In this video we're going to introduce the biochemical technique ELISA. So ELISA is actually an acronym for enzyme linked immunosorbent assay, and really it's just a biochemical technique that uses antibodies to detect and quantify proteins in a sample. Now the samples could be blood or urine from a patient or a solution from cells grown in a lab. Now ELISAs are appropriate for diagnosing many different types of diseases and notice down below in this table we have some of the diseases that can be detected by ELISAs which include human immunodeficiency virus or HIV which can translate into AIDS, chickenpox and shingles, Lyme disease, Zika virus, syphilis, and much more. And, again these are just a small handful of the diseases that can be detected by ELISAs. Now ELISAs 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 ELISAs that exist including indirect ELISAs and sandwich ELISAs. Now, indirect Eliza's are going to use the antigen first and so the antigen is going to be coated onto the surface of a well first and then it will be detected with antibodies. Whereas with sandwich ELISAs, the antibody is going to be coated 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 antigen gets sandwiched in between 2 different antibodies. And so, notice down below over here, we're showing you an 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 ELISA and then it's detected with antibodies, second. So notice that the antibodies are added after the antigen is added. And then with the sandwich, Eliza, notice that the antibody is coated to the surface first. So you can see here we have the antibody added first. And then we can sandwich the antigen in between 2 antibodies. 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, we'll be able to talk about the setup for an indirect Eliza and then later we'll talk about the sandwich Eliza. And so that concludes our introduction to Eliza and I'll see you guys in our next video.
2
concept
ELISA
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5m
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Video transcript
In this video we're going to talk about the setup for an indirect ELISA and so an indirect ELISA can be set up and performed in 5 general steps. And we have those 5 general steps numbered down below. And notice that the numbers in the text correspond with the numbers of each of the steps in the image down below. And so the very first step of an indirect ELISA is going to be to adhere the antigen of interest in a sample, to an inert surface in the wells of a microplate. And so notice down below over here, we are showing you a microplate with many different wells and so, in the images that we have down below, we're zooming in to one particular well and what's important to note is that we can actually separate different samples into different wells of the microplate. So, we can run, many different samples at one single time. And so again, the very first step of an ELISA is gonna correspond with step number 1 in our image down below, which is to coat the surface with the antigen of interest and so notice that these red dots here represent the antigen of interest. So, step number 2 is going to be to block any unoccupied sites on the surface of the well by washing with a non specific protein such as casein. And so if we take a look down below at our image right here, in step number 2, 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 3, 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 3, we're going to add the primary antibody, which is going to bind to the antigen of interest. So you can see here's 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, antigen. Now, in step number 4, we're going to treat the surface with an enzyme linked secondary antibody And so this secondary antibody, is going to be specific to the primary antibody. And so, what you'll notice is that, down below in step number 4, you can see that we have our secondary antibody here in black and notice that the secondary antibody is specific to the primary antibody. So it's binding, to the primary antibody. And, the secondary antibody is indeed enzyme linked. So you can see that it's covalently linked to an enzyme represented by e here. And so, together they form the enzyme linked secondary antibody, which again is gonna bind to the primary antibody. Now, what'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 gonna catalyze a reaction that forms a colored product. And so the yellow color that we see down below corresponds with that colored product. And so, of course, in step number 5, all we really need to do is add the substrate, for that enzyme linked antibody and then, of course, the substrate is gonna get converted into a colored product and then we can monitor the color intensity using a spectrophotometer and measuring absorbance in each of these wells. And of course, the color intensity is going to direct, be directly proportional. 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 antigen 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, the more antigen is present. And so you can see, within a plate, we can get a variety of different shades of yellow. And so in step number 5 down below, notice we're adding the substrate here, the s, and then of course the substrate can be converted into the colored product forming, the yellow color that we see here in this well. And so really this is the setup for an indirect ELISA broken up into these 5 steps and so, we'll be able to get some practice utilizing these concepts as we move forward. But in our next lesson video, we'll be able to talk about how the setup for an indirect Eliza compares to the setup for a sandwich Eliza. So, I'll see you guys in that video.
3
concept
ELISA
Video duration:
3m
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Video transcript
So now that we've covered the setup 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. That's 1, 2, 3, 4, and 5. And really, when it comes to a sandwich, Eliza, we can 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 dialysis, 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 2, we're gonna block the remaining surface with a non specific protein such as Cassie in here. And then in step number 3, we're going to add the antigen of interest. And so the antigen 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, and then, the antigen itself is going to act like this tomato that we see here. And then, of course, in the 4th 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 antigen of interest. And so you can see that, here the secondary antibody, the enzyme linked secondary antibody, is going to bind to the antigen, just like the primary antibody is bound to the antigen. So it creates this sandwich like effect where the antigen is being sandwiched by, the enzyme linked secondary antibody and the primary antibody. And so, of course, in step number 5, all we need to do is add a substrate, for this enzyme linked, secondary antibody. And of course, the substrate here is gonna be converted into a colored product, to give us this yellow color that we see in the well. Now, really what's important to know about sandwich ELISA's is that because they have 2 different antibodies, bound to the same antigen, it actually provides higher sensitivity and specificity than indirect ELISA which only uses one antibody to the antigen. However, a sandwich ELISA can present a lot more challenges to perform, especially when we're trying to select which antibodies to use, to detect the antigen. And so really this is, the end of our lesson on Sandwich ELISA setup 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.
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Problem
Problem
In an Indirect ELISA, the enzyme-linked antibody will attach to:
A
The patient's antigen.
B
The antigen binding region (Fab) of the primary antibody.
C
The constant region (Fc) of the primary antibody.
D
The wall of the microtiter well.
E
The constant region (Fc) of the secondary antibody.