An Introduction to the Lateral Flow Test: Strengths, Limitations and Applications

Diagnostic testing is key in many areas including healthcare and preventative medicine, food safety and environmental monitoring. As such, analysts need appropriate and accurate assays suited to the sample type, analyte and testing environment that respect monetary, time and operator constraints. One such test to fill this niche is the lateral flow test (LFT). In this article, we will consider what LFTs are, how they work and their applications.

A lateral flow test (LFT) is a simple, quick and cheap assay that can detect the presence of target antigen(s) or antibody(ies) in a liquid sample.¹ As a type of immunoassay, detection relies on the binding of antibodies with their target antigen combined with a detection reaction to produce a readable, often visible, result.

LFTs are referred to by multiple terms and abbreviations, some of which are interchangeable, depending on whether the testing process or the device used are being referred to and whether the test detects antigens or antibodies. Common terms are summarized in Table 1 below.

Table 1: Names and abbreviations used to describe LFTs.

LFTs are typically housed within a plastic casing with a well for sample introduction and window through which the test and control lines can be seen.

Performing a direct LFT proceeds as follows (Figure 1):

Figure 1: Diagram of a lateral flow device (LFD) indicating the key components and path taken by a sample. Credit: Technology Networks.

1. A liquid sample is introduced to and absorbed by the sample pad located immediately beneath the sample well. The volume must be sufficient to soak the pad and allow the sample to wick along the LFD membrane, but without flooding the device. A couple of drops of sample is typically used.
   
   
2. Through capillary action, the sample is then drawn along the device to the next pad, known as the conjugate pad. Here, binding occurs between the target analyte (if present) and the labeled immunoreagent. This is normally an antibody that will bind to the target antigen or antibody conjugated to some form of detection medium, such as colloidal gold. To prevent interference with test line binding, conjugated antibodies are designed to bind to different regions of a target antigen to the test line antibody. Where the assay is detecting antibodies, the conjugate antibody normally binds the constant region of the target antibody rather than the target-specific variable region.
   
   
3. The sample continues to wick along a porous membrane, typically nitrocellulose. On its journey, the conjugated sample will pass through the test and control lines on the membrane. At the test line, binding of the conjugated target analyte (if present) to the immunoreagent in the membrane occurs. This reaction is designed to be specific so that binding only occurs in the presence of the target. Where the target is an antigen, the test line will contain immobilized target-specific antibodies. Where the target is an antibody, the test line will contain immobilized antigens that are targeted by the antibodies of interest.
   
   
4. When the sample reaches the control line, binding occurs to indicate that conjugate release and sample transfer along the membrane has been successful. This binding reaction is typically between an immobilized antibody and the antibody conjugate and is independent of the sample analyte. Consequently, a control line should appear even in the absence of the target analyte.
   
   
5. Finally, the remaining sample flows into the absorption pad at the end of the LFD.
   
   
6. Once the sample has been applied, the test must be left for a specified amount of time to allow the binding and subsequent detection reactions to occur. Optical detection – in particular colorimetric detection – is most frequently used as the result can be read by eye. However, some assays have also been developed that use fluorescence detection for which a specialist reader is required. Biochemical detection reactions, such as horseradish peroxidase-based assays, surface-enhanced Raman spectroscopy (SERS), electrochemical and magnetic² label and detection methods have also been developed but are less commonly utilized.³ The conjugate label used will determine the detection modality required.

Examples of the binding reactions occurring in direct LFTs to detect an antigen or an antibody are shown in Figures 2 and 3 respectively.

Figure 2: Binding reactions at the conjugate pad, test and control lines when detecting antigens in samples. Credit: Technology Networks.

Figure 3: Binding reactions at the conjugate pad, test and control lines when detecting antibodies in samples. Credit: Technology Networks.

As the majority of tests produce a visible result with lines developing where the conjugate, with or without the target, is captured, many LFTs can be read by eye. Successful testing with direct assays is indicated by the presence of the control line and a positive or negative result indicated by the presence or absence of the test line (Figure 4). Another form of LFT, the competitive test (discussed in the next section) requires a different interpretation of results. Although not frequently deployed among the general public, it is important to establish what type of test you are assessing.

It is important to note that results should only be interpreted within the given timeframe stipulated for that specific test. If they are read too soon after the sample has been introduced, the result may not have had time to develop fully. If left too long after sample introduction, the bands may become altered over time as the reaction deteriorates or overdevelops.

