Antibodies are little proteins that specifically bind viral parts, such as viral proteins, like the spike protein. When someone’s infected, their body makes antibodies against the infector by mixing & matching constant & variable regions to find ones that specifically bind parts of the invader. This allows them to call in for backup from other immune system components when they find the virus. And neutralizing antibodies have the added bonus that they bind to the virus in such a way that the virus can’t get into cells at all – thus “neutralizing” the threat. Such neutralizing antibodies are therefore highly valued. But how are they tested for? 

text a rough (sorry) mash-up from some longer past posts I will link to. blog form also has figures 

Before I got into science, antibodies  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 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 anti-Spike antibodies and the strength of their binding using things like an ELISA, which measures binding to a protein of interest (e.g. coat the wells of a plate with an antigen (e.g. Spike protein) -> add antibodies to test -> let them potentially bind and wash off non bound stuff -> add labeled antibody that binds to the generic part of the first antibody -> wash off non bound stuff -> see if you see signal -> shows you if there was antibody there that stuck). much more here:

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. 

Antibody levels are typically talked about in terms of “titers.” Basically this refers to “how much” antibodies are in the plasma. Typically, when it comes to neutralizing antibodies, they measure how far you can dilute the plasma and still block the virus from infecting other cells. But neutralizing antibody tests are harder to do, so usually tests just look for binding, which can be done using an ELISA. The further you can dilute it, the more (and/or stronger) the starting amount of antibodies. So, for example, a 1:2000 titer would be better than 1:1000, because you can dilute the 1:2000 plasma 2X as much and still have the same antibody effect as you have with the “low titer” one. 

more on antibodies in general: 

When an animal is confronted with something unusual, like a protein from an invading virus or bacterium, it can mount an immune response to destroy the invader, but only if it knows that the thing is unusual, and the way it knows this thing is “foreign” is because it binds to proteins called antibodies that are made by a type of immune cell called B cells, which make antibodies and put them on display (sticking out of their membrane) to “keep watch.” Antibodies are made by a type of immune cell called a B cell as part of the adaptive immune response when you first encounter the thing. So that next time you encounter the thing, alarms will ring!⠀

All antibodies bind to the thing they “recognize” (often a viral protein) but if antibodies happen to bind to a virus part in such a way that they prevent that virus from infecting a cell, we call them neutralizing antibodies (they “neutralize” the viral threat). For SARS-CoV-2 (the novel coronavirus that causes the disease COVID-19) this usually means binding to the virus’ Spike protein (the one that juts out from the viral membrane) in its Receptor Binding Domain (RBD). This binding can be directly in the spot it uses to latch onto the cell’s ACE2 receptor (the Receptor Binding Motif), out-competing it – or it can bind in some way that prevents the Spike protein from undergoing the dramatic shape-shifting (conformational changes) required for the Spike protein to get cleaved by proteases, shoot out its inner parts, latch onto the cell membrane, and fuse it with the viral one. more on that here: ⠀

So if B-cells are making these, how does anyone ever get sick? Problem is, the B-cells don’t know what they’re going to have to face in the future – they can’t predict that someone with COVID-19 is going to sneeze on you. And they don’t even know it’s out there (your cells aren’t tuned in to CNN). So they don’t know what to make. Instead of stocking up on every possible antibody, which is like limitless, they wait until an infection starts and then “experiment” to find what works – different B-cells make different antibodies (they have different variable regions) and then the ones that bind the virus (and not the host) get selected for and more of those get made. This process (called clonal selection because all of the cells made from that original experimenter are clones of it) takes time (which is why antibodies don’t show up until late in infections and your immune system relies on more generic first responses). ⠀

Different B cells make different antibodies (which have different variable regions) but each B cell can only make one type because the random “experimenting” they do to decide what to make involves actually changing their DNA in a process called somatic recombination – don’t worry – the “somatic” tells you that these genetic changes are happening in non-germline-cells – the changes are permanent for this B-cell, and all of the cells that arise from it (clonal cells), but the changes don’t effect any of the other cells in your body, like the ones that would get passed on to your children. ⠀

How it works is that antibodies have a “generic” adapter region (constant region) as well as variable regions which are unique to different antibodies and are where the experimentation occurs. B-cells “randomly” choose what to make by piecing together different options for different parts of the variable regions of antibodies through a process called V(D)J recombination – more on that here:  Immature B cells have a whole menu of options to choose from, but in their maturation process they go through a form of somatic recombination, where they choose from that menu – and remove the other choices so they (and all the cells that come from them) don’t have options anymore – they’re forced to specialize in making that specific antibody. ⠀

Bottom line – different B-cells randomly specialize to make different antibodies. Those antibodies all, by chance, happen to recognize different things. These things can be different molecules all together (e.g. Antibody 1 recognizes Protein A and Antibody 2 recognizes Protein B) OR the things can be different parts of the same molecule (e.g. Antibody 1 & 2 both recognize Protein A, but different parts of it). ⠀

When someone gets infected with a virus, immune cells called “antigen presenting cells” engulf the virus, cut its proteins up into little pieces, and then display those pieces on their surface. Then, B cells that happened to have made an antibody that can bind it do bind it, leading to those B cells being selected for. Those chosen B cells start making more copies of themselves, some of which (memory B cells) continue to display the antibody sticking out from their membrane, while others (effector or plasma B cells) secrete the antibody into the serum. 

more here: &

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