How Does a Lateral Flow Immunoassay Work?

How Does a Lateral Flow Immunoassay Work?

Lateral flow immunoassays—also known as lateral flow tests, lateral flow immunochromatographic assays, rapid tests, and strip tests—are a straightforward and accessible diagnostic technique. Lateral flow immunoassays offer fast results for a wide range of diagnostic applications, from home testing to laboratory use,

The most common example of lateral flow tests is an over-the-counter pregnancy test. However, scientists and medical professionals also use these tests to identify diseases (such as Ebola or COVID-19), detect toxins in food and environmental fields, and even analyze specific target molecules and gene expression.

Lateral flow immunoassays clearly have a major impact on countless scientific and medical fields. How does a lateral flow immunoassay work, though? Learn the answer—along with everything else you need to know about lateral flow tests—with this guide.

An Introduction To the Lateral Flow Immunoassay

A lateral flow immunoassay is a simple diagnostic device that confirms the presence or absence of a specific analyte. That target component might be a pathogen or other biomarker in human or animal samples. Alternatively, the lateral flow test might target contaminants such as chemicals or toxins in water or food.

The simple function of a lateral flow immunoassay grants it the versatility to serve a wide range of applications. Moreover, lateral flow tests are relatively easy and affordable to produce, making them an accessible solution for researchers, doctors, clinics, and more.

How Lateral Flow Tests Work

The process of a lateral flow test is simple: the sample flows along the device, meeting conjugated labels and antibodies that bind to the target analyte if it’s present. These binding reagents will continue along the device to the test lines, where they bind to the test line to show a positive result. If the target analyte is not present, no binding occurs. The test lines remain blank to show a negative result.

A few key components work together to ensure the sample moves swiftly through the device and delivers an accurate result.

Sample Pad

Lateral flow immunoassays can work with a wide range of samples, including urine, blood, sweat, and other fluids. The sample pad collects the sample and begins the process of the lateral flow test. The sample pad evenly distributes the sample to ensure an accurate and controlled flow throughout the device. The pad often includes components such as buffer salts or proteins, which help direct flow and prepare the sample to interact with the rest of the immunoassay.

Conjugate Pad

The liquid moves from the sample pad to the conjugate pad. This is the area that stores conjugated labels and antibodies until the test begins. The pad contains a conjugate buffer that preserves the conjugate particles until the sample enters the pad. If the target analyte is present in the sample, then the conjugates will bind to it and move along with it as it flows through the conjugate pad toward the rest of the device.


The sample then moves from the conjugate pad to a nitrocellulose membrane. This membrane contains binding reagents at the test lines. If the target analyte has bonded with the conjugate particles in the conjugate pad, those particles will attach to these binding reagents and form the line that indicates a positive result. If there is no target analyte present, there will be no conjugate particles to bind to the test line, creating a negative result.

The membrane also includes a control line at the end. This line contains specific detection antibodies that bind with the sample to indicate that the test performed correctly.

Absorbent Pad

The absorbent pad sits at the end of the device and wicks moisture through the membrane, aiding in sample flow throughout the lateral flow test. As the name suggests, the absorbent pad absorbs the sample once it reaches the end of the device. This allows users to work with larger samples volumes, which in turn creates better sensitivity and accuracy for the test.

Different Types of Lateral Flow Immunoassays

Lateral flow tests come in two main formats: sandwich and competitive. These formats work in a similar fashion, but they have different ways of displaying results.

Sandwich Immunoassays

Sandwich assays—also known as direct format immunoassays—are the more common format. These tests follow the above explanation of how a lateral flow immunoassay works. The target analyte binds to the conjugate particles in the conjugate pad, then binds again to the reagents at the target line. As a result, a colored line at the test position indicates a positive test, and the lack of a line indicates a negative test.

The sandwich immunoassay gets its name from the way the target analyte is sandwiched between the conjugate particles and the binding reagent. This test format works best with larger analytes that can bind to multiple antibodies. Examples of sandwich format lateral flow tests include HIV tests and pregnancy tests.

Competitive Immunoassays

Competitive assays only work when testing smaller analytes that can’t bind to more than one antibody at a time. In this format of lateral flow test, the conjugate particles and the binding reagents at the test line “compete” with each other to bind to the sample.

If the target analyte is present in the sample, it binds to the detection antibody in the conjugate pad. As a result, it cannot bind to the reagents at the test line. Therefore, the lack of a colored line indicates a positive result, while a colored line indicates a negative result.

Challenges and Requirements When Designing Lateral Flow Devices

While the process of a lateral flow test is simple, the device itself is a remarkably complex instrument. Point of lab (PoL) testing device development and manufacturingis a precise science that requires extensive knowledge of how analytes, antigens, and materials interact with each other.

Lateral flow immunoassays require a clean, sensitive read in order to produce accurate results. As such, device developers must find suitable antigens or antibodies to use as the detection method.

Additionally, incompatible materials can cause problems and create inaccurate results. Developers must choose materials that perform the test efficiently without contaminating the samples or altering results in any way. From the particles that react with the sample to the backing card that stabilizes the test strip, developers must choose every single material with care.

At Gener8, we pride ourselves on our extensive knowledge and attention to detail. Our experienced team and comprehensive development services will help you achieve the results you need with better flexibility and a shorter time to market.

How Does a Lateral Flow Immunoassay Work?
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