Figure 4: Interpreting direct LFT results. The diagram indicates A) a successful test providing a negative result, B) successful tests providing a strong (left) and weak (right) positive result and C) examples of void tests in which the control line fails to appear. Credit: Technology Networks.

Some tests can also be read digitally using a handheld or benchtop scanning device or a smartphone. The first handheld scanners became commercially available in the 2000s.⁴ For tests that produce a fluorescent rather than colorimetric result, a reader of some form is a necessity. While handheld devices and smartphone systems will typically read one test at a time, benchtop devices can read batches of LFDs simultaneously but are normally restricted to lab-based settings unlike their portable counterparts. The use of these devices can remove the uncertainty of human error in reading, interpreting and, where applicable, recording results. Some readers will also measure the intensity of the test lines to give a form of quantitative result. Digital measurements can be particularly useful where lines on the LFD are faint and having a digital record of test results can help with data integrity and traceability issues. They also offer opportunities to gather data for patient monitoring by doctors, potentially leading to early interventions and better health outcomes.

While benchtop and handheld readers are not typically practical or affordable for home use, smartphone readers have filled this gap. Early forms were not widely adopted due to the need for additional customized add-ons^{5, 6, 7} to allow the phone to capture the results. However, advancements in assay and phone technology along with increased connectivity have improved this situation. During the COVID-19 pandemic, digital readers were approved for use whereby users submitted a photo of the LFT through an app on their phone and artificial intelligence was used to interpret the result. The creators hope that the app can be used more widely for other LFD-based tests. As demand continues to increase for quantitative point-of-care and point-of-use tests and sensor technologies continue to improve and reduce in size and cost, it seems likely that we will continue to see novel technological development in this area.⁸

Tests may be designed to detect multiple targets in a single run, known as multiplexing (discussed further in the next section).⁹ In these cases, the results for each target may be presented as a single shared control line with sequential test lines, each of which will indicate if the sample is positive or negative for its corresponding target (Figure 5 A). The same principle may also be applied to create an array format, where multiple test strips, each for a single target and with their own control lines, are run in parallel and results read off each strip (Figure 5 B).

Figure 5: Physical arrangements for multiplexing LFTs A) with targets in sequence on a single test strip, and B) with individual test strips in an array format. Credit: Technology Networks.

Alternatively, systems may be used that employ multiple labels, each for a different target in a single sample, which can be distinguished from one another, such as fluorescent labels with differing emission profiles (Figure 6 A). This approach requires specialist readers. If it is desirable to detect a whole class of a given target, rather than differentiate the specific target (e.g., multiple antigens on a given pathogen or multiple forms of the same pathogen), a broadly selective antibody may be used that will detect the presence of a range of molecules in a given class but report it as a single undifferentiated result (Figure 6B).

Figure 6: Immunological probe selection for multiplex LFTs where A) multiple probes are used in a single test, the signals from which can be differentiated and B) antibodies with broad selectivity bind multiple targets and report them as a single result. Credit: Technology Networks.

As with most immunoassays, there are a number of important considerations when developing an LFT or transferring a lab-based assay onto an LFD and it is important to select and optimize all the assay components carefully. These include:

Type of lateral flow test

Most LFTs we will come across, and indeed the type described above, are direct assays, however, LFTs may be in a direct (sandwich) or competitive (inhibition) format. Direct assays are typically used when testing for larger analytes with multiple antigenic sites, such as the human pregnancy test for human chorionic gonadotrophins (hCG). Here, the conjugate must be in excess to ensure that it, along with the target, is not all captured at the test line and prevented from reaching the control line.

Competitive formats are typically used when testing for small molecules with single antigenic determinants, that are unable to bind two antibodies simultaneously. Conversely to direct assays, a positive result is indicated by the absence of a test line while a control line should still appear in all cases. This is because the conjugate is already bound to purified target antigens and only when there are target antigens in the sample is the conjugate therefore competed off and prevented from binding to and developing a result at the test line (Figure 7).

Figure 7: Binding reactions in a competitive LFT that produce a positive or negative result. Credit: Technology Networks.

Antibodies

Monoclonal antibodies bind a single epitope of their target and thus provide good specificity. The use of polyclonal antibodies can increase sensitivity as they will bind multiple epitopes of the target unlike monoclonal antibodies, but risk increasing non-specific binding, reducing specificity. Whether monoclonal or polyclonal antibodies are used, each antibody must be validated and optimized for a given assay, including the amount used, to ensure accurate and sensitive results.

