Let us now focus on the disadvantages of Luminex multiplex assays.
Limitations of using Luminex multiplex assays at different stages of drug discovery and development
Luminex multiplex assays require a dedicated platform similar to flow cytometers. This requirement increases the upfront costs for acquiring Luminex systems. However, instead of laser-based units, researchers can use light-emitting diode-based units to decrease the initial cost of Luminex multiplex assays.
Also, some research suggests that Luminex assays are subjected to variability due to product lot number, assay manufacture, and assay execution. However, researchers can use the same manufacturer and maintain consistency to reduce assay variability.
Furthermore, human autoantibodies present in biological samples may cause nonspecific binding and hinder the performance of Luminex cytokine assays. Although, novel methods are available to extract these autoantibodies and maintain the integrity of Luminex testing.