 Diagnosing a disease in today's world is all about super sensitive, highly specific molecular techniques. They are faster, precise and so much more reliable than the older practices. All we have to do is submit our samples and boom results by midnight. I don't think anyone could have imagined something like that about 60-ish years ago. Take ELISA for example or enzyme-linked immunosorbent assay, one of the most highly specific and accurate tests out there. ELISA works on the principle of antigen and antibody reactions. You know these proteins that our body produces whenever there's an infectious agent inside of us? Those proteins are called antibodies and that infectious agents they are the antigens and they're super, super specific to each other. Like this antibody X, if this green colored antibody is antibody X, then it will only bind to antigen X and no one else. This pink colored blob right over here, that's antigen X. So this antibody X will only bind to antigen X and no one else. And using this unique style of interactions ELISA can detect all sorts of antigens, antibodies and even other types of proteins like glycoproteins and hormones. Now there are many types of ELISA techniques but the most common and the most specific one is the sandwich type. Yes, there's something called a sandwich ELISA and sadly it does not involve the food that we all love. So we're gonna get rid of this sandwich right here. So the most common and the most specific type of ELISA technique is the sandwich ELISA type. Okay, so let's try out something here. Let's say that we want to find out if a person has HIV or not using the sandwich type of ELISA. Now there are two ways ELISA can detect HIV. One way is to detect certain HIV proteins which will act as the antigens and the other way is to detect the antibodies that would be produced by the patient's body against the virus. Here let's try to detect the antibodies produced against HIV. In order to get started we're gonna need two things. One we're gonna need a blood sample from the patient and the second thing we're gonna need is this plate or tray in which ELISA is carried out and these tiny circles that you can see. Let's zoom in a little bit so that you guys can see it. So these tiny circles that you can see inside the tray these are called wells. Each of these tiny circles they are called wells. Now let's zoom into one of these wells and see exactly what happens during ELISA. Each of these wells is coated with certain HIV proteins. So these triangles which you can see over here these are the HIV proteins aka they are the antigens. So they are the antigens. Now what we're gonna do is that we're gonna add the blood sample into this well, the well with the fixed HIV proteins at the base. Now if the person indeed has the virus their body is going to produce antibodies against the virus against HIV. So if the blood sample contains these antibodies so these Y shaped green colored structures these are the HIV antibodies. Now if these antibodies are present in the sample they are going to bind with these antigens which are present at the base of the well. And just like that we're gonna have our very first antigen antibody binding. So it's gonna look something like this. So you can see that the antibodies have bound with the HIV proteins aka the antigens and it's gonna look something like this. So this is the first antigen antibody binding that has already occurred. In the next step we're gonna add another antigen an enzyme linked antigen. So this is now this antigen is also a HIV protein that will bind with the HIV antibody as you can see it has happened. So this square thingy that you can see this is the enzyme linked antigen. Now this antigen is also HIV protein like I said and it will bind with the HIV antibody. The only difference is that it comes attached with an enzyme. So this pink dot that you can see attached this is the enzyme. Okay so it goes something like this that we have the fixed antigen at the bottom on top of that we have that antibody the HIV antibody and then we have this enzyme linked antigen which is also attached to the HIV antibody. Now this enzyme it could be multiple different things but the most common one that we use is something called HRP or horseradish peroxidase. Why do we always use this and why is this so common because it's cheap and it's super stable. So we use this enzyme most of the time. Now in the final step we're gonna add a substrate for this HRP enzyme and that substrate is this colorless compound called TMB or its full name is tetramethyl benzidine. I know it's a huge name so we'll just stick to TMB instead of this methamethyl benzidine. Really long name. Now when this HRP enzyme acts on this TMB substrate it produces a blue colored compound and the entire solution turns from colorless to completely blue. So you see these blue dots this is the blue colored compound and this turns the entire solution blue, completely blue. The minute we get this blue solution it confirms the presence of the HIV antigen and now we can conclude that the person really does have HIV in their bloodstream. By the way did you notice the sandwich right here? The HIV antibody is sandwiched between two HIV antigen so one antibody is bound to two antigens. It cannot get any more specific than this. On the other hand what do you think would have happened if the person didn't have HIV? Well in that case if the person's blood sample didn't have these HIV antibodies then nothing would bind to these HIV proteins inside the well. No antigen antibody interaction would take place. Even the enzyme linked antigens wouldn't have anything to bind to because there are no antibodies. Then what would happen? Well the enzyme would be absent so there would be nothing present to turn the substrate blue so the solution will remain colorless confirming the absence of the HIV antigen and that is how ELISA has made detection so much more effective and efficient compared to the older techniques.