Antigen diagnostic tests detect structural features of the outside of the virus called antigens—small proteins that make up the virus—that may be present in a patient’s sample. When a sample from someone’s nose, for example, contains SARS-CoV-2 antigens, the antigen test will indicate the result through a change in color or fluorescence. Over-the-counter antigen tests usually rely on the appearance of a colored line on the test cartridge to indicate a positive result. This means of providing a diagnostic result has been likened to the color changing lines on pregnancy tests, which can indicate the presence or absence of a pregnancy. Antigen tests that rely on a change in fluorescence are performed in laboratories that have specialized readers to observe the fluorescence.
Antigen diagnostic tests work by:
- Finding larger pieces of evidence that the SARS-CoV-2 virus is actively infecting a person
- Detecting specific, 3-dimensional antigens on the outside of the SARS-CoV-2 virus
Many antigen tests are authorized to be used at the point of care, meaning they can be performed in a medical setting and the results can be available within about 30 minutes. While few antigen tests to date have been authorized for over-the-counter, at-home usage, more tests are entering the market to fill this important need.
More on antigen tests:
- What is needed to perform an antigen test?
- Types of antigen tests being developed for SARS-CoV-2
- Current antigen and molecular tests with FDA EUA Status
Antigen tests require samples—such as nasopharyngeal surface cells or sputum/saliva—that are likely to contain the virus. Viruses and other pathogens may also be detected in feces, urine, or blood. For respiratory-presenting diseases like COVID-19, most tests now available or in development use samples from a person’s nose (using either nasopharyngeal swabs or anterior nasal swabs) or mouth (using saliva collection cups) to make testing easier for both healthcare providers and patients.
Rapid antigen test: Detects easy-to-find surface markers on the outside of the virus and avoids extraction and amplification steps. Samples are collected from easy-to-reach areas (like the nasal passage) where the virus tends to replicate the most.
Pros: Can detect active production of viral proteins, fairly rapid tests (minutes to results), RNA extraction and amplification steps not needed.
Cons: Requires extensive design and troubleshooting of test antibodies that will react to viral proteins in the sample and deep knowledge of viral proteins produced in various tissue environments. The test may yield false negatives if the viral protein production is low or if there is not enough virus replication in the sampled area.
How it works: These tests can be fairly rapid, using lateral flow assays. These types of tests are common in diagnosing illnesses such as strep throat. They may seem similar to serology tests, but they are different in 3 important ways: (1) they only detect active infection by the virus; (2) they do not detect antibodies, but rather viral proteins (antigens) that the virus is producing within the human host; and (3) they are typically not blood or serum based. The main principle behind antigen detection tests is the use of designed, synthetic antibodies to probe a patient sample for the presence of viral proteins. During an infection, viral proteins are often produced and can be detected in the blood or in other tissues or secretions. A patient sample is mixed with buffer, reagents, and synthetic antibodies. Importantly, these antibodies are designed so that they bind only to certain viral proteins; they will not bind to viral proteins from other viruses. If the proteins are present in the patient sample, then the synthetic antibodies will bind, and these complexes can be detected on a lateral flow strip with a color- or light-based readout. The synthetic antibodies used must be very carefully designed so that they properly bind the protein produced by the virus during a natural infection, including any special folding or protein modifications that could be present. This method also depends on there being sufficient viral proteins in the sample tested for them to be detected. In other words, these tests sometimes have sensitivity issues and can be less sensitive than RT-qPCR or RT-LAMP methods. These tests also must be very specific, so that similar proteins produced by other pathogens (eg, other human coronaviruses, influenza, etc.) are not detected by these synthetic antibodies. While there are some shortcomings to the sensitivity of this method, it is rapid and could be used in a point-of-care setting.
While the principles of rapid antigen tests remain similar, in that the test produces a quantifiable or visible change to signal a positive test result, there are different mechanisms by which tests can accomplish this and different ways in which the results can be read.
Types of antigen test mechanisms:
- Lateral flow assays – detect active infection through directional flow of patient sample over target proteins, usually on a flat card or cassette
- Immunoassays – detect active infection through incubation of patient sample with test proteins, usually in a 96-well plate or similar
Both test mechanisms take advantage of SARS-CoV-2 antigens binding only to specific human proteins. When this binding action occurs during the test, a color or light change is produced that can then be read.
Ways antigen test results can be read:
- Visual read – can be performed at home (either supervised or over the counter) or in a laboratory/healthcare setting
- Instrument read – must be performed in a laboratory or healthcare setting
Examples of antigen tests with FDA EUA using these methods:
|Lateral flow assay||Immunoassay|
|Visual read||BinaxNOW COVID-19 Ag Card 2 Home Test||-----|
|Instrument read||Sofia SARS Antigen FIA||VITROS Immunodiagnostic Products SARS-CoV-2 Antigen Reagent Pack|
We have compiled a list of commercial and laboratory-developed tests that have received FDA Emergency Use Authorization. There are 2 main sections:
- Commercial tests: These tests (sometimes referred to as kits) have been developed by industry professionals in biotechnology. These kits are typically available for purchase by healthcare providers or by researchers, undergo internal validation, and researchers manufacture the tests themselves. These tests can be purchased and completed at labs and facilities other than the manufacturing facility.
- Laboratory-developed tests: These tests have been developed by medical and research professionals. They are for use only in the institution in which they were developed. For instance, if a test was developed in a hospital, the test can only be used in that hospital. The laboratory developing and performing the test must be certified under Clinical Laboratory Improvement Amendments of 1988 (CLIA), 42 U.S.C. §263a, to perform high-complexity tests. These kits are not for commercial sale; they undergo internal validation and list the needed reagents in the protocols.