7 Key Questions To Ask While Choosing A Luminex Multiplex Assay

Luminex assays are immunoassays capable of measuring multiple analytes in a single study sample. Luminex multiplex assay employs bead-based technology with capabilities of simultaneous detection of up to 100 analytes in a given study sample. Luminex ELISA comprises internally dyed color-coded beads in different proportions of infrared and red fluorophores. With Luminex cytokine alternatives, Luminex cytokine assays can quantify multiple cytokines in a given sample for evaluating critical diseases and biological processes. Discrete antibodies coupled to a specific bead region are incubated with the study samples. Post the initial washing step, a mixture of streptavidin-phycoerythrin reporter molecule and a biotinylated detection antibody is incubated with the study sample. The Luminex instrument emits a laser to excite the color-coded bead, whereas another laser estimates the magnitude of the reporter molecule. The estimated magnitude is proportional to the amount of bound analyte. Numerous Luminex assay kits are available for research purposes. Hence, choosing the right option may require a prerequisite understanding of these assays. Following are the seven key questions to ask while selecting a Luminex multiplex assay. Will samples stay in the dynamic range? The dynamic range should incorporate the expected assay values. If the assay contains endogenous samples, it is better to have a lower concentration range. Whereas for samples with disease state models, dynamic range towards higher concentration will be more appropriate for the assay. When the assay evaluates both kinds of samples, a broader dynamic range is suitable for the assay.

What will be the assay sensitivity?

As the level of endogenous analytes can be very low, the assay sensitivity should be high enough to detect low levels of endogenous samples.

What is the level of kit accuracy?

For accuracy data, explore the vendor-provided data that is available with all Luminex assay kits. For a spike and recovery experiment, 80-120% is the industry standard.

What about intra-assay and inter-assay precision?

Intra-assay and inter-assay precision and accuracy are the most vital aspect of any assay. Whether with one plate or multiple plates, all results must be precise and accurate. Any observed variation should solely be because of treatment effects.

What is lot-to-lot variability? How is it assessed?

As ensuring accuracy, reliability and consistency depend on lot-to-lot variability, it is crucial to evaluate lot-to-lot variability while performing preclinical and clinical studies. This is particularly crucial with large numbers of samples or during pre-clinical studies.

How much is cross-reactivity?

The multiplex analysis involves studying several analytes in one assay. Multiple analysis can result in cross-reactivity among detection or capture reagents providing inaccurate results. Vendor-provided data should mention any cross-reactivity, and high cross-reactivity kits should ideally not be used for analysis.

How does kit influence matrix effects?

All study matrices such as serum, plasma, tissue homogenates, and cell culture media can hinder the assay performances. Hence, kits providing a buffer solution to overcome these effects or kits providing mitigation strategies should be chosen for analysis.

Final thoughts

With the increase in Covid-19 infections, researchers are in the hunt for multiplex assays capable of evaluating the Covid cytokine storm. A complete understanding of study requirements will help researchers choose an ideal Luminex multiplex assay for analyzing study samples.