hello everyone. In this video, I'm going to speak about MEMS, and specifically about realize that the enzyme-linked immunosorbent assay. Now, when I say immunise, there are two types of them. There are the labeled and unlabeled in the immunoassays. I'm not going to speak about unlabeled in United States because there they are not they are not used anymore. Some people still you are still using them but not like before because now there are the labeled immunise a, which are sales, which are much more used nowadays. And we have two very known types of labelled immunoassays, the Eliza and the rear, the radioimmunoassay and the enzyme-linked immunosorbent assay. Now, in this video, I'm going to speak specifically about Eliza, and in the next video, I'm going to mention the rear. However, if you are interested in knowing more about one of these unlabeled immune essays, either the single diffusion or double stranded, you can write me in the

comments and then I was doing. I can do another video speaking about one of these techniques if you are interested in. Now let's start speaking about immunoassays in general. When I say I mean, I say you should something should come directly to your mind, which is antibody antigen. Antibody an added immunoassays depend mainly on the interaction between an antibody and an antigen, which we call them the immuno complex. I can use this interaction in order to qualify or in order to detect a particular protein in my sample or to quantify this protein in my sample. When I say antibody an antibody is a protein which is composed of four chains of amino acids. So first, we have the heavy chains, the inside ones, and the light chains. And each chain is composed of a constant region, the blue one, and the variable region. This variable region can't the binding site, which is used to bind to the antigen. So it's the antigen binding site. An antigen is a protein, is any protein that has a binding site.

specific binding site on its membrane, on its sorry, on its surface. You know that a protein is a chain of amino acid, and then this chain of amino acid can form a secondary and the tertiary, tertiary structure, and then in this tertiary structure, the protein forms specific binding sites on its surface, which are used to bind this protein, which we call an antigen. Now let's see. So this is the antibody, and antibody has a specific binding site to a specific protein, which we call the key lock mechanism. Why we call it key lock? Because it's like a key and the lock. So this key can bind only to this lock, and then this is why the immunoassays are very specific and very sensitive essays, so we can use them to detect a particular protein in a process, a mixture, or in a protein extract, depending on this key lock mechanism. Now let's speak about Eliza. Eliza is a solid-phase enzyme immunoassay, so it's performed on a solid phase, and it's

enzyme immunoassay. It depends mainly on enzymes. We will see exactly how we use it to detect a specific protein, which is an antigen, and it can also be an antibody. We can also detect an antibody using the ELISA. As I said before, we use it to detect this protein and to quantify this protein in a protein mixture. In order to apply ELISA, we usually use this 96-well plate, the polystyrene microtiter plate. And we should pay attention when we choose the plate for ELISA. The plate should have this straight bottom and not this concave one. This is what we use for ELISA. You should pay attention when you choose the plate for ELISA. Now let's speak more in detail. First, I will say generally what is ELISA as a general view. When I, this is the well, okay, this is the well of the plate, the 96-well plate. But this is a zoomed-in well, and this is the protein extract, saying that I have a protein extract.

extracted from a human cell. Okay, this extract is going to contain many proteins, like hundreds or thousands of proteins. Now, what I want is to detect the particular protein in this mixture. In order to detect this protein, first of all, I should design an antibody, a specific antibody for this protein. Then, I should immobilize this protein on the surface of the well. So, I am going to apply the protein extract. The protein is going to be immobilized on the surface of the well, and then this specific antibody, which I design, is going to be attached to this protein, the particular protein. The reason it's called the enzyme-linked is because this antibody is enzyme-conjugated or enzyme-linked, and then this enzyme can be detected by treating a particular substrate. So, when I apply this substrate to the well, the enzyme is going to treat the substrate, giving me a particular signal. This is a general view about ELISA. So, I first need to have my protein, the protein of my interest, a specific antibody to this.

