From prototype to product: validate your lateral flow device

Converting a lab-based test to a lateral flow assay is a long process but when you finally have your prototype, it’s time to validate! As we’ll find out, there are several things you should consider to smoothly and successfully validate your immunoassay.

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Designing and developing a fully-validated lateral flow assay (LFA) is not always a straight forward task. Initially, when transferring your lab-based assay onto a lateral flow device (LFD), there are a number of elements — including nanoparticles, component materials, and bioreceptors — that need to be carefully selected and optimized.

Subsequently, your prototype LFD will need validating to ensure the results are just as reliable and robust as those obtained with the original lab-based assay. In this blog, we walk you through some of the key elements you should consider to help smoothly and successfully validate your LFA.

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General considerations for assay validation

Setting out design requirements

Outlining a set of design requirements is an important preparatory step that guides your validation testing and helps you review your results. Design requirements can simply be a list of acceptance criteria that you expect your LFD to fulfil, such as assay run time, specificity limits, and limit of detection. Most importantly, they must be measurable, such that your validation test results can unambiguously show that your LFD performs as intended.

What controls do I need?

Controlling your experiment ensures that the observed result is not just a random event and can help account for errors and variability in your experiment. Therefore — as with any other scientific experiment — selecting appropriate controls can make or break the validity of your results.

Choosing the right concentration of your positive controls is critical since very low concentrations of target can become undetectable (false negatives) while very high concentrations can lead to high background, saturating your assay. Additionally, non-specific binding or cross-reactivity can generate false positives. Testing a range of concentrations will help you determine the optimal target abundance for your positive control.

Metabolites, proteins, salts, and other components in real-world samples might have an effect on your assay’s performance. Therefore, switching from lab-based controls to real-world samples early on can help you quickly identify any changes in sensitivity.

What can you do if signal intensity is lost when making the switch? Adding blocking agents, such as proteins, surfactants, and polymers, to your LFD can help recover signal intensity.

How many samples are required for validation?

When performing validation tests, the question “How many samples do I need?” is frequently asked. The answer is that it depends. Ensuring you have enough samples is critical, otherwise your results could be statistically meaningless and ultimately lead to costly re-validation experiments. On the other hand, an over-sized study can be a waste of your time and resources.

We would advise consulting a statistician, although it is possible to determine an effective sample size with a little self-tutoring. Resources such as Statistics review 4: Sample size calculations provide a good place to start.

Keeping an eye on batch variation

Further down the line, your LFD will be manufactured in batches. Some components, however, are notorious for variability, such as any biologically derived agent and some nitrocellulose membranes. These differences translate to batch variation that could affect the performance of your LFD.

Testing multiple batches is therefore an important part of your validation process, not to mention an integral part of most regulatory requirements. So, while testing batches might seem inconvenient, getting to this early could save you from unexpected issues down the line.

Assessing your LFD design

Determining sensitivity and specificity

An ideal LFD would have both high sensitivity and specificity. For a clinical diagnostics LFD, this means that it would correctly identify everyone with and without a certain disease. In reality, there is often a trade-off between sensitivity and specificity and finding the right balance is a key element to LFD design.

However, it is also worth considering your final application, given that an LFD can still be useful even if it is not both highly sensitive and specific.

For example, the D-dimer test is very sensitive for the detection of pulmonary embolism as elevated D-dimers are almost always present in patients. However, as D-dimers are associated with other diseases, it is not very specific. A positive test is used to indicate that more specific, expensive, and/or invasive tests need to be employed, while a negative test negates this unnecessary disruption.

Determining the best cut-off

You can determine the best cut-off value (the detection limit) of your test by using known negative samples and a range of concentrations of known positive samples. It is smart to consider both “spiked” positive samples, as well as real-world samples when determining the detection limits of your LFA since they can give very different results.

If you are determining the results optically, a positive test should produce a clearly visible coloured test band. Similarly, this test band should be unequivocally missing in the case of a negative result.

When using a reader output, quantitative cut-off values — that minimize false positives and false negatives while maximizing true positives and true negatives — need to be determined. These values can be programmed to deliver outputs of “positive”, “negative”, and “re-test”.

Optimal run-time

LFDs are known for providing rapid results, although time is still needed for your sample to run through the device and for the detection chemistry to work.

Figuring out how quickly a reliable result can be consistently obtained should be relatively straight forward, but how long is too long? Even negative samples may eventually show up positive, so testing a range of concentrations and run times is needed to determine an optimal cut-off time.

