A Luminex assay is a magnetic bead-based immunoassay that utilizes principles similar to traditional sandwich ELISA assays. Color-coded beads are internally dyed with distinct proportions of fluorophores, each corresponding to a different bead region or spectral signature. With its multiplexing capacities, assays such as Luminex cytokine assays can detect and quantify up to 100 biomarkers in a single assay volume.. Evaluating multiple cytokines provides a comprehensive picture of disease and normal biological functioning.
Using Luminex ELISA multiplex assays has several advantages in the process of drug discovery and development. Luminex assays save time and money, are flexible, accurate, and provide an extensive range of analyte detection. Hence, the following are ten tips for creating an excellent Luminex cytokine assay and Luminex ELISA multiplex assay.
- Use multichannel pipettes for Luminex analysis. They can accelerate the plating process and provide consistent data. Hold the pipette at an angle while pipetting. Also, avoid touching the bottom. Although the same pipette tips can be used for the same replicate, change the pipette tip for different standards or samples to control contamination.
- Bring all reagents to room temperature before beginning with Luminex elisa assay, unless samples are needed to be kept cold.
- Controls and standards are for single-use. They should not be frozen or aliquoted.
- Researchers must opt for automatic plate washers for consistent results. During the washing step, whether manually or through a plate washer, add a 30-sec soak window to allow the wash buffer sometime between washes.
- Strictly follow incubation times. As a general rule, the incubation time must vary less than +/- 5 mins of incubation time.
- Before use or dilution, centrifuge all frozen and fresh samples at 16000 * g for 4 mins. This step must be performed on the day of the assay.
- Always run samples in triplicates or at least duplicates. This replication provides enough statistical data to validate the sample results. Average each reading for control, standard, and study sample and subtract the blank medical fluorescence intensity value.
- Precision is a crucial parameter for any bioanalytical assay. Precision is the ability of an assay to produce reproducible results between and within assays. There are two primary types of precision, inter-assayand intra-assay precision. The coefficient of variation is the measure of assay precision. Moreover, the coefficient of variation for Luminex ELISA assays must be ≤ 20%.
- False-positive results can be a significant challenge for the drug discovery process. A false positive means the assay has detected an undetectable analyte. Researchers must perform linearity experiments to identify false-positive results. While running back-calculation concentration and multiple dilutions, the sample values should be consistent. If the value increases with an increase in dilutions, the assay has issues with specificity. Hence, ensure linearity of dilution is used to assess false positive signals.
- Validation in the matrix of choice is of utmost importance while developing a Luminex ELISA assay. Generally, Luminex kits are validated for plasma and cell culture supernatants. Hence, if a different sample matrix is used in Luminex analysis, researchers must develop and validate the assay in their choice of a matrix.
