On some things I like to remain neutral, but I’m gonna go out and say it – neutralizing antibodies are awesome! They’re the kind of antibodies scientists really want to see getting made after a person gets injected with a vaccine, and in the blood of recovered patients that they’re hoping to give to sick patients or even non-sick people that are at high risk of catching it (like doctors on the front lines). And making them in a lab is a potential future treatment strategy (i.e. monoclonal antibodies). They’re so valuable because they have the power to prevent a virus from infecting cells! But how do scientists find them? Unfortunately, not with the easy antibody tests – instead they need to use neutralization tests like Plaque Reduction Neutralization Tests (PRNTs) or Pseudovirus neutralization tests.
You might not have heard much about those tests, but you’ve probably been hearing a lot about antibodies in general recently. Before I got into science they seemed like these magical little superheroes that just popped up in your body outa nowhere and chased off infections. But they’re actually “just” little proteins and your immune system spends a lot of time making ones that are just right for the threat your body is facing. It involves a lot of trial and error, randomly mixing and matching unique part (variable regions) and generic adapter parts (constant regions) to make antibodies that bind specifically to some part of the invader. Once they find ones that work, they make a lot more and use them to help your body recognize the virus & call for help. That’s pretty superhero-y in and of itself, but the really really powerful antibodies are the “neutralizing antibodies” which single-handedly block the virus from getting into cells, such as by binding to the part of the virus that docks onto cellular receptors, thereby hiding it.
There’s nothing really “special” about Neutralizing Antibodies (NAbs), they just happened to have a molecular makeup that was well-suited for binding a crucial spot on the virus. For SARS-Cov-2, the novel coronavirus that causes the disease Covid-19, this crucial spot is on the Receptor Binding Domain (RBD) of the Spike protein, that protein that juts out of the virus’ oily lipid membrane “crown-like.” “Domain” is just a fancy word for a part of a protein that has some function or structure or something and you need a way in which to refer to it. So we’re just talking about a region of the Spike protein. Normally what happens is that this RBD does what it’s name suggests – it binds a cellular receptor (in the case of SARS-Cov-2, this is the ACE2 receptor). This docking then allows the virus to get swallowed by the cell, where it escapes and hijacks the cells’ machinery to do its bidding. But if neutralizing antibodies get to the virus before the virus gets to the cell, they can stop it from getting in.
Most antibodies can’t do this – these other, so-called “binding antibodies” can only bind parts of the virus – no blocking its entry. All these other antibodies are able to form because, although the Spike protein is the one that’s most obvious from the outside of the protein, antibody-making cells get exposed to the virus’ inner proteins as well because there are immune cells called antigen-presenting cells that swallow the virus, chop up its proteins, and display the protein pieces (peptides) on the surface of the cell to serve as antigens (things antibodies bind).
You can test for antigens specific to any viral protein or protein part by sticking that part to the wells of a plate and adding blood serum (blood w/cells removed). If the blood serum contains antibodies specific to the stuck part, those antibodies will stick too. And then you can detect the stuck antibodies using secondary antibodies that recognize the generic adapter part of the antibody. This is called an ELISA and you can learn more about it here: https://bit.ly/covid19testtypes
But the key thing to realize here is that it doesn’t tell you anything about whether the antibodies are neutralizing or not. Even if you had the Spike protein RBD stuck on there to serve as the antigen, and you find antibodies in the blood that stick, there’s no guarantee that those stuck antibodies are bound in just the right spot in such a way that they prevent viral entry. And, even if you didn’t find anti-RBD antibodies with the ELISA, that doesn’t mean they aren’t there – maybe the piece was just stuck on the plate awkwardly or something. Bottom line, if you want to see if neutralizing antibodies are present, you’re gonna have to put in some harder work, because you need to see if the virus can actually infect cells, not just bind something.
The traditional way to do this is a plaque reduction neutralization testing (PRNT), where you put virus & antibodies onto a layer of non-infected cells in a dish and see if the virus infects those cells. If the antibodies are neutralizing, the virus can’t get in, so the cells survive. But if the antibodies are not neutralizing, the virus will infect cells and, since these cells are just in a dish (no immune system backup), those cells will die – and they’ll infect nearby cells, so you have whole regions of cells dying, leaving dead-cell zones called plaques. The “better” the neutralizing antibodies (either stronger antibodies or higher concentration of them) the fewer plaques will be seen.
A couple details for those who are interested: the cells they use are often “Vero” cells – they’re a line of cells derived from an African green monkey’s kidney, hence the name Vero, short for “verda reno,” Esparanto for “green kidney.” The scientists take the cell-less portion of a patient’s blood (the serum), heat it to kill any virus that might already be in it, and mix it with live virus. Then they make a serial dilution of this (e.g. dilute in half, then dilute that in half, then dilute that in half…) to get a range of concentrations. Then they add these dilutions to cells in dishes and they pour on top some agarose – this is the same sugar-based gel we use to make agarose gels for separating DNA through gel electrophoresis, but here it just serves to keep the virus from spreading around willy-nilly. You want to see how many cells the virus is able to originally infect and then how well those cells are able to infect other cells. You don’t care about whether the virus was able to roll from one side of the dish to another if you accidentally tipped the dish….
In terms of detection, there are different methods to visualize the plaques depending on the virus you’re testing for, etc., and they often involve chemical stains. After counting the # of plaques in your different dilutions, you look to see how dilute you could get it and still have 1/2 the # of plaques as the no-serum-added control plate – this value is called the PRNT₅₀
This assay is typically considered “gold standard” because it’s basically as close to the real thing as you can get. But working with live coronavirus is dangerous – it’s considered a “biosafety level 3” which means if scientists want to do this sort of assay they need to really PPE up and work in special hoods, etc. And their labs have to be certified for this sort of work.
Enter the pseudovirus… If all you care about is whether an antibody can prevent the virus from getting into cells, you just need the parts of the virus that are needed for cell-getting-into, which, in the case of SARS-Cov-2, is the Spike protein. So scientists can make a “fake virus” that looks like the coronavirus from the outside, and can get into cells, but it’s not dangerous. They do this by taking a harmless virus (such as a modified version of vesicular stomatitis virus (VSV) and sticking it into cells along with a plasmid (circular piece of DNA) containing the gene for the coronavirus Spike protein. Those cells then make a pseudovirus that the scientists can mix with blood serum to see if the serum has NAbs.
The pseudovirus looks like the coronavirus from the outside, but VSV on the inside. But not just normal VSV – they can modify the VSV to contain the instructions for making a protein that glows or makes something else glow – like Green Fluorescent Protein (GFP) or firefly luciferase. Those proteins will only get made if the virus gets inside cells. So they can serve as “reporters” – if neutralizing antibodies are present, they’ll prevent that, so no (or less) glow. This offers an easier readout than counting plaques.
Speaking of easy readouts, before I let you go, be very wary of the antibody tests you might be seeing people try to cell you. They’re pretty unregulated and often pretty bad… More here: https://bit.ly/rapidantibodytests
more Covid-19 resources: https://bit.ly/covid19bbresources
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0