An enzyme-linked immunosorbent assay (ELISA) is a versatile method used to quantify the level of target antigen in a sample. While Engvall et al. originally developed the ELISA assay to measure antibody levels, scientists have since adapted it for a host of different proteins and small molecules from a variety of sample types (Engvall 1971). Scientists value ELISAs for their accuracy, specificity, and sensitivity (a good ELISA can detect picogram quantities of the target!) and use them in numerous fields including research and development, diagnostics, drug discovery, and food safety.
Part of the assay’s versatility lies in the user’s ability to modify the protocol to best fit their specific needs. Herein, we’ll give you a rundown of the different types of ELISA, advantages and disadvantages of each, and some considerations when using the method.
All ELISAs follow the same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent may be an antigen or an antibody depending on how the assay is set up. Then they block the capture reagent to prevent non-specific interactions. Next comes the binding step where the target antigen binds to antigen-specific antibodies. The antibodies are conjugated with a reporter or tag that can then be detected.
Typically, the reporter is an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Fluorescent tags may also be used, but for simplicity we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users provide a substrate that the enzyme converts into a detectable product. Scientists compare the signal intensity of the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A variety of chromogenic, chemifluorescent, and chemiluminescent substrates are available. Labs with limited equipment often opt for the chromogenic substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and chemiluminescent methods that require specialized equipment. Chromogenic methods are not as sensitive, however, so those studying low abundance antigens may want to consider alternative detection methods.
A direct ELISA is the quickest and simplest of all varieties. In this method, users immobilize the target antigen on the multiwell dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate and produces a signal that is directly proportional to the level of antigen present in the sample and is calculated by extrapolating from the standard curve. Because the primary antibody is directly conjugated to the reporter, there is no need for a secondary antibody incubation step, saving time. Moreover, since secondary antibodies can cross-react with samples, eliminating them from the procedure removes one potential source of cross-reactivity.
While the method is simple and saves time, it also has several drawbacks. Direct ELISAs generally have higher levels of background staining. Antigen-containing samples often include undesirable molecules that may bind non-specifically to assay components and increase background staining. Direct ELISAs also lack the signal amplification afforded by secondary antibodies and thus are not suitable for low abundance proteins. Because the method uses a single antibody, it is less specific than other ELISA methods. Users may also find it difficult to source a commercially available and appropriately conjugated antibody. While labs can conjugate antibodies themselves, the process can be laborious and expensive. In some cases, conjugation methods can interfere with binding, and some common antibody buffer components can interfere with conjugates. Make sure to do your research before conjugating at the bench.
The indirect ELISA process is similar to that of a direct ELISA, but the indirect method uses both an unconjugated primary antibody and a conjugated secondary antibody. Incorporating a secondary antibody increases the sensitivity of the assay, since multiple secondary antibodies will bind to a single primary antibody and thereby amplify the signal. Since the primary antibody does not need to be conjugated, and conjugated secondary antibodies are easy to find, it is often easier for labs to source appropriate antibodies for an indirect ELISA than for a direct. In addition, a lab can use the same secondary antibody for any ELISA protocol that uses primary antibodies of the same isotype.
Specificity and timing are the major disadvantages of the indirect method. The assay only uses one antigen binding antibody, making it less specific than other methods. In addition, the assay includes both primary and secondary incubation steps, increasing the workload and time commitment for users. This method also requires more optimization and controls when developing, as secondary antibodies can also be a source of cross-reactivity.
In the most common type of ELISA, the sandwich ELISA, users first coat a multiwell plate with a capture antibody and then add their antigen-containing sample. The target antigen binds to the capture antibody and a series of wash steps removes non-target proteins and molecules from the assay. The user then adds an antigen-specific detection antibody.
Importantly, the detection antibody and the capture antibody must bind to distinct, non-overlapping epitopes so that they do not interfere with each other. The detection antibody may either be conjugated directly with the reporter enzyme or used with a conjugated secondary antibody. The amount of signal produced by the enzymatic reaction between the detection antibody and substrate is proportional to the amount of antigen present in the sample and is calculated by extrapolating from the standard curve.
Scientists often opt for sandwich ELISAs because they require two independent antibodies for the capture and detection steps, which increases the specificity of the assay. The assay is also flexible, as users can develop methods with either directly conjugated detection antibodies or unconjugated detection antibodies followed by conjugated secondary antibodies. Assay development, however, can be challenging since scientists must first identify two independent antibodies that work well together. Moreover, the procedure is longer than alternative ELISA methods, especially when secondary antibodies are used.
Scientists typically use a competitive or inhibition ELISA when measuring small molecules that cannot bind efficiently to two antibodies as would be required for a sandwich ELISA. In this method, the target antigen competes with a reference antigen for binding to a primary antibody.
While the precise setup of the method can vary, typically users first coat the multiwell plate with a reference antigen and then they preincubate the antigen-containing sample with their conjugated primary antibody. Users next incubate the sample with the multiwell plate to allow the unbound primary antibody to bind to the reference antigen. The more antigen in the sample, the more primary antibody that will be bound, and the less antibody that will be left available to bind to the reporter. Unlike for direct, indirect, and sandwich ELISAs, in a competitive ELISA the signal intensity is inversely proportional to the amount of antigen in the sample: more antigen means less antibody available to bind to the reference antigen, producing a weaker signal.
The major drawbacks of the competitive ELISA method are similar to those of the direct method. Namely, since the assay requires a single conjugated primary antibody it is both less specific and can be difficult to develop if an appropriate conjugated antibody is not readily available.
Table 1: Overview of the advantages and disadvantages of each ELISA method
Higher level of background staining
Less sensitivity
Less specificity
Limited availability of conjugated antibodies
High sensitivity
Good antibody availability
Longer protocol
Less specificity
Increased chance of cross-reactivity
Longer protocol
Increased chance of cross-reactivity
Challenging to develop assay
Less sensitivity
Less specificity
Limited availability of conjugated antibodies
No matter what ELISA method you choose, it is essential to include proper controls. As a positive control, include a sample that is known to contain the target antigen. Some examples could be an endogenous sample of wild type cells, purified protein, or a peptide. The positive control will confirm that the procedure is working and should always be included.
For a negative control, include a sample that does not contain the target antigen, such as a knockout or knockdown cell line or tissue samples where the target is not expressed. The negative control will help you determine if there is non-specific binding or false positives. If testing a recombinant protein, especially one with tags, be sure to include a sample that expresses the endogenous protein, since folding of the recombinant protein may differ from the native form, which can affect antibody binding.
We hope that you now feel empowered to choose the best ELISA for your needs. Good luck and happy binding!
