Simplifying Component Selection in Lateral Flow Assay Development

Presenting highlights from the webinar “Simplifying component selection in lateral flow assay development” hosted by Klaus Hochleitner, PhD., which is now available to view on demand here.

Lateral flow assays (LFAs) are unrivalled in terms of speed and ease of use, however their development requires highly informed decision making. It can take 6–12 months to move from the initial gathering of ideas and information through to design verification, with regulatory approval taking another 1–2 years. Selecting key components—such as the correct type of nitrocellulose (NC) membrane—early on and in the correct way, is key to keeping LFA development on track, within specification and on budget. So, let’s take a look at some of the considerations of component selection in more detail.

The basics


There are two types of LFA assays. The first is a non-competitive, or sandwich test, which is used for macromolecular analytes with two antigen binding sites — binders that attach to the target and do not interfere with each other. If the target is present in the sample, there will be an increase in the signal at the test line. A well-known example of a sandwich test is a pregnancy test.

The second type is known as a competitive test and it used for small molecule analytes which do not have two binding sites. Since you cannot bind more than one binder to the target molecule, you need to use a test line that contains your target molecule. If the target is present in the sample, there will be a decrease in the signal at the test line. An example of a competitive test is one used to test for drugs of abuse.

Key considerations for the reaction membrane

Choosing the correct membrane often comes down to the nature of the test being designed. It is worth testing many variants, while considering the following:

Capillary rise time

This is known as the time it takes for a liquid (typically water) to flow across a defined distance on the membrane surface (the industry standard is 40 mm). All suppliers manufacture their membranes to a defined capillary rise time. The speed that the liquid passes both the test line and the control line determines the sensitivity and specificity of the test. LFAs for aqueous samples such as water and urine typically use slower membrane speeds (170–185 seconds). Tests with faster membrane speeds (80–100 seconds) are often used for more turgid samples, such as saliva and resolubilized solids.

All membrane capillary rise time specifications have an upper and lower limit. While most productions of the membrane will fall in the middle of the range, you may occasionally get reels which fall near the upper and lower limits. This can cause issues for example, with particularly fast membranes you will tend to see false negative results as the sample runs too quickly across the membrane, whereas for slow membranes, you will have false positive signals or problems with membrane clearance. In rare cases you may see issues at both edges of the specification limit. You should test membrane samples from both edges of the specification (called limiting samples) during your development. In order to do this you may have to select a membrane with a different speed rating (which overlaps with your capillary rise speed of interest) or ask for a customized membrane that has its own capillary rise time specification.

Membrane backing

In the manufacture of nitrocellulose (NC) membranes, the raw materials are dissolved in organic solvents with boiling points lower than water and poured onto a solid surface (this can produce either unbacked or backed membranes). Unbacked membranes have an air side and a belt side. The structure of the air side is coarse and random, which can increase variability and reduce protein binding capacity compared to the belt side. The structure of the belt side is denser and more regular and thus has a high protein binding capacity. Backed membranes have increased mechanical strength when compared to unbacked membranes, but they only have one usable side — the air side. If you are unsure what to select or are new to LFAs it is best to select backed membranes due to the ease of handling.

Membrane blocking

NC matrices are preferred due to their protein binding capacity, however any nitrocellulose that is not on the test line is likely to bind to the target molecule, severely impacting test sensitivity. To avoid this, you can block free nitrocellulose that is adjacent to the test and control line in two ways: by coating the membrane with blocking reagents during manufacturing (called ‘blocking on the fly’), or by integrating blocking reagents into either the sample or conjugate release pad, so that they migrate along the membrane ahead of the samples. The latter is more cost- and time- efficient, however you have less control over the amount of blocking reagents that you apply to the membrane.

Membrane surfactant

The surfactants found in NC membranes can influence antibody binding by making the membrane more hydrophilic—which helps to fine-tune the flow rate—or by partially denaturing the protein molecules, which helps to immobilize antibodies. Different suppliers use different surfactants and apply them at different stages of the manufacturing process, so it is best practice to test these membranes for your specific application before using them. The effect of immobilization on target binding depends on whether the epitope used is linear (it binds targets to a single linear peptide chain) or confirmational (it only binds targets when multiple peptide chains are in close proximity). Even with successful immobilization there is the chance that the epitope is not available due to the antibody orientation. You should consider whether there is a preferential orientation that blocks the binding sites, which will impact the antibody-antigen interaction. Secondly, you should consider how close the binders are to each other once bound to the target; if they are too close this will cause steric hindrance. Epitope mapping by surface plasmon resonance (SPR) can be done to check for antibody interference.

What about pads?

The pads on both sides of the membrane are just as critical for performance. For example, mistakes that occur during the preparation of conjugate pads are the most likely cause of problems with your apertures.

Lateral flow pads	Purpose	Tip
Sample pad	Prepares the sample prior to mixing with the conjugate.	Typically contain reagents maintaining key parameters of the sample, but can also be coated with buffers containing blocking reagents.
Blood separator pad	Used only with blood samples, to reduce coloration and interference (e.g. from DNA) by removing red and/or white blood cells.
Conjugate pad	Delivers the detector particles onto the membrane in a consistent volume.	It is important not to soak the pad with the detector reagent. When drying the conjugate pad, ensure it remains stable and undamaged.
Absorption pad (wick)	Captures and retains the sample at the end of the assay.	The absorption capacity must be larger than the volume of the sample liquid applied, to avoid a backflow of the sample from the wick to the membrane.

In summary, there are many important aspects to consider when developing LFAs. Getting these parameters right requires detailed information about each component, the sample, and the antibodies. We’d always advise developers to work closely with manufacturers to avoid the common pitfalls that affect the development process.

Ask the expert
What are the common reasons why people fail to develop a viable test?

There are many different factors. It could be anything from the choice of detection reagent, pads, and membrane, to the backing card selected or equipment setup. Every consideration within the development process can affect the results of the test, so it is important to test all these items.

Why does a test take so long to develop, for example the COVID-19 test? 

It can take 2–3 years for an LFA to move through development and clinical trials to reach the market. A good robust test will take a minimum of one year in the R&D phase. Although everyone is working as quickly as they can during the pandemic, you need to allocate enough time for development and optimization to fulfil the test requirements.

How do you evaluate a membrane based on the surfactant they use?

Not all monoclonal antibodies (mAbs) work well in the presence of a given surfactant, and 2%–3% of mAbs do not work at all. Antibody testing in the presence of various surfactants during early development stages is critical to optimizing performance. Different surfactants can affect test results (e.g. false positives, or insensitivity).

If you missed the webinar, you can view the on-demand video here!