Rapid Antigen Test: The Sidekick Of PCR In The Battle Against Covid-19

By Nguyễn Anh Khôi (Kinzhal)

Overview

In the midst of the COVID-19 pandemic in Ho Chi Minh City, the Ear – Nose – Throat Hospital is full of patients waiting at the front gate because, unlike humans, otorhinolaryngologic diseases (i.e ear – nose – throat illnesses) do not fear or care about COVID-19. Crowds in such closed spaces easily spread SARS-CoV-2, the causative agent of COVID, so the hospital wants to test for any COVID positive cases and put them in quarantine as quickly as possible. But given that the standard PCR test takes one day, how does this hospital test everyone without sending them home for the next day? The answer is the rapid antigen test (RAT), which gives a result in 15 minutes.

Thanks to its affordability and simplicity, up to the point an untrained person can easily test oneself, RATs do more than helping hospitals deal with massive crowds. Companies can test their employees regularly without help from healthcare facilities, while people can test themselves at home if they want. Combining acceptable accuracy with features unheard-of for diagnostic tests in a RAT requires a few design subtleties, which will be described in this article.

Basic principles

When the body is fighting against infections, a special type of immune cell produces antibodies. These Y-shaped proteins act as molecular glues targeting the surface of microbes, binding, neutralizing and tagging them to be destroyed by other immune cells. However, an antibody is not a universal tool; each type is tailored to be effective at binding to one and only one type of molecule, which is called an antigen (antibody generator). This special specificity between an antibody and its corresponding antigen forms the core principle of a RAT: if something binds to a SARS-CoV-2 antibody, it should be SARS-CoV-2 itself.

However, a naked eye cannot see the interaction between nanometer-sized antibody particles and their antigen molecules. To solve this problem, designers attach an indicator to each antibody particle, which gives off clearly visible light and helps a test user interpret the test result. In many tests, the light indicator is gold nanoparticles.

Structure of a rapid antigen test

From front to back, a typical test device consists of:

• A sample pad, where the test sample is placed on at the beginning of the test.
• A conjugate pad, holding antibody specific to SARS-CoV-2 and chicken antibody, denoted as IgY. These antibodies are conjugated to gold nanoparticles.
• A reaction pad, where the magic works to bring about the result in several minutes. There are two immobilized lines of antibodies on this pad:
• The first line is the test line, coated with antibodies specific to SARS-CoV-2 antigen.
• The second line is the control line, coated with antibodies specific to the IgY on the conjugate pad.
• A wicking pad, also called absorbent pad, is located at the end, wicking excess fluid and preventing backflow.

Other axillary components in one test kit include a sterile swab, a tube containing a suitable buffer for the reaction and a nozzle cap to drop the analyte onto the test device (COVID-19 Ag Rapid Test Device, 2021).

Test designs of each manufacturer may vary, but the general principles are the same.

Operation

First thing first, a test sample must be taken by inserting the swab into a nostril or a throat, depending on the instructions from the manufacturer. The swab is then dipped into the buffer solution in the tube with some stirring and squeezing to release viral material, if present, into the solution. The role of the buffer solution, apart from dissolving viral particles, is to maintain the correct pH, or acidity, for the reaction to proceed correctly. Next, the tube is closed with a nozzle cap and a few drops of the test solution is applied to the specimen well on the test device. Results should be obtainable after 15 minutes.

After the drops are placed on the specimen well, or a tiny hole at the sample pad, the fluid is pulled across all pads by capillary action, similar to a sponge sucking fluid around it. The test sample first reaches the conjugate pad, which contains anti SARS-CoV-2 antibody and chicken antibody. If the sample is from a COVID-19-positive person, then there should be SARS-CoV-2 antigen in the sample, and it binds to the corresponding antibody in the conjugate pad.

Everything is dragged along to the test line at the reaction pad. The whole antigen-antibody-gold conjugate, previously formed at the conjugate pad, should be trapped at this line due to the presence of immobile SARS-CoV-2 specific antibody. Meanwhile, IgY moves a bit further until stopping at the control line, which is coated with IgY specific antibody. The nano-sized gold conjugate, now present at both lines along with the antibodies, make them appear red. If you are interested in how nano-sized gold particles are red instead of yellow, I suggest you read into surface plasmon resonance (Koczula & Gallotta, 2016).

If the test sample holds no antigen, then everything would happen the same, except that SARS-CoV-2 antibody – gold conjugate would not stop at the test line. Therefore, only the control line should appear red.

Although the IgY and test line do not directly participate in detecting the antigen, they serve as an indicator of a functional test. If the control line appears colorless at the end of the test, then the antibodies somehow fail to move across the reaction pad, so the test result should be invalid regardless of the test line’s color.

As a side note, this type of test’s principle, called lateral flow assay because of the movement of fluid across the pads, is used to test for a plethora of diseases and compounds. For example, if SARS-CoV-2 antibody in the COVID RAT is replaced by a human chorionic gonadotropin (hCG) antibody and with a few minor modifications, we get the well-known pregnancy strip test.

Pros and Cons

The accuracy of a diagnostic test is indicated by two values. They are sensitivity, or the reliability of a negative result, and specificity, or the truthfulness of a positive result. For a rapid antigen test, sensitivity claimed by approved manufacturers ranges from 90 to 99 percent, while specificity is usually very close to 100 percent (European Commission Directorate-General for Health and Food Safety, 2021). This is actually very good for such a simple test. However, please note that these numbers are obtained from test subjects with relatively high viral load (i.e having a lot of viral particles), while figures for a standard PCR test are consistent even at low viral load (Centers for Disease Control and Prevention, 2021).

The strengths of this test are pretty obvious. In addition to being simple and quick, RAT kits are also cheap. According to Ho Chi Minh City Department of Health, a standard PCR test costs 734.000 VND, while most RAT test prices are a bit more or less than 100.000 VND (HIẾN, 2021).

Summary

By harnessing the power of antibodies, the capillary action of fluids and some form of light indication, designers of rapid antigen tests have combined good accuracy and simplicity into a single package. This allows RATs to fill the gaps left open by the complex PCR tests and strengthen our efforts against this pandemic. So, if you suspect that you might have got a COVID infection, feel free to buy an approved RAT kit and test yourself at home.

References

Centers for Disease Control and Prevention. (2021). Interim Guidance for Antigen Testing for SARS-CoV-2. https://www.cdc.gov/coronavirus/2019-ncov/lab/resources/antigen-tests-guidelines.html

COVID-19 Ag Rapid Test Device. (2021).

European Commission Directorate-General for Health and Food Safety. (2021). EU health preparedness: A common list of COVID-19 rapid antigen tests and a common standardised set of data to be included in COVID-19 test result certificates. Public Health, Country Knowledge, Crisis Management, Health Security, March. https://ec.europa.eu/health/sites/default/files/preparedness_response/docs/covid-19_rat_common-list_en.pdf

Hiến, T. (2021). TP.HCM: Công khai giá xét nghiệm COVID-19 tại các cơ sở y tế - Tuổi Trẻ Online. https://tuoitre.vn/tp-hcm-cong-khai-gia-xet-nghiem-covid-19-tai-cac-co-so-y-te-20211028145601863.htm

Koczula, K. M., & Gallotta, A. (2016). Lateral flow assays. Essays in Biochemistry, 60(1), 111. https://doi.org/10.1042/EBC20150012