Label type and corresponding detection system

Optical readouts are most commonly used in LFTs and can be broken down into colorimetric and fluorescent groups. The choice of corresponding conjugate label will be guided by how the test is intended to be read, where and by whom. The most frequently employed labels in colorimetric tests include colloidal gold, nanoparticles, cellulose and polymer beads, while fluorescent labels include fluorescent beads¹⁰ and quantum dots.¹¹

Sample pad

The sample pad¹ must be able to soak up the sample and release it in a form that is compatible with the LFD. Therefore, it must be sufficiently absorbent but at the same time have properties suited to the specific sample type. This may mean it is required to filter out red blood cells or particulates, change the pH or disrupt matrix components, which can be achieved through appropriate pretreatment of the pad.

Conjugate pad

The conjugate pad¹ must be able to hold the conjugate and keep it stable for the lifetime of the LFD, releasing it efficiently and reliably when the LFD is used. Poor or uneven drying and release can be important sources of variability in assay performance. It is therefore important that the material, coating and drying process when loading the conjugate pad are optimized. It is also important that the material chosen does not impede the flow of the sample.

Membrane

The membrane¹ must be able to allow the sample/buffer and conjugate to flow consistently through it and at the same time hold the immunoreagents stably at the test and control lines for the lifetime of the LFD. It must also not interfere with the binding reactions or chosen detection methodology. Nitrocellulose is a popular choice of material; others have been tried but generally with little success.

Absorbent pad

The volume that the membrane can hold is finite and so it is the role of the absorbent pad¹ to increase the amount of sample, and thus the target if present, that passes through the membrane over the test line, improving test sensitivity and specificity. It achieves this by soaking up the sample (and buffer if used) as it reaches it, causing more to be wicked through the membrane. The absorbent pad must have a bed volume in excess of the sample/buffer to be run in order to be able to hold the excess volume until the result can be read without allowing potentially interfering backflow. Therefore, it is important to choose the correct thickness, density, strength and material. Cellulose is a popular choice thanks to its absorptive capacity.

Sample

The sample itself is an important consideration in a successful LFT. Samples that are very viscous may not wick through the membrane or cause clogging that leads to test failures while inhomogeneous samples can produce unreliable results. Sample concentration is an important consideration too. Too dilute and positive results may be missed, too concentrated and it may interfere with test performance. Therefore, some sample types may need pretreatment (e.g., mucolytics), separation (e.g., extracting serum from whole blood) or dilution to make them amenable to LFD-based testing and produce results that are within the dynamic range of the test.

Multiplexing

While detecting a single target may suffice, some applications benefit from multiplexing to enable the detection of multiple targets or multiple parts of the same overall target in one assay. This avoids the need for repeat testing on the same sample, thus reducing time and cost. However, the inclusion of multiple testing reactions on a single device can increase the complexity of assay optimization and it is important to avoid cross-reactivity or interference between the different targets. Spatial separation of multiple test lines is a popular way to multiplex, although it can complicate the interpretation of results, require more materials and sample and increase the time to result compared to a single target assay. Array formats overcome the issue of combined optimization, run time and reading closely spaced results but also require significantly more materials and sample. Multiplexing strategies that employ differentiable labels also typically require specialized readers to interpret the results.

There are a host of strengths associated with LFTs that have led to their widespread use, not least in applications such as pregnancy testing and in the COVID-19 pandemic. However, as with most assays there are associated limitations¹² to their use as summarized below.

Strengths

• Cheap
• Quick time to result (normally minutes)
• Portable, especially those that can be read by eye
• Easy to use
• Can typically be kept at ambient temperature, very useful for point-of-care and home testing or testing in remote areas where refrigeration may be difficult or inconsistent
• Typically have a long shelf life
• No power required for most
• Array of different digital readers offer options to suite different settings
• Small sample volumes needed
• Readers can increase accuracy of result interpretation/recording
• Can be multiplexed

Weaknesses

• Samples with particulates/viscous samples can cause clogging or inconsistent running of the device
• Pretreatment of some sample types may be necessary
• Can be less accurate (lower sensitivity/specificity) than lab-based assays
• Once tests have been run, they must be disposed of appropriately while potentially containing infectious material, which can be more challenging and less enforceable outside of a care setting
• Although some benchtop readers can take multiple devices, LFTs are generally less well suited to high-throughput analyses
• Normally need to be read within a restricted timeframe as result will fade or overdevelop if left too long
• Give a “yes” or “no” answer, generally not quantitative (other than digitally read devices) so can hinder interpretation of stage of infection/risk posed to others etc.
• Readers add complexity to assay development and cost for users where benchtop or handheld devices are used instead of smartphones
• Reproducibility can vary from batch to batch
• Cross reactivity can be a challenge, especially for multiplex devices

The applications for LFTs are so numerous and diverse that it would be impossible to cover them all here, but below are a selection of the most popular areas for use.

