What is a Lateral Flow Immunoassay?

What is a lateral flow immunoassay?

The following article describes what a lateral flow immunoassay is used for. In addition, the article explains the different component parts used during manufacturing in order to make them work.

Common names for a lateral flow immunoassay

Different industry sectors and different countries use varying terminology to describe a lateral flow immunoassay. 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

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.

LFDs 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 used in human health for point of care testing. They can be performed by a healthcare professional or by the 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 developed and manufactured and Abingdon Health can guide you through this process.

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.

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.


Sample matrice

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, the Abingdon Health product Seralite® – FLC, a rapid test for the quantitative measurement of kappa and lambda immunoglobulin free light chains (FLCs), uses serum and requires a buffer for sample dilution. FLCs are used as an aid in the diagnosis and monitoring of a type of blood cancer, Multiple Myeloma.

A rapid plant disease tests, however, require the mixing of plant material samples, such as leaves and wood, with a running buffer in order to detect commercially damaging plant disease.

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

MilkWhole bloodSerum
SalivaUrineTissue samples
FoodDrinkAnimal feed
Plant materialWaterFuel

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 assay 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.

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.

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.

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, click the play button on this image to watch a video.

  • ˃ 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.