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Image of microplates and antibodies reactions for article on ELISA.
Published On: April 5th, 2022

There are 3 different types of ELISA that are widely used: direct ELISA, indirect ELISA, and ELISA sandwich assay. ELISA tests have been used to detect and diagnose HIV, Covid-19, food allergies, and many other conditions. You’ll learn how ELISA tests work, how different types of ELISA are used, and why scientists would choose one type over another.

What is an ELISA?

ELISA is an acronym for Enzyme-Linked ImmunoSorbent Assay. It is a plate-based immunoassay used to detect and measure soluble molecules. The target molecule is called an analyte, and can be a protein, antibody, peptide, or hormone. An ELISA measures the antibodies or antigens that respond to the analyte. ELISA tests are used for screening and diagnosing many viral and bacterial infections. ELISA can also detect certain forms of cancer, allergies, and blood diseases.

ELISA immunoassays have 4 steps: coating, blocking, detection, and reading. To conduct an ELISA, a plate is coated with an antigen or antibody. In between each of the 4 main steps, the plate is washed to rinse away any remaining reagents. The plate is then blocked with a substrate like Bovine Serum Albumin (BSA) to ensure that the only binding sites available are on the antigen or antibody. An enzyme-linked detection antibody binds the original antigen or antibody. Finally, a substrate is added which changes color as it interacts with the enzyme. The intensity of the color shows how much of the antigen is present. Automated plate readers can help quickly calculate the values for each well of the plate.

Each of the different types of ELISA use each of the 4 main steps, but indirect ELISA and ELISA sandwich assays add additional steps to the process. Let’s explore the first of the 3 different types of ELISA: the direct ELISA.

Direct ELISA

Direct ELISA uses only a primary antibody in the detection step. The plate is coated with an antigen. If the antigen is present in the plate, the primary antibody will bind to it. The enzyme binds to the primary antibody in direct ELISA. Direct ELISA is the simplest of the different types of ELISA, and it’s also the fastest type.

Indirect ELISA

Indirect ELISA uses a secondary antibody in the detection step. The primary antibodies bind to the antigen, and then the secondary antibodies bind to the primary antibodies. The substrate interacts with the enzyme on the secondary antibodies. Because the primary antibodies can have more than one binding site for the secondary antibodies, this technique can provide a stronger signal. Sometimes this use of a secondary antibody is called an amplification step.

Indirect ELISA is more time-consuming than direct ELISA. It’s also more sensitive, which makes it a good technique to choose when you need to detect even a small amount of an antigen. Indirect ELISA is the most commonly used test for HIV detection for this reason. One potential risk with indirect ELISA is cross-reactivity. If the enzyme reacts with both the antigen and the secondary antibody, the results will be inaccurate. When choosing between different types of ELISA, scientists choose indirect ELISA when they need to amplify the signal.

ELISA Sandwich Assay

The ELISA sandwich assay adds a step before the coating with the antigen. An antibody is added to the plate, called the capture antibody because its purpose is to capture the antigen. Then, the primary antibody is added for detection. Finally, just like in direct ELISA, the enzyme bound to the primary antibody reacts with the substrate to produce a color change.

The “sandwiching” of the antigen between the capture antibody and the primary antibody allows for the antigen to be detected with high sensitivity without requiring the sample containing the antigen to be purified. Because the capture antibody will only bind with the antigen, other proteins in the sample won’t remain on the plate after washing. This leaves only the antigen to bind with the primary antibody.

The sandwich assay has the highest sensitivity of all of the different types of ELISA, yet it doesn’t require a purified sample. That’s why the ELISA sandwich assay is used for at-home tests, like pregnancy tests or rapid Covid-19 tests. The biggest disadvantage in the sandwich assay is in the choice of antibodies: it’s key to make sure the capture and primary antibody are targeting different binding sites of the antigen or else the 2 antibodies will interfere with one another.


Now that you’re familiar with the different types of ELISA, you can appreciate how scientists detect diseases and immune responses. Direct ELISA is the simplest procedure of the different types of ELISA, but it has the weakest sensitivity. Indirect ELISA is more sensitive than direct, but it’s more time-consuming to run the technique. ELISA sandwich assay is the most sensitive but it requires careful selection of the capture antibody and primary antibody.

Each of these is an example of the trade-offs that microbiologists make when designing lab tests to get the best results.