Rapidly awaiting rapid antibody tests! Everyone wants to know a date at which they can un-isolate, and we keep getting told it depends on testing, testing, testing. There are a lot of kinds of tests and it can get confusing, but they can be divided into 2 main categories: Diagnostic tests look for evidence of active (likely contagious) infection (e.g. is my sore throat corona?!) and are important for figuring out where current outbreaks are, who needs to be contact-traced, quarantined, etc.. Antibody tests look for evidence of past infection (or late-stage current infection)(e.g. did I have Covid-19 without even knowing?!) and are important for determining the true extent of disease spread, accurate mortality figures, etc. And they might be able to tell us who could be immune from re-infection. But this is a big might! Especially if you’re relying on rapid, qualitative, antibody tests (the ones that look kinda like pregnancy tests) instead of lab-based quantitative ELISA tests (which are done in plates). Let me explain…

Whereas diagnostic tests look for the virus itself (typically from nose or throat swabs), antibody tests look for evidence of a person’s immune response to the coronavirus (typically in the blood, so they’re a type of “serological test”). Rapid antibody tests use lateral flow immunochromatography to look for little proteins called antibodies that an infected person made to specifically target parts of the coronavirus and call in the immune system to attack it. Those terms make it sound a lot more complicated than it actually is. It’s really a lot like a pregnancy test, except you bleed on it instead of peeing on it and you’re looking for evidence of past disease instead of future babies.

In both situations, a liquid sample from a patient is applied to one side of a paper strip (usually encased in plastic). The sample is then wicked horizontally (flows laterally) to the other side of the paper. Along the way, the molecules in the sample (antibodies?!) meet molecules that are stuck onto the paper strip. If they “recognize” each other, they stick, while everything else keeps moving. And then you look to see if stuff stuck. Because you’re separating based on differential interactions between a mobile phase and a stationary phase, we call it “-chromatography.” And, the use of antibodies (aka immunoglobulins) leads to the “immuno-“ part in the name. 

Before I dive into the details of how these tests work, it’s really important to note that these antibody tests CANNOT tell you if a patient is still contagious. For that, you need a test that looks for evidence of the virus itself, not the host’s response to the virus. So, diagnostic tests are often run in addition to the antibody tests. 

Another really important caveat to antibody tests is that these sort of rapid tests CANNOT tell you for sure if a person is immune to re-infection. Scientists still aren’t certain if people who have been infected have protection against re-infection. And, if so, how long that protection lasts – and this can differ from person to person. Because each infected person has to mix and match to find successful antibodies, they can come up with different solutions. Some antibodies aren’t as strongly-binding as others, and different people can produce different levels of antibodies, which may also vary over time, etc. 

Scientists can use lab-based tests called Enzyme-Linked ImmunoSorbent Assays (ELISA) tests to get quantitative information about what types and how much antibodies someone has. This can give a better sense of how strong a person’s immune guard is, but even then, they still can’t tell us for sure about re-infection risk.

Those tests do however, give a better idea than you can get from the rapid tests which can only provide qualitative information (no actual numbers, more of a yes/no or a descriptive, subjective readout (e.g. “somewhat darker band”)). Similarly to how one of those at-home pregnancy tests doesn’t tell you “how pregnant” you are, you just see a plus or a minus or whatever, rapid antibody tests don’t tell you “how immune” you are. Another way to think of it is like sunscreen. You’re going on a trip (oh, the good old days…) so you ask your friend if they packed sunscreen and they say “Yes.” But then when it’s time to go out for your hike you realize that they only packed a pea-sized amount of SPF 5… https://nyti.ms/3b46aZk 

With these caveats in mind, rapid antibody tests are still a really important epidemiological tool – they help scientists better understand the disease spread and how we can best combat it & return to normal. 

So, let’s meet this enemy we’re combatting… Covid-19 is the disease caused by the “novel coronavirus” SARS-Cov-2. It’s a single-stranded RNA virus, meaning that it holds its genetic blueprint (genome) in a single strand of  RNA. It wraps this RNA up with some nucleocapsid proteins and surrounds that with a fatty, membranous coat. Sticking out of that membrane are the “spike proteins” that give it a halo or crown-like appearance and allow it to dock onto and get “swallowed into” human cells. Once inside, the virus is able to get the host cells’ machinery to make viral proteins based on the viral RNA’s instructions, allowing it to make lots of copies of itself, which can bud out, infect new cells, and do it all again. 

The virus will keep doing its thing until it kills the host or the host kills it. And the way the host kills it is through an immune system response that involves the host’s body learning that the virus is “foreign” and attacking it. This “learning” takes some time because it relies on the host’s immune cells using a “trial and error” approach to create little proteins called antibodies that bind to the viral protein but not the host’s own proteins. Once a successful antibody is found, the cell that made it gets “selected for” and more and more of that antibody is made – when these antibodies encounter the viral proteins, they’re able to recruit help to destroy the virus. And then, once the virus (with its genetic information) is gone, some of these antibodies stick around to keep watch.

So, antibody evidence of infection takes longer to appear than genetic evidence, but sticks around even after the genetic evidence is gone. Thus, tests that look for SARS-Cov-2-specific antibodies aren’t very useful for diagnosing active disease,  BUT they can tell us if someone had an infection in the past, which is also really useful – especially since many people with the disease are asymptomatic, so they don’t get diagnostic tests when they’re infected, and lots of people with the disease AND symptoms haven’t been able to access diagnostic tests. Having antibody tests is critical to better understanding the true extent and nature of Covid-19. 

