ELISA - Online Tutor, Practice Problems & Exam Prep

ELISA, or enzyme-linked immunosorbent assay, is a biochemical technique utilizing antibodies to detect and quantify proteins in samples like blood or urine. It is effective for diagnosing diseases such as HIV, Lyme disease, and Zika virus. There are two main types: indirect ELISA, which involves coating wells with antigens, and sandwich ELISA, where wells are coated with primary antibodies. Both methods consist of five key steps: coating, blocking, adding antibodies, enzyme-linked secondary antibodies, and substrate addition, leading to color change proportional to antigen concentration.

ELISA

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. The samples could be blood or urine from a patient, or a solution from cells grown in a lab. 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. ELISAs are also appropriate for screening many different samples at once, so they can save a lot of time.

There are several different types of ELISAs that exist, including indirect ELISAs and sandwich ELISAs. Now, indirect ELISAs 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, followed by the addition of a secondary antibody so that the antigen gets sandwiched between two different antibodies.

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 ELISA, 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 two antibodies. So, the antigen is here in the middle and notice that it's sandwiched between a second antibody, and so this is the sandwich ELISA. In our next lesson video, we'll be able to talk about the setup for an indirect ELISA and then later we'll talk about the sandwich ELISA.

And so that concludes our introduction to ELISA, and I'll see you guys in our next video.

ELISA

In this video, we're going to talk about the setup for an indirect ELISA, and an indirect ELISA can be set up and performed in five general steps. We have those five general steps numbered down below. Notice that the numbers in the text correspond with the numbers of each of the steps in the image down below. The very first step of an indirect ELISA is going to be to adhere to the antigen of interest in a sample to an inert surface in the wells of a microplate. Notice down below, over here, we are showing you a microplate with many different wells, and in the images, we have down below, we're zooming into one particular well. 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. Again, the very first step of an ELISA is going to correspond with step number 1 in our image down below, which is to coat the surface with the antigen of interest. Notice that these red dots here represent the antigen of interest.

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. If we take a look down below, at our image right here, in step number 2, we can see that we're going to block the remaining surface with a non-specific protein, and 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. The primary antibody is going to be specific to the antigen of interest. 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. You can see here our primary antibody here in blue, and it's binding specifically to the antigens of interest. We will also wash away any unbound primary antibody that doesn't bind to any antigen.

In step number 4, we're going to treat the surface with an enzyme-linked secondary antibody. This secondary antibody is going to be specific to the primary antibody. Notice 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. It's binding to the primary antibody, and the secondary antibody is indeed enzyme-linked. You can see that it's covalently linked to an enzyme represented by "e" here. Together they form the enzyme-linked secondary antibody, which again is going to bind to the primary antibody. 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 going to catalyze a reaction that forms a colored product, and the yellow color that we see down below corresponds with that colored product.

In step number 5, all we need to do is add the substrate for that enzyme-linked antibody, and the substrate is going to get converted into a colored product. Then we can monitor the color intensity using a spectrophotometer and measure absorbance in each of these wells. The color intensity is going to be directly proportional to the amount of antigen present in the sample. We will be able to detect the presence of an antigen and also quantify the amount of antigen that's present in the sample. The less color there is, such as this well right here, the less antigen there is, and the darker shade of yellow, the more antigen is present. You can see, within a plate, we can get a variety of different shades of yellow. In step number 5 down below, notice we're adding the substrate here, the "s", and then the substrate can be converted into the colored product, forming the yellow color that we see here in this well. This is the setup for an indirect ELISA broken up into these five steps, and we'll be able to get some practice utilizing these concepts as we move forward. In our next lesson video, we'll be able to talk about how the setup for an indirect ELISA compares to the setup for a sandwich ELISA, so I'll see you guys in that video.

