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Deciding which ELISA technique is right for you

Bio-Rad
Jul 27, 2016

The enzyme linked immunosorbent assay (ELISA) was first described by Engvall and Perlmann (1971), and used initially for the detection of immunoglobulin G. This immunoassay was a welcomed change from the then common radioimmunoassay, which utilized radioactively labeled antibodies and antigens. Development of the ELISA was based on the observation in the 1960s that antibodies or antigens can be adsorbed to a solid surface and still participate in high-affinity binding. The term ELISA now refers to a wide range of immunoassays some of which do not involve enzymatic reactions. However, the commonality among all ELISAs is the use of antibodies, which play a major role in determining the sensitivity and specificity of the assay.

ELISAs are used across several areas of biomedical research and discovery as well as for diagnostic purposes. As its widespread use suggests, there are many ways to do an ELISA, and determining which one is right for addressing your specific research question depends on several factors including the desired sensitivity or exactly what is being detected. Here, we describe the different types of ELISAs, focusing on the advantages and disadvantages of each as well as what to consider before choosing an ELISA technique.

What types of ELISAs are there?

There are four ELISA techniques that can be applied to your research. Choosing which is appropriate depends on the antibodies available, the results required and the complexity of your samples. The ELISA techniques include: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competition or Inhibition ELISA.

Direct ELISA

How it works:

Figure 1 demonstrates the typical set up of a direct ELISA. Generally, an antigen is immobilized to a multiwell plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as horse radish peroxidase (HRP).

direct ELISA technique
Figure 1. Overview of the direct ELISA technique

Advantages:

  1. It is much faster than other ELISA techniques since fewer steps are required.
  2. Because less reagents and steps are required, the assay is less prone to error.

Disadvantages:

  1. Because the method of immobilizing the antigen is not specific, this may cause higher background noise than indirect ELISA (described below). This is primarily because all proteins in the sample, including the target protein, will bind to the plate.
  2. Less flexible since a specific conjugated primary antibody is needed for each target protein.
  3. No signal amplification, which reduces assay sensitivity.

When you typically use it:

This ELISA technique is typically used when the immune response to an antigen needs to be analyzed.


Indirect ELISA

How it works:

In this ELISA technique, antigen that is immobilized on a multiwell plate is detected in two steps.  First, an unlabeled primary antibody (typically monoclonal) binds the specific antigen. Second, an enzyme conjugated secondary antibody (typically polyclonal) that is directed against the host species of the primary antibody is applied.

indirect ELISA technique
Figure 2. Overview of the indirect ELISA technique

  Advantages:

  1. High sensitivity since more than one labeled secondary antibody can bind the primary antibody.
  2. Economical since fewer labeled antibodies are needed.
  3. Allows flexibility since different primary antibodies can be used with a single labeled secondary antibody.

Disadvantages:

  1. Possibility of cross-reactivity due to use of secondary antibody, which could increase background noise.
  2. Longer procedure than direct ELISA technique since an additional incubation step for secondary antibody is required.

When you typically use it:

The indirect ELISA is suitable for determining total antibody concentration in samples.


Sandwich ELISA

How it works:

Sandwich ELISAs require the use of matched antibody pairs (capture and detection antibodies). Each antibody is therefore specific for a different and non-overlapping region or epitope of the antigen. It is important that matched antibody pairs are tested specifically in ELISA to ensure that they detect different epitopes, for accurate results. The procedure for a sandwich ELISA involves coating a polystyrene plate with a capture antibody. The analyte or sample is then added, followed by a detection antibody. The detection antibody can be enzyme conjugated, in which case this is referred to as a direct sandwich ELISA. If the detection antibody used is unlabeled, a secondary enzyme-conjugated detection antibody is required. This is known as an indirect sandwich ELISA.

Both monoclonal and polyclonal antibodies can be used for sandwich ELISAs. However, a polyclonal antibody is often used as the capture antibody in order to pull down the greatest amount of antigen possible.

sandwich ELISA technique
Figure 3. Overview of the sandwich ELISA technique

Advantages:

  1. High sensitivity; it is 2-5 times more sensitive than direct or indirect ELISA.
  2. High specificity since two antibodies are used.
  3. Offers flexibility since both direct and indirect methods can be used.

