Has anybody seen the ANTIBODY? Or do I need to use a secondary antibody to find it? 🤓Antibodies, or immunoglobulins, are little proteins that recognize specific parts of foreign proteins (or other molecules) by their “shape.” Our bodies use them to recognize and direct the immune system at things that don’t belong in our bodies (like proteins from bacteria or viruses) – things we *don’t* want to find there. And scientists use them to recognize & measure or isolate specific things – often things we *do* want to find there! When we use antibodies outside of bodies “in vitro”, there’s no immune system to alert, but the antibody can still bind. And this makes the thing you’re looking for easier to find!
Some terminology before we talk about how they work. The “thing” that an antibody recognizes is called an ANTIGEN and the “specific part” of that thing is an EPITOPE. An antigen can be *anything* perceived to be “foreign” that triggers an immune response (even something “harmless” like a peanut) whereas a pathogen is a disease-causing microorganism like a virus or a bacterium). Inside the body, antibody binding can trigger an immune response, which involves the body directing destruction machinery to attack the invader and making more of the antibody that bound.
In the lab, we take advantage of antibodies’ specificity to help us find antigens we’re interested in. One place antibodies are used in the lab are in an experiment called a Western Blot, where the antigen is a protein you’re looking for on a membrane, and the epitope is a specific site on that protein. Much more on Western Blots starting here: http://bit.ly/2Iy5b6r
But basically you separate proteins by size in an SDS-PAGE gel, transfer (blot) them onto a membrane, then use antibodies to probe the membrane to detect specific proteins.
But how do antibodies get their specificity? Organisms have to “learn” what doesn’t belong, so that they can recognize & respond to threats more effectively if the invader tries again. and this learning process is called ADAPTIVE IMMUNITY. There’s so much to learn in science it can get overwhelming – thankfully this type of learning we can let organisms do for us! We let organisms do the selection, then we isolate and use the antibodies we want.
But how does that selection occur? The adaptive immune system (aka acquired immunity) is like natural-selection based evolution of antibodies on a really tiny scale happening *inside* your body – and staying in your body (or at least in your cells & bodily fluid) – it occurs in somatic cells (cells that just “make you”) NOT germline cells (cells that can be used to make children from you)
Our adaptive immune systems work by a sort of trial & error approach – immune cells recombine DNA sequences to make lots of different antibodies. Most of these won’t work, but, when one does, it gets added to your body’s “permanent collection” – the cell that has that “winning lotto ticket” specializes in making it and it makes more copies of itself. This puts the pathogen on the immune system’s “watch list.” Now, if that same pathogen tries to invade you, you’ll have antibodies that recognize it as foreign and call in the molecular assassins
A bit more detail: the “experimenters” are progenitor B-cells. They’re like “blank slates” – gene rearrangement occurs (they mix & match different genetic “options” for the antibody’s final form) & they start making a unique antibody receptor that sticks out from its membrane. Now it’s a MATURE B CELL. But it’s still “naive” – it hasn’t encountered its matching antigen.
If it does, it’ll DIFFERENTIATE into 2 kinds of B cells – EFFECTOR B CELLS (aka plasma cells) & MEMORY B CELLS. Effector B cells make lots of that antibody (each can make millions of the antibody molecule) &, instead of displaying them on their surface, they secrete them into the bloodstream for a wider-reaching response
The memory B cells display the antibody on the surface like the original naive cell, but now in the “permanent collection.” This “permanent” memory isn’t stored in our genome, and it isn’t passed down in our genes (in contrast to the bacterial immune system, CRISPR which I discuss below). Only that subset of cells knows it. Those memory B cells live in the bone marrow (which is why bone marrow transplants “swap” someone’s immune memory) & can circulate between lymph nodes – they secrete a low level of antibodies to keep watch.
So if the body encounters that invader again, it doesn’t have to search through billions of potential antibodies to find a match – it has one in the “permanent” collection, it just has to make more of it.
Instead of messing around with the entire antibody protein, experimentation is restricted to specific “variable regions” which serve as unique parts that recognize different antigens. Throughout the experimentation, a “generic” adapter region (constant region) is kept constant – but that generic adapter part’s only generic for the particular animal that made it (i.e. the adapter part’s slightly different in mice & rats).
