How-to-guide: Multiplexing your lateral flow assay

Learn what you need to consider before multiplexing your lateral flow assay.

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You have identified a need for a point of care detection assay, you know what your test medium is, what you are hoping to detect, and a lateral flow assay (LFA) fits the bill. You may even have developed an LFA to meet the initial need. But there’s still something missing. What if identifying multiple targets could vastly improve detection, sensitivity and specificity, or perhaps there are multiple causes or markers you wish to screen for simultaneously?

There are numerous reasons why combining detection of multiple targets within a single assay is desirable. This guide outlines some of the important factors to consider when developing a multiplex LFA, be it from scratch or building on previously developed singleplex assays.

Why would I want to detect multiple targets with a lateral flow assay?

If you are testing for targets that can be lost or mutated, which can be an issue with detection of pathogens, screening for multiple targets is highly desirable. Delivering a negative result only to find that it was just missing your target could have devastating consequences. Likewise, not everyone responds to and produces antibodies to all antigens within a bacterium, for example. By having multiple targets, the chances of false negative results are reduced.

Additionally, the sensitivity and specificity of a test designed to detect a single cause e.g. a bacterial species, can be greatly enhanced by detecting multiple targets.

For some samples, the available quantities can be limited.

Being able to test for multiple targets in a single assay may therefore mean extra information can be acquired rather than picking to test for one thing or another. Increasing the likelihood of a correct diagnosis.

The same sample may routinely be individually screened for the same set of targets (e.g. a respiratory screen), therefore combining them all on to a single assay could significantly reduce processing time and cost for the user. Using a single device in place of multiple devices has environmental benefits too.

When developing a multiplex lateral flow assay, optimize, optimize… oh and optimize!

The more targets in the assay, the more possible combinations of lateral flow device (LFD) components and reagents. This increases the risk of errors therefore it is important that you pick reagents and components that work in harmony. As with any LFA, it is important to include a suitable control to ensure the test has run correctly.

Even if you have developed and optimized independent assays that use the same membrane, sample pads and reagents, this does not guarantee that, once combined, they will be the best performing components. Therefore, you should also optimize the system once the components are combined.

Optimizing flow rate is very important. If the flow rate is too low, by the time the sample reaches the last test line the first may be fading or bleeding, too fast and the sensitivity may be reduced.

Beware of cross-reactivity and interference, as one conjugate may bind non-specifically to the test line of another target.

10 Top Tips for lateral flow assay development

For a successful multiplex assay compromise is key

What if the best performing components are different for the different targets? Whilst it would be ideal to have the peak performance for all targets, when creating a multiplex test compromise is key. The benefit gained from having three sub-optimal targets may outweigh having a single perfectly optimized target.

The dynamic ranges for the assay targets, sample and other components may not be the same. As they must all work with a single sample, choose a dilution that can work for all proposed targets and adjust the target specific components accordingly.

Likewise, peak signals for individual targets may occur at differing run times – compromise!
You must remember that some targets are not compatible.

It is important to know when to draw the line and rethink the targets that you are combining. Targets that may have been discarded individually during development may, when combined, display enhanced sensitivity and specificity so end up being a better choice.

I can put the test lines in any order, right?

Wrong, peak signals from different targets and conjugates may appear at different times following exposure to the sample. It is important that all your targets can be read within the same timeframe e.g. if one produces its best signal at 5 minutes post sample application but another is at 35 minutes, then it is likely you will need to rethink your choices or optimization. Identify the peak signals from your different targets and arrange the them in different conformations to determine the optimal arrangement of your test lines.

How many targets can I detect on one assay?

There are obvious physical limitations to the LFD itself, the test lines need to be sufficiently far apart that they do not interfere with one another (line bleed). Attempting to wick the sample along the membrane also becomes more challenging the longer the device becomes, flow rate decreasing non-linearly with distance. Therefore, there are demonstrable physical constraints.

As with all LFAs, the control line(s) is essential. Beyond that, at least three test targets are certainly achievable.
Remember the more targets that are added, the more complicated optimization becomes and the more there is to go wrong.

What are the implications for the end user?

Multiplex assays can be a double-edged sword for the end user. They can speed up the testing process and reduce the number of points at which mistakes can occur as the user only needs to run a single assay.

However, the interpretation of a multiplex result is inevitably more complex than a yes/no answer. It is vital to make it clear which test line is which and where the control line is to avoid misinterpretation.

Consider who your end user will be, if guidance instructions will be sufficient for them to correctly interpret the results, or if a reader system that performs this for them might be more appropriate.

What happens if an assay is negative for one target but positive for the other? Make sure users have sufficient information to be able to interpret the importance of the result and know when retesting in necessary.

Cost is another important consideration. Inevitably, the more targets you add to the test, the higher the cost will be. Is your target market going to be willing to pay more?

Will users still want to test only for one of the targets on your multiplex test (so will you offer a singleplex option) or does multiplexing offer enhanced detection that would be desirable to most users? Each question is just as important as the next, and all are critical to the development of your multiplexed LFA.

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