protein and enzyme. So, the antibody should be enzyme-conjugated. And then the substrate, which is treated by this enzyme. Of course, the higher the concentration of the protein in my sample, the higher the signal I will get out of this. So, it's a colorimetric assay. It will give me, at the end, a signal that is gonna be detected by a spectrophotometer. We're gonna see this at the end of the video. Now, this is a general view. Now, let's speak more into details when I have to perform it, how to perform Eliza. As I told you before, when I say Eliza, I directly think about antigen-antibody. This is the plate used to perform Eliza, and this is my protein extract extracted from a particular cell. When I want to perform Eliza, I have several types of Eliza. I should choose one of them. I have simple Eliza, the sandwich model Eliza, and then I have the direct and indirect Eliza. Let's speak about each one of these in details.

and then you will find yourself able to understand. To fully understand the technique of Eliza, so let's start speaking about simple direct Eliza. So simple Eliza and direct simple direct Eliza. You will see what what do I mean by this. As I told you before, this is the well, this is a zoomed in well from the plate from the Eliza plate. First of all, in order to detect my protein, I should apply the buffered protein sample in the well. When I say buffer proteins some plate that will it's because when I extract the proteins from from the cell, the proteins will be in a buffer. Normally it's the PPS or another buffer, but the most-used buffer to extract proteins or the P is the. So first of all, I apply the protein sample in the well. Of course, the protein sample doesn't contain only my protein, but it contains hundreds or thousands of proteins. So all these proteins are going to be attached to the surface of the well. As we said before, in order to

detect the protein of my interest. I should have a specific antibody for this protein, but I cannot apply the antibody directly because otherwise this antibody may bind to the bottom of the well, and then it can give me a false signal. So, before applying the antibody, what I do is something called surface blocking. To block the surface, I use a non-reacting protein. Usually, we use BSA or casein proteins just to block the surface of the well. In this, when I block the surface, then I don't have any more space for any more proteins to be attached to the surface of the well. In this moment, I apply the antibody, which is the specific primary antibody. This antibody is going to detect the specific binding site in my protein of interest, and it's going to bind to it. So, as you see here, this antibody can bind specifically to this binding site on the protein of my interest. In simple direct ELISA, the primary antibody is specific to the protein of my

interest is enzyme-conjugated. What I do next is that I wash out. So, all the antibodies which are not bound will be washed out using buffer or anything. Yeah, usually we use buffer to wash out. And then I will have the primary antibodies, specific primary antibodies attached to my protein of interest and conjugated with a specific enzyme. What I do next is to apply the substrate, and this substrate is going to be treated by this enzyme to give me the particular signal which is going then to be detected, as I told you before, by a spectrophotometer. Now, this is simple ELISA and what we call direct ELISA. Now, we're gonna see the difference between simple, direct, and indirect ELISA. So here I'm gonna tell you what's the difference between the direct and indirect ELISA. In indirect ELISA, the beginning of the process is the same. So I apply the buffered protein sample, the proteins in my extract is going to be attached to the membrane, and then I block the surface of the membrane.

using a non-reacting protein, as we saw before, and then I applied the specific the primary antibody, which is specific antibody to the protein of my interest. The primary antibody is going to bind specifically to the protein of my interest. Now, there is something I want to speak about here is that how we prepare or we design the primary antibody. Of course, we can buy the primary antibody, specific primary antibody, from special companies. There are companies who design primary antibodies, we combine the primary antibodies from them, but how do they prepare a primary antibody? Primary antibody is prepared by injecting the protein of my interest in a certain animal, like in a mouse, for example, or in a monkey, or in a rat, doesn't matter, and then we extract the species, and then we let the mouse preview the immune system of the mouse to produce the primary antibody specific for this human protein, and then we extract this primary antibody from the animal. So what we have here is that we have a human protein bound to a primary like

mouse antibody. Let's say, then we wash out this unbound antibodies, washing out, and then we apply something called the secondary antibody. The secondary antibody is enzyme-conjugated, so the secondary antibody is going to detect the primary antibody, and it's going to bind to it. And then I apply the substrate. Then I wash out, just we always wash out between the steps, and then I apply the substrate, which is going to be treated by the enzyme and give me the signal. Now the question is, why did the secondary antibody detect the primary antibody? Is that because the secondary antibody is species-specific. So, as I told you before, we have here human proteins, and then we have this mouse protein. The secondary antibody can be anti-most like can be an antibody that can detect any mouse protein, and in this, well, we don't have any Mouse protein, but the antibodies, the primary antibodies, and so the secondary antibody is going to detect the primary antibody. Now you may ask yourself, why did I add this extra step? We I could have just