You can think of it kind of like different versions of an iPhone that can all charge with the same charger (until Apple decides to create a new “species”) – Apple experiments with changes to the iPhone that don’t interfere with it’s “iPhone-ness” and compatibility with iPhone stuff. But you can’t use a charger that you’d use for your Android phone.
I’m guessing that most of the things they try never make it to market, but their experimenting’s not “random” like the experimenting of our immune system, so there’s a lot less “failure” but also less potential for “creativity”
Even though antibodies are pretty small proteins, they still have multiple peptide chains – antibodies have 2 copies of a heavy chain (~440 amino acids long) & 2 copies of a light chain (~ half that length) & there are different choices for how to make the chains – they can’t mess with the constant regions – the generic part that allows the unique part to be displayed, but they can “experiment” with variable regions. Both the heavy & the light chains have variable regions & they both contribute to the antigen-binding site that forms when the chains link up through disulfide bonds.
The “experimentation” occurs through a process called V(D)J recombination, which rearranges Variable, Joining, and (in the heavy chain but not the light chain) Diversity gene “pieces.” It’s like at a restaurant where you can choose 1 appetizer, 1 entree, and 1 dessert. But after you choose, the other options get cut out of the menu so that every time you go back to that restaurant you have to order the same thing.
The recombination process involves stitching together pieces of DNA and cutting out in-between parts. The recombination process changes the DNA, but since these cells are somatic cells, not germline cells, they won’t get passed down to your progeny, and they only affect the cells that come from them. So the other cells in your body still have all the possible parts to recombine (full menu), but cells that come from that particular mature B-cell can only make that one type of antibody.
This whole recombination thing might remind you of alternative RNA splicing – this is where the messenger RNA (mRNA) copies of DNA genes can be edited in different ways to make different products. more here: http://bit.ly/2AsVdjG
That kind of recombination is at the RNA level, not the DNA level, so it can’t even be passed down inside your body – it’s every cell for itself – if they want to coordinate, they have to rely on external cues like splicing factors that only get expressed at certain times & in certain tissues.
Now that we’ve looked at how antibodies are made, let’s look at how we can use them for “unintended” purposes in the lab. In Western Blots, we usually use 2 antibodies – a primary antibody that recognizes the specific thing we’re looking for and binds it but it doesn’t have anything “seeable” about it & a secondary antibody that recognizes the primary antibody and has something detectable about it. The secondary antibodies usually recognize the generic part (constant region) so you can use things like “goat anti-rat” which is a secondary antibody that recognizes the generic adapter part of a primary antibody made by a rat.
We use this 2-tiered strategy so that we don’t need “fancy” (detectable) antibodies for every single protein we want to look for. Plus, it increases sensitivity because multiple secondary antibodies can bind each bound primary one so it amplifies the signal
You add the primary antibody after you block the membrane with a “generic” protein like BSA or milk proteins so that antibodies don’t just stick to it nonspecifically (the same protein-stickiness that lets the proteins you want stick to the membrane can also let proteins stick to the membrane that you don’t want stuck, leading to high background signal (false alarms)). More here: http://bit.ly/2wIULM2
Then you wash off non-bound primary antibody & add SECONDARY ANTIBODY – this recognizes your primary antibody and has some “detectable” quality like a fluorophore so it will emit light or an enzyme like horse radish peroxidase (HRP) that will convert an uncolored compound you add to a colored compound (chromogenic method) or light (chemiluminescence). Then you do whatever you need to do to detect the detectable thing (e.g. shine light on it or add substrate) & see where you see signal.
Another type of adaptive immune system scientists take advantage of in the lab is the bacterial CRISPR system. Unlike our adaptive immune systems, bacterial immune systems rely on CRISPR guides, which are pieces of RNA that recognize parts of foreign DNA by their sequence.
Both systems rely on “unique parts” that recognize specific invaders and “generic parts” that allow the unique parts to be processed & displayed. In our adaptive immune systems, the unique parts are the variable regions of antibodies, and they come from “trial and error” – randomly producing antibodies and then making more of the one(s) that, by chance, match.
Instead of this random but effective approach, bacterial adaptive immune systems go straight to the source – they take a bit of the invader’s DNA and use that as the unique part. So it, by design, will bind the invader if it tries again – if it survives that initial attack. Much more on this here: http://bit.ly/2JQ1Baq
Immunology’s not my field of expertise, but if you want more info, I found this great blog post: https://epomedicine.com/medical-students/vdj-somatic-recombination-made-easy/
more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0