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Posted by star on 2017-11-21 11:19:16 Hits:447
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Today, I am going to be talking a little bit about the concept behind Elisa which are very common laboratory technique and its going to be helpful to know how it works for a number of reasons whether its a class you are taking or a procedure that youre trying to master. 
So basically what were going to start out with is looking at some samples. If we were to zoom in on them we would see that theres a mixture of proteins inside of each of these tubes and each different type of protein is represented by a different color or different shape of the squiggly line. What I want to do is measure the concentration of a particular protein. So in this case I want to know how much of the green protein I have in each of these tubes and so basically I want some way to measure that. We can graph it for each of the samples. Elizas are really a powerful way of doing that. In order to understand how the Eliza works we have to know what an antibody is in the basic way that antibody works Basically speaking, antibodies are proteins that the immune system makes and whats special about an antibody are they have these really sticky regions that sticks specifically meaning that they only can stick to certain things or only certain shapes and these things which normally stick to viruses and bacteria. They stick to our protein of interests. When our protein of interest comes into proximity of the antibody it sticks irreversibly to the other proteins because they have a different shape. They dont stick to this particular antibody, they might stick to a different one but this one only stay that protein so we can use that antibody or one like it to detect how much of the protein there is in these samples and in order to analyze, one the thing we really have to start with is something called an Eliza plate. Eliza plate is basically a 96-well plate that has a special surface which binds protein really strongly. If we take our proteins from our sample and put it into one of the wells of this plate then what will happen is over time the proteins will sort of settle and stick to the surface of each well of that plate. Then what we can do is adding one of these antibodies and were going to call it a detection antibody because what it has is this little green thing representing an enzyme that we can stick to these antibodies and the enzyme can produce a color change which allows us to measure how much of our sample there is. What we do then is adding the detection antibody which the proteins have stuck to the surface of each well. After adding the detection antibody and over time, the antibody will stick specifically to the protein of interest and then after wash away the extra unbound detection antibody. Next we can do is adding a certain chemical which is clear and but when it reacts with the special enzyme stuck to the detection antibody, it changes to a blue color and then if we add some acid, the blue color changes to a yellow and in the end by measuring how much yellow color we have, we can then determine how much protein there was. Theres a direct correlation between the amount of the yellow color that is produced and the amount of our original protein of interest in the sample. So this technique is called a direct Eliza and the word “direct” refers to the fact that the detection antibody sticks directly to the protein of interest. 
The next thing we want to talk is whats a indirect divisor and an indirect allies. It basically works the same way. So we just have to back up a couple steps to when we stuck our proteins to the dialyzer plate and the indirect Eliza makes use of two antibodies instead of one. The first is the primary detection antibody and that antibody is just like in the last slide with the direct Eliza. It sticks directly to the protein of interest. But theres no enzyme stuck to it now. what we have to do then is introduce a second antibody called the secondary detection antibody and then antibody will stick to the first antibody. It doesnt stick to anything else so basically this gives us a little bit more flexibility in terms of choosing antibodies that we can use to detect different proteins. There are some advantages to using two antibodies instead of one which Im not going to go into right now but the basic idea is that we had to use two antibodies and the eventual chemical reaction and the enzyme that changes the color from clear to blue and then eventually with acid to yellow. All that is happening farther away from our protein of interest but this idea is the same in that the amount of yellow is still correlating directly with the amount of our original protein. So the term indirect for indirect allies that comes from the fact that the secondary detection antibody is sticking indirectly to the protein of interest as opposed to sticking directly to it. So one thing that I did mention is that theres a lot of empty space between the protein. So one thing that will happen is if we dont somehow cover up that empty space, our detection antibodies will stick to those empty spaces and give us a false measurement meaning that we think that theres protein there when its really just empty space. So the step that I didnt mention in order to avoid confusion before is basically called a blocking step where you add a little bit of a reagent. Usually its just a protein, like bovine serum albumin which is not the protein of interest for your study, so you just add some of that extra protein in and it covers up all the empty space and then from that point forward, you can continue with the Eliza as I told you before. 
I want to talk about is something called sandwich Eliza and thats actually what this article is going to be covering. So what exactly is a sandwich Eliza? Well basically, the concepts are all the same, the only difference is before we add our sample, we first add something called the capture antibody and the capture antibody is the same as before it just sticks specifically to our protein of interest. Now the reason we add the capture antibody first is because we want it to be the only thing that sticks to the Eliza plate. After the capture antibody sticks, were going to wash away all the excess and then we can block the surface just like before. The reason that this is powerful is because now we can add in our sample and just our protein of interest is going to stick to that capture antibody, so all the other proteins are just going to wash away and this gives us a lot more sensitivity because now we can really capture all of the protein of interest in our sample. Were going to get a lot more sensitivity with this technique. From this point forward everything is pretty much the same as the direct and the indirect Eliza in that you just add some detection antibodies and the color change is produced and that correlates with the original amount of protein in the sample. The only difference between the sandwich Eliza is that we started with coding a capture antibody and so the protein of interest really gets sandwiched between these two proteins which is why its called the sandwich Eliza. I hope that can clarify how these different Elizas will work 
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