Testing under a range of conditions

In what environment is your LFD going to be used? One thing is for sure: it is unlikely to resemble the relatively stable conditions of your lab. Factors such as temperature and humidity fluctuations can change the effectiveness of your detection chemistry or how quickly your sample runs though the device. So, validating your assay under conditions that resemble their intended use environment is a necessity.

Optimizing sample loading

While optimization of sample loading should be considered prior to validating a prototype LFD, it is worth revisiting the (a) sample type and (b) sample volume parameters at this stage, especially if previous optimization was not performed with application-relevant samples or design criteria have not been met.

Sample type

A lab-based assay might behave perfectly with artificial samples but switching to clinical samples could result in unexpected complications.

For instance, high viscosity or particulates in a sample could restrict its ability to travel through the membrane. Similarly, highly pigmented samples — such as blood — could make results interpretation challenging.

Similarly, you need to consider the intended use of your device. If your LFA is meant to be used by the patient themselves at home, then samples must be both easy to obtain and require little to no preparatory steps or specialized equipment.

Sample volume

The volume of sample required for your LFA will not only depend on the sensitivity of your assay, but also the natural range of target concentration in your chosen sample type. If the target abundance falls out of the sensitivity range of your assay, there are several things you can do (Table 1).

Table 1. Challenges, solutions, and considerations for optimizing LFD sample loading

Determining shelf life

Light, humidity, temperature changes, and simply the course of time will have an effect on the performance of your LFA. Therefore, it is important to determine how long your LFA will last under various environmental conditions.

LFDs typically have a shelf-life of approximately 2 years. However, waiting years to evaluate the performance of your LFD is clearly time-consuming and wastes valuable resources. So, what is the solution?

Accelerated shelf-life testing induces chemical and biochemical changes in a shorter amount of time by exposing your product to elevated stress conditions. This data can be used to predict the degradation at recommended storage conditions using established relationships between the acceleration factor and the degradation rate.

There are three main components that can affect the shelf-life of an LFD: the bioreceptors, the membrane, and the label. If your shelf-life data does not live up to expectations, it is worth considering whether the stability profiles of these three components can be improved.

10 Top Tips for lateral flow assay development

Is my LFD valid and suitable?

If you have taken the time to write down a comprehensive set of design requirements, then determining whether your test is valid should be relatively straightforward. Simply ensuring your results meet your acceptance criteria will give you all the answers!

My LFD does not meet my design requirements

Finding yourself in the unfortunate situation where your results are not quite good enough can be disheartening, but all is not lost. Ask yourself “where is my LFD failing to meet acceptance criteria?”.

From here, you can re-visit optimization of specific components and start the validation process again. For example, if your assay is failing to meet sensitivity criteria, you could consider increasing the concentration of nanoparticles or using a slower flow membrane. Remember, producing an effective LFD is an iterative process that can take some time.

My LFD satisfies my design requirements

Your LFD satisfies your acceptance criteria: it’s time to celebrate! But while your design requirements should be written with your end-user in mind, it is important to continually re-visit the suitability of your test, especially when reviewing your final LFD.

Since most end-users will not be scientists, minimizing the number of steps needed to perform your test while maximizing simplicity is key to a successful LFD. Could you make things even simpler? While optical interpretation of results is often the most cost-effective, using a reader output can remove any and all ambiguity.

Finally, it is time to look at your competition. Presumably, your LFD fills a gap in the market but how does it compare with lab-based assay, or even your own initial assay?

LFD’s offer several advantages over lab-based assays — such as cost and ease of use — but if your assay underperforms relative to its lab-based counterpart, take a moment to examine your space in the market or any repercussions for the intended user./p>

What next for commercializing an LFD?

After checking all the boxes, it is very likely you’ll be asking “what next?”. Before moving on to manufacturing and commercialization, there may still be some work to do.

For example, if your device is intended for clinical diagnostics, there are strict regulatory requirements that must be adhered to for all products developed and manufactured. Additionally, if you haven’t done so already, it is critical to address the patentability of your device since this can be challenging for an established technology, such as LFAs.

Are you facing any LFD design challenges? Or do you have a great idea for a LFA but lack the resources to make it a reality? At Cytiva, we combine 30 years of diagnostics test development experience with access to laboratory infrastructure to help you overcome any challenges, minimize costs, and accelerate time to market.

Find out more about our Lateral flow immunoassay development services