ELISA

in this video, we're going to introduce the biochemical technique. Eliza. So Eliza is actually an acronym for enzyme linked immune assortment Ass A. And really, it's just a biochemical technique that uses antibodies to detect and quantify proteins and a sample. Now the samples could be blood or urine from a patient or a solution from cells grown in a lab. Now, Eliza's are appropriate for diagnosing many different types of diseases and noticed down below. In this table, we have some of the diseases that can be detected by Eliza's, which include human immunodeficiency virus, or HIV, which can translate into AIDS, chicken pox and shingles, Lyme disease, Zika virus, syphilis and much mawr. And again, these are just a small handful of the diseases that can be detected by Eliza's. Now Eliza's are also appropriate for screening many different samples at once so they can save a lot of time. Now there are several different types of allies is that exists, including indirect Eliza's and Sandwich Eliza's now in direct allies. US are going to use the antigen first, and so the antigen is going to be coded onto the surface of a well first and then it will be detected with antibodies where, as with sandwich allies, is the antibody is going to be coded onto the surface of the well first, and then the antigen will be added and followed up with the addition of a secondary antibody so that the Auntie Jen gets sandwiched in between two different antibodies. And so notice down below. Over here, we're showing you a image of an indirect ELISA on the left hand side, where you can see that the antigen, which is in red here, is added first in the indirect Eliza, and then it's detected with antibodies. Second, so notice that the antibodies air added after the antigens added. And then with the sandwich, Aleida Eliza, notice that the antibodies coded to the surface first, so you can see here we have the anybody added first, and then we can sandwich the antigen in between two antibody. So the antigen is here in the middle and notice that it's sandwiched in between a second antibody. And so this is the sandwich Eliza. Now, in our next lesson, video will be able to talk about the set up for an indirect ELISA, and then later we'll talk about the sandwich, ELISA. And so that concludes our introduction to Eliza, and I'll see you guys in our next video.

in this video, we're going to talk about the set up for an indirect Eliza and so an indirect ELISA could be set up and performed in five general steps. And we have those five general steps number down below and noticed that the numbers in the text correspond with numbers of each of the steps in the image down below. And so the very first step, if an indirect ELISA is going to be to adhere the anti jin of interest in a sample to an inert surface in the wells of a micro plate. And so notice down below. Over here, we're showing you a micro plate with many different wells. And so, in the images that we have down below, we're zooming into one particular well. And what's important to note is that we can actually separate different samples into different wells of the micro plate, so we can run many different samples at one single time. And so again, the very first step of in Eliza is gonna correspond with step number one in our image down below, which is to coat the surface with the anti gen of interest and so notice that these red dots here represent the antigen of interest. So Step number two is going to be to block any unoccupied sites on the surface of the well by washing with a non specific proteins such as Casey in. And so, if we take a look down below at our image right here, uh, step number two, we can see that we're gonna block the remaining surface with a non specific protein. And so you can see all of these green proteins in between represent the non specific protein that the antibody is not going to interact with. Now, in step number three, we're going to treat the surface with our primary antibody. And so the primary antibody is going to be specific to the antigen of interest. And so notice down below. In step number three, we're going to add the primary antibody, which is going to bind to the antigen of interest. So you can see here is our primary antibody here in blue, and it's binding specifically to the antigens of interest. Now, we will also wash away any unbound primary antibody that doesn't bind to any, um, antigens. Now, in step number four, we're going to treat the surface with an enzyme linked secondary, uh, antibody. And so this secondary antibody eyes going to be specific to the primary antibody. And so what you'll notice is that down below in step number four, you can see that we have our secondary antibody here in black and notice that the secondary antibody is, ah, specific to the primary antibody. So it's binding, uh, to the primary antibody. And the secondary antibody is indeed enzyme linked, so you can see that it's co violently linked to an enzyme represented by E. Here on DSO. Together they form the enzyme linked secondary antibody. Uh, which again is gonna bind to the primary antibody. Now it's important to note about this enzyme that's attached to the secondary antibody is that the enzyme that's linked to the secondary antibody is going to catalyze a reaction that forms a colored product. Uh, and so the yellow color that we see down below corresponds with that colored product. And so, of course, in step number five, all we really need to do is add the sub street for that enzyme linked antibody. And then, of course, the substrate is going to get converted into a colored product. And then we can monitor the color intensity using a spectra fa Tom Attar and measuring absorb INTs in each of these wells on. Of course, the color intensity is going to die. Be directly proportional, um, to the amount of antigen that's present in the sample. And so not only will we be able to detect the presence of an Auntie Jen, but we can also quantify the amount of antigen that's present in the sample. And so the less color there is such as this well, right here, the less antigen there is, and the darker color there is the darker shade of yellow. There is, um the mawr antigen is present, and so you can see within a plate, we could get a variety of different shades of yellow. And so in step number five down below notice, we're adding the substrate here. Uh, the s. And then, of course, the substrate can be converted into the colored product forming uh, the yellow color that we see here in this well, and so really, this is the set up for an indirect Eliza broken up into these five steps. And so we'll be able to get some practice utilizing these concepts as we move forward. But in our next lesson, video will be able to talk about how the set up for an indirect ELISA compares to the set up for a sandwich. ELISA. So I'll see you guys in that video.

So now that we've covered the set up for an indirect Eliza in this video, we can see that a sandwich Eliza is performed with similar parallel like steps to the indirect Eliza. And so notice that we have the same number of steps. Steps 1234 and five. And really, when it comes to a sandwich, Eliza, we could see it's almost like we're actually putting together a sandwich. And so the first step, which is gonna be different from an indirect allies, is that we're gonna coat the surface with a primary antibody rather than coating the surface with an antigen first. And so here you can see in blue, we have our primary antibody. Then in step number two, we're gonna block the remaining surface with a non specific proteins such as Cassie in here and then in step number three. We're going to add the anti gen of interest. And so the antigens is going to bind to the primary antibody just like what we see here in red. And it's almost like adding the tomato to a sandwich. And so the bottom bread of the sandwich is gonna act as the primary antibody on, then Theme. The antigen itself is going to act like this tomato that we see here. And then, of course, in the fourth step, what we're gonna do is add the enzyme linked secondary antibody. And this time, the secondary antibody is going to be specific to the anti gen of interest. And so you can see that, uh, here, the secondary antibody, the enzyme linked secondary antibody, is going to bind to the Auntie Jen. Just like the primary antibody is bound to the antigens. So it creates this sandwich like effect where the Auntie Jen is being sandwiched by the enzyme linked secondary antibody and the primary antibody. And so, of course, in step number five, all we need to do is add a substrate for this enzyme linked, um, secondary. Anybody. And of course, the substrate here is going to be converted into a colored product to give us this yellow color that we see in the wealth. Now, Really, what's important to know about sandwich allies is is that because they have two different antibodies bound to the same Auntie Jen, it actually provides higher sensitivity and specificity than indirect ELISA, which Onley uses one antibody to the antigens. However Ah, sandwich. Eliza can present a lot more challenges to perform, especially when we're trying to select which antibodies to use, uh, to detect the antigens. And so, really, this is, uh, the end of our lesson on sandwich Eliza set up. And we'll be able to get some practice utilizing the concepts that we've learned in our next few videos, so I'll see you guys there.