Disadvantages:

If not using a standardized ELISA kit or tested antibody pair, antibody optimization can be difficult since it is important to reduce cross-reactivity between the capture and detection antibodies.

When you typically use it:

Sandwich ELISAs are particularly suitable for the analysis of complex samples, since the antigen does not need to be purified prior to measurement using this method.


Competition/Inhibition ELISA

How it works:

The competition/inhibition ELISA, also known as a blocking ELISA, is perhaps the most complex of all the ELISA techniques. However, each of the above assay types can be adapted to a competitive format. The competitive/inhibition ELISA is predominantly used to measure the concentration of an antigen or antibody in a sample by detecting interference in an expected signal output. Essentially, sample antigen or antibody competes with a reference for binding to a limited amount of labeled antibody or antigen, respectively. The higher the sample antigen concentration, the weaker the output signal, indicating that the signal output inversely correlates with the amount of antigen in the sample.

An example of a competition ELISA to test for antigen based on the direct detection method is shown in figure 4. In this example, a known antigen is used to coat a multiwell plate. Following standard blocking and washing steps, samples containing unknown antigen are added. Labeled detection antibody is then applied for detection using relevant substrates (e.g. 3,3’,5,5’-Tetramethylbenzidine or TMB).  If there is a high concentration of antigen in the sample, a significant reduction in signal output will be observed. In contrast, if there is very little antigen in the sample, there will be very little reduction in the expected signal output. In the example shown in figure 4, there would be a reduction in signal output.

direct competitive ELISA for testing antigen concentration
Figure 4. An example of a direct competitive ELISA for testing antigen concentration

Advantages:

  1. The main advantage of the competitive ELISA is that no sample processing is required and crude or impure samples can be used.
  2. Less sensitive to sample dilution and sample matrix effects than the sandwich ELISA.
  3. Less variability between duplicate samples and between assays.
  4. Maximum flexibility in experimental setup since it can be based on direct, indirect or sandwich ELISAs.

Disadvantages:

Because each ELISA technique can be adapted to a competitive format, the same limitations as described for each ELISA technique above would be applicable to the respective competitive/inhibition ELISA technique.

When you typically use it:

This ELISA technique is commonly used when only one antibody is available for the antigen of interest. It is also suitable for detecting small antigens that cannot be bound by two different antibodies such as in the sandwich ELISA technique.


Things to consider when choosing an appropriate ELISA technique

As described, there are many factors that determine which ELISA technique is appropriate for addressing your research question. Here are a few questions to consider before deciding which is right for you (Crowther 2009).

What do you need to detect?

For example, if a large protein with multiple epitopes, such as a cytokine is being detected, then a sandwich ELISA would be most appropriate. However, if a small molecule such as a hapten is being detected then a competitive ELISA would be more appropriate in that instance.

What reagents are available for your antigen or antibody of interest

If only one antibody is available for an antigen of interest then a direct or competitive ELISA can be applied.

Do you want to measure an immunological response or analyte?

If you need to detect or quantitate an analyte, then a sandwich or competitive ELISA can be utilized. However if you need to measure an immunological response, then a direct or indirect ELISA is most suitable for your needs.


Other ELISA resources to help you master the technique

To learn more about ELISA, check out our comprehensive ELISA guide that discusses ELISA sensitivity, detection options and more. We have also compiled a list of common controls and tips to help you master this immunoassay. If you’re struggling to create your own ELISA protocol, we also have detailed, optimized and standardized ELISA protocols as well as troubleshooting tips and advice.

We also offer an array of matched antibody pairs for ELISA, particularly for veterinary research. Discover them here for bovine, porcine, and canine species.


References

Crowther JR (2009). The ELISA guidebook (2nd edition) Methods in Molecular Biology (New York: Humana Press).

Engvall E and Perlmann  P (1971). Enzyme-linked immunosorbent assay (ELISA). Quantitative assay for immunoglobulin G. Immunochemistry 8, 871-874.

 

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