Enzyme-Linked Immunosorbent Assay: Definition & Application

Instructor: Bridgett Payseur

Bridgett has a PhD in microbiology and immunology and teaches college biology.

The enzyme-linked immunosorbent assay is a tool used by scientists to study protein interactions. Read on to learn about the different types of assays and how they work.

Enzyme-Linked Immunosorbent Assays

The enzyme-linked immunosorbent assay is a helpful tool used by immunologists and other scientists. It's also a long, cumbersome name, so we call it an ELISA for short. Let's break down the components of the full name to try to figure out what ELISA is.

Enzyme-linked means simply that an enzyme is used to help show the results. An enzyme is attached to an antibody. At the end of the assay, a substrate is added. The enzyme will perform a chemical reaction that results in a color change. The color change can be directly visualized, or sometimes read with the help of a computer.

Immunosorbent means that something from the immune system is being linked to something else. In the case of ELISA, an antibody is directly or indirectly attached to the plate being used.

An assay is a term for a qualitative test. When you put all the terms together, you see that an ELISA is a qualitative test that uses antibodies and enzymes to see a color change.

What is the Basic Protocol of an ELISA?

No matter what type of ELISA is performed, there are a few steps that are similar. ELISAs are performed in 96-well plates. These are plastic trays that have 12 rows and 8 columns of small wells, where the sample is placed. The wells are also 'sticky' so that protein (antigen or antibody) can bind to it. The size plates allows for a lot of samples to be run simultaneously, so different concentrations can be compared easily.

The first step for most ELISAs is to coat the plate. This means it is incubated overnight with an antigen or antibody in a special coating buffer. The bottom of the well is then covered with protein. The plate is washed to remove excess material after this and every subsequent step.

The next step is called blocking. The plate is incubated with a non-specific protein solution. This takes up all the extra binding sites, to help prevent the next step from binding to the bottom of the plate.

Next comes detection, when an enzyme-linked antibody is added to the well. The antibody should only bind to the material added in the first step since the rest of the well had been blocked.

Finally, the substrate is added and used to see the result. The darker the well, the more interaction there was between the enzyme-linked antibody and whatever was being measured.

Types of ELISA

There are various setups of ELISA that can be used. Each has pros and cons.

Direct ELISA

A direct ELISA is very simple and straightforward. The antigen being studied is coated on the plate. Then, after blocking, an enzyme-linked antibody is added to the wells. The substrate is finally added, and results are recorded.

Direct ELISAs are quicker than other methods, and because there are only a few steps, there are fewer chances for human error. They may be more prone to showing non-specific binding or having higher background noise. They also require a specific enzyme-linked antibody for every antigen being studied.

In a direct ELISA, the well is coated with antigen (blue) and detected with an antibody (red) attached to an enzyme (green).
direct ELISA

Sandwich ELISA

A sandwich ELISA is helpful for assaying if a specific antigen is in a solution. In this assay, the antigen is sandwiched between two antibodies - a capture antibody and a detection antibody.

First, the plate is coated with the capture antibody. After blocking, the solution being measured is added. Then, the detection antibody, which is linked to an enzyme, is added. The substrate is finally added so that the ELISA can be read.

A sandwich ELISA uses two antibodies, the capture antibody (red) and the detection antibody (purple).
Sandwich ELISA

Competitive ELISA

A competitive ELISA is used to determine the antigen concentration in a solution. First, a specific antigen is incubated with its antibody. This antibody is unlabelled and is called the primary antibody. Then, the plate is coated with the antigen and blocked as normal. The primary antibody, bound to antigen, is added to the wells. Finally, a secondary antibody, linked to an enzyme, is added to help

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