Is monoclonal or polyclonal the way to go when you’re playing antibody “Where’s Waldo?” Whether in the body or in the lab, if you want to identify something specific (Waldo) you can use antibodies to help you find it! And bind it! But you want to make sure you find the real Waldo and don’t get confused by the fakers! A discussion on MONOCLONAL & POLYCLONAL ANTIBODIES in a Where’s Waldo analogy – any takers?

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”

The way they “keep watch” is through their antigen recognition site (paratope). An antigen is just the sciencey name for the thing that an antibody recognizes. If an antigen has a part (epitope) that matches the antibody’s antigen binding site, it can bind and trigger an immune response. B cells that make antibodies that recognize things that are supposed to be there (self things) don’t make it past quality control so you don’t just constantly set it off. 

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 a specific strain of cold is about to sneeze on you. And they don’t even know what potential options are out there. So they don’t know what to make – so they “experiment” – they recombine pieces of DNA to make antibodies. Antibodies have a “generic” adapter region (constant region) and 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 in yesterday’s post 

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 I say “recognize” it’s not that the antibody can go – “oh hey that’s Waldo!” – all it means is that the antibody binds it – and when it does, it still doesn’t even know the identity of the thing it’s bound to, just that it likes to bind it. And they bind because of the same reasons any molecules bind – intermolecular forces (shape, charge distribution, etc. complement each other)

It’s not like a fingerprint, where it’s recognizing something totally unique just something that happens to have certain qualities that happen to complement its own certain qualities – it’s not looking for a soulmate, just something that meets its minimal standards. 

As a result, you can get cross-reactivity -> antibodies that “recognize” more than one thing if they “look” similar -> a promiscuous antibody that’ll bind Waldo OR Joe (whose ID they still don’t know).

And, since the part they’re “recognizing” is just a little part of Waldo or Joe, and different antibodies “recognize” different parts, they can get “confused” by different proteins. (remember, they’re not really “looking” for anything – they’ll just bind what fits)

Say, for instance, you’re interested in detecting Waldo. You have 1 antibody that recognizes Waldo’s hat, 1 that recognizes his striped shirt, and 1 that recognizes his shoes. The 1st antibody might “confuse” someone with a similar hat as being Waldo whereas the other 2 don’t care about the hat. But the 2nd could get “confused” by someone with a similar shirt and the 3rd by someone with similar shoes.

A mix of antibodies that recognize the same antigen, but different parts (epitopes) of that antigen is called POLYCLONAL – they’re derived from cells made from (clonal) many (poly) different parent B cells, whereas MONOCLONAL is where all the antibodies recognize the same epitope because they come from a single (mono) parent B cell

In the body, it can be good to have a mix of antibodies that recognize something because you can then get multiple antibodies binding to different parts of it, giving you a stronger response. And, in the lab, polyclonal antibodies can give you a stronger signal, which is good if you have a small amount of protein. But, the more different antibodies are in a mixture, the more chances for cross-reactivity

Polyclonal antibodies are easier to produce and therefore cheaper to buy – but you may pay the cost in cross-reactivity. Monoclonal antibodies are harder to produce & thus more expensive – but there’s less risk of cross-reactivity and you know you’re always getting the same product when you buy it

Typically, antibodies are produced for lab use by injecting an animal with some protein or part of protein or whatever you want to detect. That thing goes to the animal’s spleen, where B cells that happened to have made an antibody that can bind it bind it, leading to those B cells being selected for.

The 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.

Scientists can collect the secreted antibodies from the serum (the cell-less part of blood you get when you spin blood in a centrifuge to separate out the cells). What they initially collect contains antibodies against your antigen, but also antibodies against other things – what you want is only ~2-5% of the antibodies. So they have to purify them, which they can do by selecting only things that really do bind the antigen.

If you want a polyclonal antibody, you can stop here. But if you want a monoclonal antibody, there’s still more work to do – after purification, you have antibodies that recognize your antigen – BUT you have a MIX of antibodies that recognize different parts of the antigen (different epitopes)

If you want a single one, you need to go straight to the source – the B cell that’s making that single antibody, not just the antibody it’s making. If you can isolate that B cell, you can be assured that all the antibody that comes from it and its progeny (clonal line) is the same antibody because they have the same edited menu.

Speaking of the cell’s progeny, you’re going to need a lot of them – a single cell won’t give you nearly enough. Problem is, these cells don’t like to grow outside of the body. So they need some help. So scientists fuse them with cells that DO like to grow outside of the body – and inside of the body – cancer cells. They often use a type of cancer cell called myeloma cells

B cells from the spleen of the immunized animal (good at making antibody but bad at growing in a dish) + myeloma cells (can’t make antibody but good at growing in a dish) = hybridoma cells (good at making antibody AND growing)

Now you have an “endless” source of a specific antibody. The antibody can be produced in cell culture, or the hybridoma cells can be injected into the peritoneal cavity (abdomen outside of the organs) of a mouse or rat, where they’ll grow and secrete into the abdomen fluid (ascites) that can then be harvested & usually gives you more antibody than in a dish. So every time you order it you get the exact same thing -> better reproducibility. But you’ll likely get lower signal because there’s only 1 place on each protein that can get bound.

Polyclonal antibodies are usually produced from bigger animals like rabbits, donkeys, sheep, and goats, which have more serum that antibodies can be purified from.But you don’t have an “endless supply” and each batch will be different. 

more on topics mentioned (& others) #365DaysOfScience All (with topics listed) 👉 

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