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The 5 best tips for
successful multiplex cytokine analysis 

by Alex Szabo, Ph.D.

Multiplex cytokine analysis offers enormous cost and time advantages over ELISA and westerns, which is why so many labs are adopting these powerful methods. 

As Australia’s leader in multiplexing technology, I am pleased to share with you our 5 very best tips to ensure your success in cytokine multiplexing.

Cytokine Multiplexing Technology

Most platforms for multiplex cytokine analysis are based on the traditional sandwich ELISA with the addition of an extra element to distinguish multiple analytes in a single reaction vessel. In the Luminex method, antibodies specific for different cytokine analytes are coupled to different colored beads. The cytokine concentration in a sample can be determined by measuring the fluorescence intensity of a detection antibody labeled with phycoerythrin.

5. Overnight sample incubations are better

For researchers seeking to maximize the sensitivity of their assays, our first suggestion is a simple one. Standard protocols for most multiplex assays usually recommend a 2-hour incubation of the samples with the primary antibody reagent (either beads or arrays). However, our view is that this should be considered a minimum incubation period. Sensitivity can be enhanced by allowing the binding of the analyte to reach equilibrium. To ensure this is the case, we strongly recommend researchers to carry out their sample incubations overnight. In our experience, this can increase the limit of detection of multiplex assays by severalfold. And what could be more efficient than enhancing your result while you catch some Zs?

4. Reserve samples for comparison in future experiments

Researchers often want to carry out comparisons of data obtained at different time points, such as in longitudinal clinical trials. To minimize inter-assay variation, it is desirable, wherever possible, to utilize the same lots of kits. An additional measure that can greatly bolster confidence in your data is to reserve a few samples to include in each group of samples being run. Ideally, the calculated values for all targets will closely track over each analysis. If they do not, it can be an indication that the samples are compromised or the performance of the kits has changed in some manner.

3. Research the expected concentrations in your samples

One of the most common tech support questions we receive goes something like this: “I expected the cytokine levels in these samples to be much higher than they are reading. Is there something wrong with the assay?” Based upon their understanding of the biology of a system, scientists often expect that there will be significant changes in cytokine in a given experiment, but don’t have specific values in mind. With so much data provided by multiplex assays, there is an increased likelihood that your measured values of particular cytokines will clash with expectations, creating some uncertainty – is that the real value, or something wrong with the method? To place your results in context, we strongly recommend a thorough literature review to determine the values other researchers have obtained in similar samples or cell types. Unfortunately, there is no good single reference for cytokine levels in different media and with different methods.

2. Include spike controls to measure recovery

How do you know if the measured concentration of a cytokine in a sample is the actual concentration? As is the case for all other immunoassays, the measured concentration in a multiplex assay may be either greater than the actual concentration due to cross-reactivity with other compounds in the sample, or less than the actual concentration due to interference. The difference between the actual and the measured concentration is called the recovery. Immunoassay kit manufacturers attempt to validate their kits in common media such as serum, plasma and tissue culture supernatants, but it is impossible to guarantee that these tests will transfer to your own samples. To address this, we strongly recommend that you include spike controls in your experiment. To do this is simple: Spike some of the standards into your kit into samples (preferably normal samples with low cytokine levels). Then compare these values to your standard curve and the identical, unspiked samples. The ratio of these values, represented in percent, is the recovery.  Ideally, recovery should be between 80-120%. 
For more detail on determining recovery,
we are happy to send you a copy of

Practical Guide for Immunoassay Validation” by Andreasson et al. 

1. Choose the right multiplexing format (Luminex or Array)

Lastly, the most important one – choose the right assay format. Luminex’s bead-based technology is probably the best known, but slide-based formats also exist. Both methods offer similar sensitivities, but there are other features that create important distinctions. If you choose the right format to suit your research aim, you can obtain significantly more data with less work and cost. 

  1. Luminex Pros: Automated data acquisition and analysis. The biggest advantage of Luminex is automation, which simplifies the process and reduces the work burden.  Luminex kits are available as 96 well plates suitable for 40 unknowns in duplicates.  And both the sample processing and data analysis are highly automated. So if you have hundreds of samples, Luminex is the way to go.
  2. Luminex Cons: Equipment required. Luminex requires a specialized reader – called a Magpix or Luminex 200.  So it requires an upfront capital cost.  There’s also typically training required, as well as time for a technician to manage the instrument.  Arrays also require a scanner for data acquisition.  However, this type of instrument has many applications and typically can already be found in most genomics core facilities.
  3. Array Pros: Large selection of targets available.  Arrays are an open-source platform.  As a result, the number of targets available for purchase in the array format is significantly larger than on Luminex beads.  So if you are working with “exotic” targets, other than common interleukins, they are much more likely to be available on arrays.  More economical for small sample numbers.  A second advantage is that array kits are typically available for as few as 2-8 samples (as opposed to 40.  So if you have an experiment where you want to look at changes in the expression of many proteins in just a few conditions or samples, arrays are likely to be the cheaper way to do the work.
  4. Array Cons: Manual sample handling. There is currently no automated sample handling instruments for the wet-lab steps in using protein arrays.  So the incubation and washing steps, which are similar to an ELISA, need to be performed by hand.  As a practical matter, the number of samples a researcher might practically process in a day is probably limited to approximately 60.


When to choose Luminex – Assuming you have access to a Luminex instrument, if you have more than 30-40 samples (and kits the targets you are interested are available) you would probably find it easier to use Luminex.

When to choose arrays – If you are working with fewer than 30-40 samples or unusual targets, you will likely find arrays to be more economical, if not your only option.

If you would like to learn more about multiplexing, please check out this episode of our video series, “At the Bench