How Does the SARS-CoV-2 Rapid Antigen Test Kit Work?
Since the onset of the pandemic, assays employing nucleic acid amplification such as real time polymerase chain reaction (RT-PCR) test have been widely used to detect infections with SARS-CoV-2. However, with the upsurge of positive cases and the demand for testing, access to more convenient, affordable, and faster testing options has become critical. The development of SARS-CoV-2 rapid antigen tests offer the potential to reduce turn-around time and cost, facilitate widespread screening, and enable people to test themselves whenever they might need. Thanks to these benefits, although PCR testing is clinically the more sensitive option, rapid antigen tests may have superior utility for the public health. Getting familiar with the logic and mechanism of SARS-CoV-2 antigen testing may help you better understand how these tests work and what exactly they might have to offer.
Rapid antigen tests detect proteins specific to a certain pathogen present in human samples and deliver results in real-time. This technology has been employed in the detection of many pathogens and rapid antigen tests for SARS-CoV-2 are very similar to those designed for the detection of strep, influenza, malaria, and HIV.
In a nutshell, SARS-CoV-2 rapid antigen tests are rapid chromatographic immunoassays which qualitatively detect SARS-CoV-2-specific proteins, or antigens, present in human nasal, nasopharyngeal, or saliva specimen. these tests usually employ a sandwich immunodetection method in lateral flow test format and target the nucleocapsid protein of SARS-CoV-2, as it is one of the predominantly expressed structural proteins of the virus.
Unlike molecular tests like PCR which detect the viral genetic material, rapid antigen tests for SARS-CoV-2 do not require laboratory equipment and personnel to collect and process samples. In fact, some rapid antigen tests have been deliberately designed as at-home tests and for self-administration. Moreover, while PCR tests may take up to a few days to return results, rapid antigen tests deliver results in 15 to 30 minutes.
The molecular mechanism of a SARS-CoV-2 rapid antigen test consists of three parts of antibodies. The first part is composed of mobile labeled primary antibodies, which have been conjugated to particles such as gold. These antibodies can bind the surface protein of the virus. The second part is composed of immobile primary antibodies that can also bind the surface protein. The third part is composed of secondary antibodies that can binds to parts of the primary antibody.
After the sample is transferred to the assigned spot, the liquid flows along the surface of the kit through capillary flow. If the virus is present in the sample, the first line composed of mobile and labeled primary antibodies bind the surface protein of the virus. As the sample continues to flow, the second line composed of immobile primary antibodies captures some of these labeled antibodies bound to the viral protein by binding the viral protein. The rest of these labeled antibodies bound to the viral protein is captured by the secondary antibodies which bind parts of the primary antibodies. If these labeled antibodies are bound, the label becomes visible as a red line. If both immobilized primary antibodies and secondary antibodies capture these virus- antibody compounds, both the control (C) and the test (T) line will appear on the test kit. If the virus is not present in the sample, only the control line (C) will still appear as the labeled primary antibodies will only bind to secondary antibodies.
How to perform a SARS-CoV-2 rapid antigen test
SARS-CoV-2 rapid antigen test kits usually use a cassette containing sample and buffer wells, a nitrocellulose test strip, and sterile sample collection swabs. All the required materials and instructions are provided within the kit.
Before you start the test, make sure to check the expiry date of the kit, and confirm that none of the components are broken or missing. In order to prevent contamination, wash or sanitize your hands and lay the components of the kit on a clean flat surface. If you will use a nasal sample, it is generally recommended that you blow your nose before sample collection. Likewise, if you will use a saliva sample to perform the test, it is recommended that you stop eating and drinking 10 minutes before testing.
Following the given instructions carefully is crucial to get accurate results. Therefore, make sure to perform each step as instructed.
A SARS-CoV-2 antigen test typically requires the collection of a nasal or saliva sample by inserting a sterile swab into the person’s nasal cavity, mouth, and throat. After the swab is rotated three to four times as instructed, it is put into an extraction buffer tube and stirred a few times as instructed. Then, the swab is removed, and the extracted sample is applied to the test strip.
The result should be ready in 15 to 30 minutes depending on the device. If the test is positive, both the control line (C) and the test line (T) will be visible as a colored line. If the test is negative, only the control line (C) will appear. If the control line (C) does not appear at all, the test is invalid. However, the results should never be read after the period specified in the instructions as it may lead to false results.
The overall performance of a specific SARS-CoV-2 rapid antigen test depends on the sensitivity and specificity of the test to identify a SARS CoV-2 infection in relative to a reference standard, typically RT-PCR.
Sensitivity refers to how well a test identifies SARS-CoV-2 in samples where the virus is present. It is measured as the percentage of positive cases reported by a reference standard that the rapid antigen test in question detects positive.
Contrarily, specificity refers to how well a test identifies samples that do not contain the virus. It is measured as the percentage of negative cases reported by a reference standard that the rapid antigen test in question reports negative.
Factors influencing test performance
The overall performance of the SARS-CoV-2 rapid antigen tests may be affected by several factors. The timing of the test in relation to the illness onset, the concentration and duration of viral shedding, cross reactivity with other viruses, and the immune status of the patient affect the test performance by shaping the viral load in the time of testing. The performance may also depend on specific qualities of a given test, such as the sample it uses and the protein it targets. The design of the kit along with the quality of the materials and chemicals may also have an impact on performance. Last, the performance of the test may be affected by the storage and administration of the kit. The test may give false or invalid results if the kit is not stored and performed as instructed.