conjugated the enzyme with the primary antibody and detected directly. Why did I add this extra step in the indirect Eliza? The question: there are two reasons why would I use indirect Eliza. First of all, this is much cheaper than direct Eliza. Why? Because these 2D to design a primary antibody is very expensive. Why? Because this primary antibody is very specific to only one protein. So, this primary antibody can detect only one protein, and it's expensive to produce. So, imagine if I want to conjugate every primary antibody with an enzyme, this is gonna be very, very expensive. While this secondary antibody is species-specific, so this secondary antibody can detect any protein coming out of a particular animal. So, this indirect Eliza is much, much cheaper than direct Eliza. The second reason why one would I use indirect Eliza: it's because primary antibody can bind several secondary antibody like this. So, this is one primary antibody. It can bind several secondary antibody, and then the signal coming out.

of it. When I have like low concentration of the protein, the signal coming out of this is going to be higher. And then this indirect Eliza is more sensitive than direct Eliza. Now let's compare this indirect Eliza and this direct Eliza. As we saw before, a primary specific antibody conjugated with a product conjugated with an enzyme, which is going to give me the signal. And this is the indirect Eliza with that with one extra step. Now everything we saw here is the simple Eliza. In simple Eliza, we have the proteins, we have the antibodies, and then either primary or secondary conjugate enzyme conjugated antibodies. There is another type of Eliza called the sandwich modal Eliza. As we saw, so here I told you, we have this simple Eliza and the sandwich mother Eliza. Definitely, you've heard about this sandwich mode Eliza because it's very well known nowadays. It's a very good technique that is used nowadays. So let's see how or what is sandwich model Eliza.

in sandwich model Eliza, we don't apply our sample directly in the well, but first we apply something called the capture antibody. This capture antibody is going to specifically capture our protein of interest. So what we do then is to block the surface as we saw before, and then we apply our sample. So when we apply our sample, the buffered protein sample, only our protein of interest is going to be bound to this capture protein, and the others, the other antibodies, are not going to be attached to this capture plot of antibody because this capture antibody is specific for the protein of our interest. So when we wash out, then we will have only the protein of our interest bound or attached to the surface or to the capture antibody. What we do then is to apply detective until detection antibody we call it, and this detection antibody is going to be bound to the protein of our interest. And then what we do is to detect this antibody. So this on the detection antibody is going to be detected either directly as we saw before, so either this detection antibody

can be enzyme-coupled, and then we apply the substrate, which is going to give us the signal. Or we can apply a secondary antibody, the indirect way. We can apply the secondary antibody, which is enzyme-conjugated, and which is going to give us the signal. As we can imagine here, the signal coming out of the sandwich model ELISA is much, much higher than the signal coming out of the simple ELISA. Because here we only have our protein of interest attached, and we don't have any other protein attached in the well. So the signal coming out of this is much higher. And so the sandwich model ELISA is much more sensitive than the simple ELISA, especially if we use this indirect detection. So the signal coming, as I told you, the secondary antibody, multiple secondary antibodies can bind to one primary antibody. And then we can get a very high signal, even if we have a very low concentration of protein in our sample. This is simple and sandwich model.

Eliza, now there is another type of Eliza called the competitive Eliza. I'm going to tell what is competitive it Eliza just in case you heard you for you heard about yeah you hear about it that you know what is competitive Eliza. Competitive Eliza is a completely different type of Eliza. In competitive Eliza, what we have is we have our protein sample. We mix our protein sample with the specific antibody with an excess amount of specific antibody. We mix them together in one tube and then what's going to happen is that the antibody is going to detect the antigen, which is the protein of our interest, and it's going to form this immuno complex, the antigen-antibody complex. And we will also have some excess amount of antibody swimming in our mixture. What we do now is that we apply this mixture containing the antibody antigen complex and the three antibodies to we apply it to an antigen-coated well. So this well is an antigen-coated well coated well. We can bring this well.

