Weighing up the advantages and disadvantages of multiplex assays

With the need to increase analytical throughput in biotherapeutic R&D and cell and gene therapy, multiplexing assays can appear to be an obvious approach. There are certainly potential advantages, but also disadvantages and considerable challenges involved in multiplexing immunoassays – assays must be carefully matched to function in the same buffer and in the same analytical range. An alternative approach – to miniaturize singleplex ELISAs and run them in parallel – may be even more attractive.

To multiplex or not to multiplex, that is the question

When it comes to generating data as quickly as possible for different analytes in a single sample, multiplexing immunoassays looks like a given solution. The possible advantages of multiplexing are as follows:

+       More data points/unit sample

+       Reduced cost/data point

+       Less sample handling means less error

+       Fewer wells and/or plates to handle

+       Increased throughput

It is certainly true that a well-designed multiplex immunoassay can deliver, but there are many hurdles to overcome before the advantages of multiplexing can be realized. In the end it can be more productive to run time- and volume-efficient to run singleplex assays in parallel. Let’s look at the downside of developing multiplex immunoassays.

- Cross-reactivity

As we saw in a companion blog post (Solutions to immunoassay interference, cross reactivity and other challenges), the performance of an immunoassay is only as good as the specificity of the component antibody reagents – where specificity is the ability of each antibody to distinguish between the analyte and other structurally similar components. Identifying antibodies with high specificity is a challenge. Doing this for multiple analytes that are to be analyzed in the same assay is even more difficult and time-consuming. Cross-reactivity, which is the opposite of specificity, occurs when an antibody raised against one specific antigen binds to a different antigen (non-target proteins) in the matrix. This issue is ubiquitous and widespread for antibodies and is one of the biggest obstacles to developing high performance multiplexed immunoassays (1).

- Dynamic range

As assay panels often contain targets that are present at radically different concentrations, linearity over a wide range is essential for reliable data. ELISA is known to have a dynamic range that is limited to a few orders of magnitude, which means that multiplexing ELISA’s to cover the range of individual analytes can be a real challenge. Having to dilute the sample two or three times to get everything in range defeats the purpose of having a multiplex assay.

- Assay development time

The decision to develop a multiplex assay involves careful consideration of the balance between development time and the time required to develop and run singleplex assays efficiently and in parallel to get the same level of reliable, robust data. With multiplex assays, the number of parameters can increase exponentially, and assays must be carefully matched to function in the same buffer, in the same analytical range, and without losing selectivity, precision or accuracy.

- Commitment to vendor-supplied assays

The shear complexity of multiplex assays means that they can take considerably longer to develop than singleplex assays, and it is for this reason that researchers regularly turn to external providers rather than developing such assays in house. This in turn limits the possibilities of meeting specific in-house analytical challenges for specific analyte combinations.

Gyroplexing on an open platform – a unique alternative

Having considered the downsides of developing multiplex immunoassays, let’s look in turn at each of the potential advantages of multiplexes that were so attractive in the beginning. This time through the lens of an automated miniaturized immunoassay platform:

1. More data points/unit sample
   Miniaturization reduces the sample consumption, which means more data points per microliter of precious sample. Running singleplex assays for different analytes on the same sample at the same time means that you can get all the data you need at once.
2. Reduced cost/data point
   Miniaturization reduces reagent consumption, which is a considerable cost. Add to that factors that greatly reduce hands-on costs, such as broad dynamic assay ranges that reduce repeats, and automation. And developing assays on the platform we have in mind is well known to be much faster (and therefore less costly) than, for example, what is required to develop an ELISA.
3. Less sample handling means less error
   Automation, in combination with a system that defines volumes through precision microfluidics greatly reduces error.
4. Fewer wells and/or plates to handle
   Automation solves this problem as well.
5. Increased throughput
   Automation again. And the possibility to do overnight runs can boost throughput to approximately 1700 data points/24 h.

All this is possible using the Gyrolab platform, which runs multiple singleplex assays in parallel with no cross talk to deliver accurate high-precision data for multiple analytes with minimum sample and reagent consumption. We call it Gyroplexing.  This approach also offers several more advantages:

• There is no need to compromise in assay performance when running Gyroplex assays compared to regular Gyrolab assays.
• Capture concentration is not reduced since only part of the surface area or fraction of the beads are functionalized with a particular antibody, in contrast to a multiplex assay.
• Different buffers can be used for different assays.
• The wide dynamic range of Gyrolab assays enables the same sample dilution to be used for several analytes.
• Gyroplex assays can be modularized by adding or removing assays in the panel depending on specific analytical needs.
• Increased throughput can be achieved by running 5 CDs (up to 112 datapoints each) with 3 runs in a day (including overnight).

And the Gyrolab platform is open to development of in-house assays, which means your analytical possibilities are only limited by your imagination and access to suitable reagents.

 References:

1. Cross-reactivity in antibody microarrays and multiplexed sandwich assays: shedding light on the dark side of multiplexing. Juncker, D. et al. Current Opinion in Chemical Biology 2014, 18:29–37.