Pregnancy test target

Probably one of the best-known examples of LFT is the home pregnancy test. It has been around since the 1980s¹³ and detects hCG, produced by the placenta during pregnancy, in urine. Sensitivity has increased over the years and tests will now typically provide a positive result from 10- or 11-days post-conception.

Target home COVID test

Most people have probably experienced one or more of the home SARS-CoV-2 LFTs¹⁴ firsthand. The majority of the devices people use at home are designed to test for the presence of the virus itself (i.e., might they be infectious?) rather than for antibodies indicating exposure or vaccination. As such, they are directed to bind to the exposed and accessible antigenic parts of the virus such as the spike, envelope, membrane or nucleocapsid proteins.¹⁵

Human pathogens

Although SARS-CoV-2 may be the human pathogen that first springs to mind when we think about the applications of LFTs for infectious disease, they have also found utility for detecting a range of targets including the Plasmodium parasites that cause malaria, Mycobacterium tuberculosis, the causative agent of TB,¹⁶ hepatitis B virus¹⁷ and human immunodeficiency virus (HIV).¹⁸

Animal pathogens

It’s not just human medicine that benefits from LFTs either. As well as pregnancy testing in animals, a number of veterinary pathogens can be tested for with this format too. It is particularly useful for practitioners working away from the clinic or testing by farmers, keepers or owners. This includes African swine fever¹⁹ and bovine enteric pathogens.²⁰ LFT testing was pivotal in the eradication of rinderpest.²¹ On-farm testing can also help to prevent pathogens in food-producing animals from reaching the consumer. There have, however, been criticisms of the quality of some tests offered for diseases such as rabies.²²

Food contaminants

Food contaminants come in many forms, from pathogens and pesticides to heavy metals and toxins. Aflatoxins, secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus found in grain-based foods, nuts and spices, are one such example.²³ They cover a group of chemically related compounds, for which numerous LFTs have been developed, targeting the toxins. Due to their small size, LFTs for aflatoxins are typically of an indirect format. Multiplex tests that check for aflatoxins along with other mycotoxins have also been developed.²⁴ Salmonella species are common food pathogens for which rapid diagnosis can help to expedite treatment, identify sources of contamination and help to prevent further spread.²⁵ LFT technology has offered a means to do this utilizing phage technology to determine effective binders for test strips,²⁶ even distinguishing live from dead Salmonella Enteritidis.

Environmental pollutants

The detection of environmental pollutants, including pesticides, bisphenol A (BPA) and heavy metals, is important for protecting people, animals and the environment. Portable testing solutions allow analysts to identify issues and take swift action whilst on site. LFTs able to detect mercury,²⁷ chromium²⁸ and cadmium²⁹ ions have been developed. BPS, an endocrine-disrupting compound of concern, can also be detected with this technology and showed favorable results compared to lab-based testing.³⁰ Organophosphorus pesticides can be detected using indirect LFTs,³¹ while a test for the simultaneous detection of carbofuran and triazophos in water has also been developed.³²

Allergens

Those with allergies must be careful to ensure they avoid their triggers. Likewise, food producers, for example, also must ensure that products declared as “free from” really are. LFTs offer a quick and easy way to keep people safe and have been developed for a whole host of common allergens including gluten,³³ casein,³⁴ soy,³⁵ mustard and various nuts.³⁶

Therapeutic drugs that require close monitoring

For some therapeutic drugs, there is a fine margin between an ineffective dose, an optimal dose and exceeding safe levels where serious adverse effects may be experienced. To ensure patients remain within the optimal dose range, close monitoring is required and companion diagnostics such as LFTs can provide that. Digoxin, a cardiac glycoside used for the treatment of tachycardia, is one such drug and scientists have developed an LFT that can be read using a smartphone app to help with patient self-monitoring and dosing.³⁷

Drugs of abuse

Urine analysis, for which LFAs are well suited, is still one of the most common testing methods for illicit drugs in the UK, providing rapid results for situations like workplace testing or law enforcement. A test for cocaine has also been developed that, unlike tests for many drugs of abuse that follow a competitive format due to their small molecular size, uses a non-competitive format by employing a biomimetic material.³⁸ LFAs have even been developed that can detect Δ9-tetrahydrocannabinol, cocaine, opiates and amphetamine in the sweat of a fingerprint.³⁹

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