Selecting Your Lateral Flow Device Components

When designing a lateral flow assay (LFA), there are many factors to consider to achieve the most sensitive and specific results – and ultimately deliver an easily usable test that gives a correct, reliable diagnosis. In this guide we discuss the important factors you need to consider when selecting lateral flow device (LFD) components to design an LFD to fit your needs.

Sample pad selection – Not all analytes are made equal

The very first point to consider is the type of sample you are hoping to test. If it is something like urine or water, then this may make life easier. However, if you aren’t lucky enough to be testing a fluid free from cells or other debris, then you will need to consider how you can prevent the test falling at the first hurdle and becoming clogged before the sample has even reached the membrane.

For more solid samples like leaves or food, pre-treatment by shaking in a buffer solution (buffered saline solutions with a detergent like Triton™ X or Tween® are a popular choice) with ball bearings, for example, may be required to release molecules for detection into solution. Some samples may then require separation, e.g. by centrifugation, to remove large lumpy bits before being applied to the test. However, if you want to design a test that can be used in the field then this kind of requirement is out of the question – think about where you are designing your test to be used. Separators can be incorporated into the sample pad that remove particulates or red blood cells, but even then, clogging can prove problematic. If using a sample like blood, consider if serum from clotted blood could be used instead. Chemicals can also be incorporated in the sample pad that help break down things like mucus in sputum samples. Alternatively, parameters such as the pH of the samples can be adjusted to deal with interferences caused by sample-to-sample variability.

If the target you are aiming to detect is sufficiently abundant in the sample, then diluting the sample can help its passage through the membrane by reducing the concentration of clogging particulates. If the available sample volume is very low, it may also be necessary to dilute the sample to give sufficient volume for it to run all the way to the end of the test strip. Another option is to ‘chase’ the sample with an appropriate buffer that pushes the sample liquid through the assay.

The volume of sample you need to apply to make the test work is also an important consideration at this stage. The sample 

pad must be able to absorb all of the sample applied to it and release it into the conjugate release pad in a controlled manner.

The meeting of matter – Conjugate selection

To differentiate positive and negative samples, they must first be combined with an agent that makes them visible. The first obvious point is that the conjugate must efficiently bind your target sample type – if not then it’s heading straight for the trash.  There are then a number of choices for conjugate labeling systems to consider, with some of the most popular options being colloidal gold or monodisperse latex, tagged with either a visual or a fluorescent dye.

No matter which conjugate you choose, to give reproducible results, it is important that its release from the conjugate pad is consistent and matches the viscosity of the sample liquid. Poor release and mixing with the test sample can reduce assay sensitivity. The material chosen for the conjugate pad can also have a bearing on the stability of the conjugate. It is therefore worth trying different conjugate pad materials (common options include glass fibre, polyester and rayon) to see which work the best with your conjugate.

The main event – The membrane has the answers

So, your sample has successfully made it into the LFD membrane. This is where the magic happens, but it also means there are lots of points to consider.

For a membrane to do its job correctly, it must allow the sample, which is now mixed with the conjugate, to flow evenly through the membrane slowly enough that the test and control lines have sufficient time for reactions to take place, but fast enough that you don’t put down roots waiting for it. Overly long run times can produce false positive results. For optimal sensitivity, it is imperative that the passage of the sample through the membrane is at an optimal rate too. Flow rates will also be influenced by the viscosity of your sample. This is another point where choosing whether to dilute your sample can be an influential factor.

Sensitivity and specificity are the holy grail of any test, and it is important to balance both. Increasing sample volumes or concentrations may help to pick up those weak positives but be careful as it may also increase the background and false negatives.

Both your test and control proteins must be able to bind efficiently to the membrane and retain their ability to bind with the correct epitope in the sample if it is present. Some proteins just will not bind to some membranes, but don’t despair, there are a multitude to try that have all been treated in different ways - it may just take a little trial and error!

It is also important to keep in mind how many proteins you wish to combine in a single assay. Many tests rely on just one target but if you have a lab-based assay that detects multiple targets to improve test sensitivity and specificity, then consider multiplexing the LFA too and choose a membrane that can accommodate this.

Nitrocellulose has historically been a popular membrane material choice because of its wicking capabilities, protein binding properties and low cost. However, it also has drawbacks including problems with batch-to-batch variation and stability over time. It is therefore worth investigating different membranes and different batches of the same membrane to ensure that you get one that consistently works well.

The capture and detection reagents that you want to use (or  have to use if there are no suitable alternatives) may restrict the properties of the membrane. Try to test your assay with samples that push the limits of the reagent and membrane properties to ensure you get the best match. Bear in mind that if you are using a strongly colored sample, it could impact the appearance of the test and control lines, especially in the case of a weak positive.

Detection is another area to consider—will the end user be interpreting the results by eye, from looking at the indicator strips, or will an LFD reader be used? A reader can remove the objectivity of personal interpretation and potentially give more consistency, especially if your LFA is less than clear cut. However, if using an LFD reader, it also relies on the end user purchasing or having access to a reader unit.

Absorption pad choice – The sample journey doesn’t end at the membrane

What happens to the sample after it has run through the LFD membrane is a factor that is easily overlooked but is 

nonetheless important. The absorbent pad at the LFD end is the powerhouse that draws the sample through the test membrane and will influence the test run-time. It must also have the capacity to take up all of the sample that flowed through the device. Choose a product that is right for your needs—if you are only loading 100 µl then you probably wouldn’t select the same pad as an assay that requires a larger volume of sample to be loaded for successful detection. The pad must also be able to retain the sample and, most importantly, unbound detector conjugates safely, as they may cause false positives if allowed to seep back into the LFD membrane.

Cost – Consider your target market

It is fine to design a fantastic test that gives great results, but if the cumulative cost per test when all components are taken into account is prohibitively high, no one will use it. The assay you spent many months creating will sit on the shelf gathering dust. Is the test aimed toward health professionals, government workers or enforcement officers, or maybe to the end user such as a farmer, grower or the general public? Different target audiences will have differing budgetary constraints. For compliance officers, safety may be paramount even if it comes at a high cost, whilst margins for farmers are often tight and cost must be minimal to make it worthwhile.

Shelf life – Users might tolerate all conditions but will the LFD?

The shelf life and temperature tolerance of the test components are really important considerations. It’s great if you can you can keep the test in a consistent, temperature-controlled environment but if this is going to be a field test, many of your end users will not. We’ve seen some users ‘store’ test components in a car trunk, exposed to extreme temperatures, or on a windy barn shelf between the weed killer and hoof trimmers. It is therefore vital to consider the temperature tolerance of the components, and what impact this has on shelf life. This may vary depending on the specific membranes chosen or types of conjugates used, so it is worth doing the research first. No one wants to discover they get fantastic results, but only if the test is stored at 4°C.

For those venturing into the diagnostics market, some useful information from an assay design perspective can be found here, and this article provides one expert’s personal advice for anyone thinking of setting up a diagnostics start-up.