and apply first this antigen. So instant antigen coated well, and then we apply our mixture into it. What's going to happen here is that the antibodies, which are already bound to an antigen, are going to stay in them in the solution, and the three antibodies, which are not bound to the antigen, are going to bind to this antigen, which is attached to the bottom of the well. Now what we do is that we wash out everything, and then what's left here is the antibody, which is attached to the antigen, which is attached to the surface of the well. We apply then, we then apply the enzyme-coupled secondary antibody, which is going to detect the primer, this primer, or this antibody, and then the substrate. This thing here, it that the, that the, the more the signal I get out of this, it means the less the protein I have in my sample, because when I have less protein in my sample, then more free antibody will be in my mixture, and then more signal will be coming out of this. But when I have more proteins in my

sample then much of the antibodies will be bound to the protein, and then a very few amount of antibodies will be bound to due to this antigen, and then I will get less signal out of this. This is competitive, this is what we call competitive Eliza. In case you hear about this, I just wanted to tell you what is competitive Eliza. Now let's move to the detection. How do I detect the signal from Eliza, and how do I the results? In order to detect the signal coming out of Eliza, as I told you before, the very important thing in the enzyme, we always have in Eliza, we always have an enzyme because it's an enzyme-linked immunosorbent assay. This enzyme can be either HRP, for example, the most, these are the most two used enzymes nowadays, there is the HRP enzyme, and there is the AP enzyme, the alkaline phosphatase. As we said before, or as we saw before, every enzyme has a specific substrate they eat. The substrates we can use with HRP can be

either the OPD, which gives me an amber color. The color you're seeing here, this is in this plate. We used HRP enzyme and OPD substrate, so this is the amber color. Or at CMB, which gives a blue color. Or the a BTS, which gives a green color. The substrate we used with alkaline phosphatase is B MPP, which gives yellow color anyway. So, as I told you, in order to detect the presence or the quantity of the protein in my extract, I should use an enzyme with a substrate. We saw that before. Now, how to quantify the amount of protein or the concentration of protein in my sample. In order to do this, I should first design my plate. In order to design my plate, the plate should contain the following things. First of all, the samples. The sample should be applied in duplicate or better in triplicate. It depends on how many samples do I have. As an example, in this play, they had 40 samples. So, a lot of samples. And this is why they applied every sample twice. But if I have for

example the samples I would apply every sample three times. Triplicate is better than duplicate because then you eliminate the chance of the error chance when you have triplicate of the sample. Okay, second, which is very important in the standard curve. The standard curve is a mixture or different dilute solutions of a certain protein. Usually we use BSA or any other protein. We prepare a serial dilutions of this protein and then we apply it in order to prepare the standard curve. So here for example we have different concentrations of the proteins: 0.5, 1, 2, 4, 8, 16, so on. Yeah, like as I told you, the protein can be BSA or any other protein. So we add. Also, the standard curve is applied in the placate. I can also apply it in triplicate. Then I need a positive control. The positive control is. So the positive control is treated exactly at the other samples. The positive control is a known concentration sample. So it's a sample of a known concentration and I treated exactly at the other samples.

it's just to ensure that the concentration. I'm getting out of this positive control is it's right, you know. Just a positive control. And then the blank samples, you see here, the blank samples contain everything but proteins. Is just to use it. We use it to as a blank for the spectrophotometer. What we do then after applying all the steps of Eliza, it that we measure the absorbance coming out of every single well. And then what we do is that we draw a standard curve. Depending on these standard curves, the standard curve is absorption versus concentration. As you know here, we know the concentrations of the standard curve. So concentration versus absorption. And then when we, when I have the standard curve, I can use the absorption of every sample here in order to measure the concentration of the protein in the sample. Of course, if I have a very concentrated sample, what I should do is to dilute the sample in order to fit in the standard curve. This is everything I wanted to tell you about Eliza. I hope

now you know better about Eliza. You know better how does Eliza work. If you find so, if you liked this video, don't forget to Like, share, and to subscribe the channel. If you have any question, don't forget to write it in the comments. If you want to know more, as I told you before about unlabeled immunoassays, write me in the comments. And then see you in the next video. Bye bye.

The Principle of Immunoassays/ ELISA (Enzyme Linked Immunosorbent Assay)

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