Ever wonder how rapid antigen tests work? Firstly, Do NOT confuse this with an antibody test! Finding a viral antigen (viral “piece”) in someone tells you the virus is in that someone – now. It’s like catching a robber red-handed. But finding an anti-viral antibody in someone is more like finding a fingerprint the robber left on his way out – it tells you someone was infected in the past (or is currently recovering). Biochemically, antigen tests works similarly to an antibody test, BUT in REVERSE. They look for a viral protein, typically the viral N (nucleocapsid) protein. Here’s how…

Antibody tests are used to detect little proteins called antibodies that your body makes to specifically bind to viral parts to help fight off infections and prevent re-infection. Antigen tests look for those viral parts, which are called antigens and are usually parts of viral proteins. So, these tests give you very different information. Antibody tests tell you if someone “has been” infected – in the past or currently (though antibodies don’t show up until later in the infection). Antigen tests tell you if someone is currently infected – they detect the virus itself which is only there when you’re actively infected, so these tests don’t tell you anything about past infections. Antigen tests are thus a form diagnostic test, along with tests like RT-PCR tests.

Format-wise, they’re “lateral flow assays” – strips in a cassette similar to pregnancy tests, but instead of peeing on them, you “snot on them”. It’s not quite as simple of just blowing your nose (but wouldn’t it be awesome if in the future they had Kleenex that changed color in response to specific infections?…). Instead, how it works is that you stick a swab in your nose (one of those giant not-q-tips), swirl it around a bit to get a good sample, and then swirl that around in some “magic liquid” that has chemicals that break open the viral particles (lyse them).⠀

These “viral particles” consist of the virus’ genetic blueprint (genome), which is a single strand of RNA, and is what the RT-PCR tests look for. The RNA is wrapped up a coating of a “nucleocapsid” protein (N), and enclosed in a protein-studded lipid membrane. One of the proteins studding this oily membrane is the spike protein (S), which is the one that juts out “crown-like.” Because this S protein is responsible for binding to cell receptors and allowing the virus to get into our cells, it’s been getting most of the attention. But the most abundant viral protein is actually that N protein, and it’s this N protein that the new antigen test looks for.  ⠀

The exact contents of the magic liquid are proprietary, but it contains detergents – these look similar to the lipids making up the membrane, so they’re able to wedge their way in between those lipids and break up the membrane, allowing the viral contents to spill out. (this is also why soaps and detergents (which are just synthetic soaps) are able to kill the virus: https://bit.ly/soapsanddetergents )⠀

Now that N’s out, the test needs to capture it and show it to us. ⠀

This capturing is actually done with anti-N antibodies. But these aren’t antibodies that come from the person being tested. Instead, they’re antibodies that have been made in a lab, attached to a colored or fluorescent thing and put onto that strip of paper in the cassette. ⠀

These tests use antibodies for the same reason your body does – they’re good at specifically binding viral pieces, helping the body do things like flag infected cells for destruction, call for backup, and keep watch after the virus has been conquered in case it tries to return (which is why antibody tests can detect past infection). ⠀

At the molecular level, antibodies (aka ImmunoGlobulins) are “just” little proteins which have generic adapter parts (constant regions) as well as unique parts (variable regions) that allow them to bind specifically to different viral parts (antigens). “Antigen” is just a shorter way of saying “that specific thing that the antibody binds to” and it typically is part of a viral protein. Terminology-wise, we typically specify the antigen after “anti-“ So, an anti-N antibody is an antibody whose antigen is the N protein. And these are the ones the test uses – in a couple different ways.⠀

Note: There are different layout strategies for doing these lateral flow tests & I don’t know exactly how each specific one’s organized inside the cassette since they keep those details inside the company as far as I can find. So I’m just going to describe one of the common schemes that is used. https://www.jacksonimmuno.com/technical/products/applications/elisa/lateral-flow/immunoassays-introduction 

When you stick that  sample of spilled-out viral “guts” onto one end of the strip, the viral gut parts get a chance to interact with labeled beads or particles attached (conjugated) to anti-N antibodies. If there is N protein present, the anti-N antibodies will latch on. And then, thanks to capillary action, the mix gets wicked through the paper strip to the other end, similarly to how water spreads across a paper towel. (capillary action seems like magic but it’s really just because water’s super sticky but also likes to spread out, so it pulls itself through spaces in the paper’s fibers, dragging its friends with it).  ⠀

So, now you have your N protein bound to an anti-N antibody that’s bound to a fluorophore. All that’s left is to catch it. And we need to catch it separately from any antibody that is unbound, so we don’t confuse them. This is where the second set of antibodies comes in. A line of stuck-on anti-N antibodies is waiting to greet them when they reach that part of the strip. These bind to the (now-labeled) N protein – if it’s present. If the N protein isn’t present, the labeled antibody will keep flowing through to the wicking pad. So the labeled antibody will get stuck on the T line if N is present, but not if it isn’t. Now you just need to look to see if it’s there.

Because, for a negative test, you’re looking an absence of a signal, you want to make sure that there’s not just no signal because the test is defective. This is where the control line comes in. The details vary by test but usually there’s a second labeled antibody present on that conjugate pad. It’s not going to stick to anything in the sample, but it will stick to antibodies on the second, control line (C) that are specific to it. So you should always see a line on the control line, regardless of whether the test is positive or negative. 

Antigen tests are less sensitive than PCR tests, but that’s not necessarily a problem if what you’re using them for is to detect whether a person is infectious. More here: http://bit.ly/reallyrapidtests

more coronavirus resources (including details on how the other tests work) here: https://bit.ly/covid19bbresources

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