ELISA

So now that we've covered the setup for an indirect ELISA, in this video we can see that a sandwich ELISA is performed with similar parallel steps to the indirect ELISA. Notice that we have the same number of steps: 1, 2, 3, 4, and 5. When it comes to a sandwich ELISA, we can see it's almost like we're actually putting together a sandwich. The first step, which is going to be different from an indirect ELISA, is that we're going to coat the surface with a primary antibody rather than coating the surface with an antigen first. Here you can see in blue we have our primary antibody. Then, in step number 2, we're going to block the remaining surface with a non-specific protein such as casein. And then in step number 3, we're going to add the antigen of interest. The antigen is going to bind to the primary antibody just like what we see here in red. It's almost like adding the tomato to a sandwich. The bottom bread of the sandwich is going to act as the primary antibody, and then the antigen itself is going to act like the tomato. In the 4th step, we're going to add the enzyme-linked secondary antibody. This time, the secondary antibody is going to be specific to the antigen of interest. 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. It creates this sandwich-like effect where the antigen is being sandwiched by the enzyme-linked secondary antibody and the primary antibody. Of course, in step number 5, all we need to do is add a substrate for this enzyme-linked secondary antibody. The substrate here is going to be converted into a colored product, to give us this yellow color that we see in the well.

What's important to know about sandwich ELISAs is that because they have two different antibodies bound to the same antigen, it 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. 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. I'll see you guys there.

In an Indirect ELISA, the enzyme-linked antibody will attach to:

• A. The patient's antigen.
• B. The antigen binding region (F_ab) of the primary antibody.
• C. The constant region (F_c) of the primary antibody.
• D. The wall of the microtiter well.
• E. The constant region (F_c) of the secondary antibody.

Here’s what students ask on this topic:

What is ELISA and how does it work?

ELISA, or enzyme-linked immunosorbent assay, is a biochemical technique that uses antibodies to detect and quantify proteins in samples such as blood or urine. The process involves coating a microplate with either antigens (indirect ELISA) or primary antibodies (sandwich ELISA). After blocking unoccupied sites with a non-specific protein, a primary antibody (in indirect ELISA) or an antigen (in sandwich ELISA) is added. This is followed by the addition of an enzyme-linked secondary antibody. Finally, a substrate is added, which the enzyme converts into a colored product. The color intensity, measured using a spectrophotometer, is directly proportional to the antigen concentration in the sample.

What are the differences between indirect ELISA and sandwich ELISA?

Indirect ELISA and sandwich ELISA differ primarily in their initial steps. In indirect ELISA, the wells are first coated with the antigen of interest, followed by blocking, and then the addition of a primary antibody specific to the antigen. In sandwich ELISA, the wells are initially coated with a primary antibody, followed by blocking, and then the addition of the antigen. Both methods then add an enzyme-linked secondary antibody and a substrate to produce a color change. Sandwich ELISA generally offers higher sensitivity and specificity because it uses two different antibodies to bind the antigen, creating a 'sandwich' effect.

What diseases can be diagnosed using ELISA?

ELISA is a versatile diagnostic tool used to detect a wide range of diseases. Some of the diseases that can be diagnosed using ELISA include human immunodeficiency virus (HIV), which can lead to AIDS, chickenpox, shingles, Lyme disease, Zika virus, and syphilis. This technique is effective for diagnosing these diseases because it can detect specific proteins or antibodies associated with the pathogens causing these conditions. Additionally, ELISA can screen multiple samples simultaneously, making it a time-efficient method for disease diagnosis.

What are the five steps involved in setting up an indirect ELISA?

The five steps involved in setting up an indirect ELISA are: 1) Coating the wells of a microplate with the antigen of interest. 2) Blocking unoccupied sites on the well surface with a non-specific protein like casein. 3) Adding a primary antibody specific to the antigen. 4) Adding an enzyme-linked secondary antibody that binds to the primary antibody. 5) Adding a substrate that the enzyme converts into a colored product. The color intensity, measured using a spectrophotometer, is directly proportional to the antigen concentration in the sample.

Why is sandwich ELISA considered more sensitive and specific than indirect ELISA?

Sandwich ELISA is considered more sensitive and specific than indirect ELISA because it uses two different antibodies to bind the antigen. In sandwich ELISA, the antigen is 'sandwiched' between a primary antibody coated on the well and an enzyme-linked secondary antibody. This dual-antibody approach increases the likelihood of capturing the antigen, thereby enhancing sensitivity. Additionally, the use of two specific antibodies reduces the chances of non-specific binding, thereby increasing specificity. However, selecting appropriate antibodies for sandwich ELISA can be more challenging, which is a trade-off for its higher sensitivity and specificity.