Last week I told you about the lab-based type of antibody test called an ELISA https://bit.ly/covid19testtypes 

Today I want to tell you more about the lateral flow rapid tests. First off, as of today (April 13, 2020) there are NO FDA-approved commercial at-home tests! So if someone tries to sell you one, it’s a scam! There are, however, according to the CDC, over 70 groups working to develop antibody tests, there are many tests already approved in other countries, and on April 1, the FDA approved (under an Emergency Use Authorization, EUA) the first antibody test for diagnostic use in the US. https://bit.ly/3b3rI8j 

This test (qSARS-CoV-2 lgG/lgM Rapid Test) is sold by Cellex Inc. If you want the nitty-gritty details (you know I did…) you can find more about the test in their fact sheets:  https://www.fda.gov/media/136625/download ; https://www.fda.gov/media/136623/download 

But here’s the gist – first, some quick terminology – antibodies are aka Immunoglobulins and they have unique “variable regions” and generic “constant regions.” In the “R&D stage” of antibody development, different immune cells make different versions of the “variable region” part of the antibody. Once a variable region part is found that specifically recognizes the virus, that unique part can be attached to different types of generic adapters to give you different types of antibodies, which play different roles that I’m not an expert in.

What I do know is that the antibody tests on the market/in development often look for 2 such types: IgM (Immunnoglobulin M) and IgG (Immunoglobulin G) antibodies. IgM antibodies are part of the initial antibody response. They show up mid/late infectionbut then “disappear” as the immune system switches to relying mostly on IgG antibodies, which don’t show up until really late – and they stick around after the fact (but for how long we don’t yet know)

So, if a test detects IgM but not IgG, it’s likely a mid-stage infection. See both IgM & IgG? Likely late-stage. And only IgG? Likely a past infection. Nothing? Either the patient hasn’t been exposed to the virus or your test is faulty – thankfully, as I will explain in a minute, the tests also include a control to make sure that the sample really did flow through. 

How it works is that a sample of the patient’s blood is added to sample well, spotting it on the special paper strip (a nitrocellulose membrane). Then a solution called a buffer is added – this buffer contains salts, pH stabilizers, and other things to help get the sample flowing. And then capillary action (the same thing that makes water spread across a paper towel) wicks the sample/buffer mix to the other end of the strip. But there are several obstacles it encounters on its way…

The first thing it runs into is a “conjugate pad.” “Conjugate” is just a fancy name for chemically-attached, and this pad contains SARS-Cov-2 protein parts conjugated to colloidal gold to give them color. These protein parts are there to serve as antigens (the things antibodies recognize and bind to). Unlike the molecules our sample will run into later in its journey, these antigens aren’t “glued on” to the strip, so if there are matching antibodies in the sample, those antibodies they can bind the colored antigens and take them with them on their travels – until they get stuck…

The next thing they’ll run into are the “test lines” which have “glued-on” animal antibodies that recognize and bind to the generic parts of the human antibodies, “trapping them.” First is a line coated with anti-human IgM. The now-colored IgM antibodies will get stuck here, producing a burgundy colored line, while the rest of the stuff will flow through. Next up is a line coated with anti-human IgG, which will do similarly for the now-colored IgG antibodies, so if a sample has those you’ll get a colored line there. 

A cool thing about this approach is that, since it’s detecting the generic parts of the antibodies, it’s able to capture all those different variants that different people have (since that whole antibody production is a trial and error approach). It’ll also capture a lot of other antibodies for other things, but unless those antibodies are holding onto a labeled antigen, you won’t see it, so no need to worry. 

But there are a couple places where you do need to worry about “false results.” There’s a chance that people who’ve been infected by a similar virus in the past might have antibodies that “cross-react” and bind the SARS-Cov-2 antibodies even though these antibodies weren’t made because of SARS-Cov-2. That could lead to a false positive. And, there’s a chance that people have antibodies against SARS-Cov-2, but not against the protein pieces used, so you could get a false negative. 

Speaking of negatives – how do you know if lack of bands is a real negative or a defective test? The company provides positive and negative control samples that they recommends running each time you open a new lot of the test cassettes, etc. Problem is, once you run the controls, you can’t use that same cassette for your sample. You have to use a fresh one for each test. 

Thankfully, there’s also a control built into every test strip. In addition to the gold-conjugated SARS-Cov-2 antigens, the conjugate pad has gold-conjugated rabbit IgG antibodies and, at the far end of the strip, past the anti-human IgM & anti-human IgG lines there’s a control line coated with anti-rabbit IgG. No matter whether the sample has anti-SARS-Cov-2 antibodies in it or not, you should see a line show up here. And this tells you that at least the sample flow part worked okay. 

The tests only take 15-20 minutes to develop and, while the current tests are only authorized to be administered by healthcare workers, they have the potential to be approved for much wider use (including home-based tests).

A few last notes about diagnostic tests:

Diagnostic tests look for the virus’ genetic information in swabs taken from a patient’s nose or throat (usually). The tests work by taking the RNA in the sample, trying to make lots of DNA copies of portions of the viral genome, and seeing if/how fast copies get made. Traditional tests do this using a method called RealTime-Polymerase Chain Reaction (RT-PCR), which has to be done in a lab but can run lots of samples at a time. The Abbott ID NOW test does it using something called isothermal amplification. It can be done in a doctor’s office or clinic (no lab required) & give you results inn less than 15 minutes, BUT it can only run 1 sample at a time.  Both tests are able to detect infection really on because the evidence they’re looking for “comes with the virus” – there just need to be enough original copies of the virus there that they reach the test’s limit of detection (LOD). What “enough” is tells you the sensitivity of the test. You can learn a lot more about them here: https://bit.ly/idnowrapidtests 

more Covid-19 resources: https://bit.ly/covid19bbresources⠀

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0

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