How Do Rapid Antigen Tests Work?

Learn how rapid antigen tests, or lateral flow immunoassays, identify COVID-19

Sherri Fraser, PhD
Published:Feb 28, 2022
|Updated:Jun 16, 2022
|4 min read

For decades, lateral flow immunoassays have been used in home pregnancy tests. Less well known, though, are the rapid antigen detection test (RADT) developed in the 1980s for the identification of group A streptococcus (GAS), and more recently, influenza and malaria. All these tests share their technology with the rapid antigen tests now being used to test for COVID-19 infections.

Sample collection for rapid antigen tests

COVID-19 nasal swab sample locations.
Clinical Lab Manager

A lateral flow test contains a nitrocellulose testing strip on a nonreactive substrate, encased in a plastic case. The strip is designed to wick the sample from the loading well upwards toward an adsorbent pad opposite from the sample pad.

Each type of test requires a different type of sample:

For COVID-19 or influenza, a sample is taken with a nasopharyngeal swab. The swab is then agitated in a small amount of liquid to suspend the sample and any virus particles. After a couple of minutes, that liquid, called the analyte, is placed in the well at the bottom of the testing cartridge.

For a strep test, a throat swab is used, but for the malaria test, a fingerstick blood sample is required.

Conjugate components of rapid antigen tests

As the sample moves up the testing strip, the liquid component of the analyte rehydrates the test components on the conjugate release pad as it passes through them.

The dehydrated test components include antibodies conjugated to a visualization component (usually gold colloid), which bind to the target antigen. For example, the Abbott rapid antigen test has human IgG specific antibodies to SARS-CoV-2 conjugated to gold colloid.

The other component on the release pad is a test antibody, such as chicken IgY-gold colloid, which is used as a control.

Control line: this line is used to show that the test is working. It should always appear when a test is used. If it doesn't appear, discard the test and take another one. Test line: this is where you find out if you are positive for COVID-19. Any line here, faint or otherwise, means you are positive for the coronavirus.
Clinical Lab Manager, KOCZULA KM, GALLOTTA A. LATERAL FLOW ASSAYS. ESSAYS BIOCHEM. 2016;60(1):111-120.

Sample migration and visualization

Test line

As the sample travels up the strip, both the test and control antibodies in the analyte may bind to targets in the detection zone of the test. 

Antigen targets in the sample form a bridge between immobilized test antibodies and free-flowing gold-colloid conjugated visualization antibodies. If the antigen is present in the analyte as it passes over the test line with the immobilized antibodies, the visualization antibodies will become stuck at that line. If enough gold conjugated antibodies get stuck, they become visible. If there is no antigen in the sample, these gold-conjugated antibodies simply flow past the test line toward the end of the strip.
Clinical Lab Manager

For the Abbott COVID-19 test, mouse monoclonal anti-SARS-CoV-2 antibodies are immobilized along the test strip. As the analyte passes across the test line, any SARS-CoV-2 antigen targets in the analyte will bind to the conjugated test antibodies. This means that at the test line, the SARS-CoV-2 antigens act like a bridge between the immobilized test antigens and the gold-conjugated visualization antibodies from the conjugate release pad. This multi-part complex gets stuck in place at the test line and the accumulation of the gold-conjugate antibodies becomes visible, indicating a positive result.

If there is no COVID-19 antigen in the sample, there is nothing to bind the conjugated antibodies. Those conjugated antibodies are then carried past the antibodies bound to the test line with the analyte, so no line appears, indicating a negative result.

Control line

After passing the test line, the sample continues to be wicked up the strip, where a similar reaction happens at the control line. The only difference is that the antibodies fixed at the control line are to the test antibody itself, and no bridging occurs. The gold colloid stuck to the test antibodies will become visible confirming that the test works.

Interpreting rapid antigen test results

Positive test results

If, after the instructed time has passed, any line is visible on the test line, the result is considered positive. The specificity of lateral flow tests is generally very high (meaning there is a small chance of false positive results).

The U.S. Centers for Disease Control and Prevention quotes a specificity of 90–95 percent for Rapid Influenza Diagnostic Tests. For SARS-CoV-2 rapid antigen tests, a 2021 study by Patriquin et al. indicated that false positives typically only occur when users fail to follow the manufacturer’s instructions, such as using improper buffer conditions, etc.

Negative test results

Unlike positive results, where you can be quite confident in the result, negative results don’t necessarily mean you are truly negative. With any of these tests, two major aspects influence the possibility of receiving a negative result: the way the sample was acquired and the timing of the infection.

If the sample was poorly taken, a negative result may be returned due to insufficient antigen for the antibody to bind to and produce a visible result.

The biggest factor, though, is the timing of the sample. For COVID-19 tests, if the sample is taken too early (before symptoms are present), or too late (beyond the first week of symptoms), a negative result may occur. In other words, receiving a negative result doesn’t mean you are free of infection.

Sherri Fraser, PhD

Sherri Fraser, PhD, has been a freelance writer for more than 10 years, but she also has many years of genetics, molecular biology, and developmental biology research experience. Her unique life story has engendered a strong passion for science communication, especially science writing.

Top Image:
Each type of test requires a different type of sample.
iStock, D-Keine