What is a lateral flow test?

Learn how lateral flow tests are developed and what is needed to manufacture tests accurately.

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Common names for a lateral flow test

Lateral flow tests have become synonymous with COVID-19 (SARS-CoV-2). However, lateral flow technology has been existence long before COVID. Therefore, different industry sectors and different countries use varying terminology to describe what lateral flow tests are. Common names include:

  • Lateral flow test (LFT)
  • Lateral flow device (LFD)
  • Lateral flow assay (LFA)
  • Lateral flow immunoassay (LFIA)
  • Lateral flow immunochromatographic assays
  • Dipstick
  • Express test
  • Pen-side test
  • Quick test
  • Rapid test
  • Test strip
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What is a lateral flow immunoassay?

So, what is a lateral flow immunoassay? Basically, it is a simple to use diagnostic device used to confirm the presence or absence of a target analyte, such as pathogens or biomarkers in humans or animals, or contaminants in water supplies, foodstuffs, or animal feeds. The most commonly known type of lateral flow rapid test strip is the pregnancy test.

LFAs typically contain a control line to confirm the test is working properly, along with one or more target or test lines. They are designed to incorporate intuitive user protocols and require minimal training to operate. They can be qualitative and read visually, or provide data when combined with reader technology, such as AppDx®.

Lateral flow tests are widely across many industries for point of care testing. They can be performed by professionals, trained lay users or a patient, and in a range of settings including the laboratory, clinic or home. In the medical diagnostic industry, there are strict regulatory requirements which must be adhered to for all products.

Due to their versatile nature, lateral flow rapid tests are used across a number of other industry sectors including pharma, environmental testing, animal health, food and feed testing, and plant and crop health.

How does a lateral flow test work?

LFDs use immunoassay technology using nitrocellulose membrane, coloured nanoparticles (or labels), and typically antibodies, to produce results.

When a sample is added, the sample will flow along the test device passing through the conjugate pad into the nitrocellulose membrane and then onto the absorbent pad.

The bullet points below demonstrate how a sandwich assay works. Alternatively, WATCH a video: click the button below.

WATCH: See inside a lateral flow test
Sample-pad
  • The sample pad acts as the first stage of the absorption process, and in some cases contains a filter, to ensure the accurate and controlled flow of the sample.
  • The conjugate pad, which stores the conjugated labels and antibodies, will receive the sample. If the target is present, the immobilised conjugated antibodies and labels will bind to the target and continue to migrate along the test.
  • As the sample moves along the device the binding reagents situated on the nitrocellulose membrane will bind to the target at the test line. A coloured line will form and the density of the line will vary depending on the quantity of the target present. Some targets may require quantification to determine target concentration. This is where a rapid test can be combined with a reader to provide quantitative results.
  • The sample will pass through the nitrocellulose membrane into the absorbent pad. The absorbent pad will absorb the excess sample. The specification of the absorbent pad will have an impact on the volume of sample the test can incorporate.



Types of lateral flow tests

Lateral flow assays can be developed to be used in a dipstick format or in a housed cassette. Both dipsticks and housed tests will work in a similar way, it is just dependent on the industry, sample matrix, and the market requirement, as to which format is suitable.

Sandwich assays – A positive test is represented by the presence of a coloured line at the test line position.

Competitive assays – A positive test is represented by the absence of a coloured line at the test line position.

To learn more about these assay types and the advantages and disadvantages, please read our article about immunoassay formats for lateral flow.

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Sample matrices

The target analyte and the market requirements will determine the type of sample that will be used in the assay.

Some samples require running buffer to aid sample delivery e.g. animal feed. Other samples such as blood, serum, urine, or saliva may be able to be placed directly onto a test, while there are occasions where a dilution buffer is required.

For example, AbC-19TM, a COVID-19 rapid antibody test that detects IgG neutralising antibodies that target the SARS-CoV-2 spike protein, uses whole blood with a buffer.

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However in the case of Pocket Diagnostic, a range of plant disease tests, the user needs to mix plant material samples with a running buffer in order to detect commercially damaging plant diseases.

LFTs are versatile enough to be developed to detect target analytes in sample matrices including:

Milk Whole blood Serum
Saliva Urine Tissue samples
Food Drink Animal feed
Plant material Water Fuel

Label types

Typically, lateral flow assays utilise conjugated gold, carbon, or coloured latex nanoparticles within the conjugate pad. Other labels include magnetic beads or coloured polystyrene beads.

Regardless of the label types, they all perform the same function to create a three-way bond with antibodies and targets in order to make visible the control and test lines.

Labels will be chosen during lateral flow development depending on several factors such as the target, sample matrix and antibody. The optimisation of the assay will ensure the label interacts correctly with the antibody and antigen to ensure efficiency and accuracy of results. This is vital for achieving a successful transfer and scale-up into routine manufacturing.

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Multiplexed lateral flow assays

Both sandwich and competitive assays can be developed to include one or more test lines. For example, our nucleic acid lateral flow immunoassay, PCRD, is a multiplexed sandwich assay containing two test lines and one control line.

In addition, by using complementary reader technology, sandwich and competitive multiplexed assays can produce quantitative results.

A multiplexed assay may be used for:

  • Detecting multiple targets in a single test rather than using many individual tests. In situations where only a small sample volume is available a multiplex assay allows you to maximise its use;
  • To assist diagnosis where the presence of a number of markers together is required;
  • Confirming the presence of multiple contaminants during high volume food and feed testing;
  • Offering cost-saving benefits to end-users in a laboratory or in-the-field by testing for different targets simultaneously; and
  • Remote or agricultural areas where resources are limited and where multiplexed testing will save time.
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Quantitative rapid lateral flow devices

Using names such as rapid test or quick test can lead to myths about lateral flow devices that they are limited in their capability. However, lateral flow devices are compact, easy-to-use, and offer considerable flexibility.

Early versions of LFDs were predominantly qualitative assays. However, improvements in reagents, component materials, and reader technologies along with manufacturing processes mean quantitative results are achievable.

In addition, the developments in reader technology and advancements in raw materials, such as labels, means a lateral flow rapid test can match the sensitivity of an ELISA assay.

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