Direct, Indirect, Sandwich and Competition
or Inhibition ELISA
All ELISAs rely on the specific interaction between an epitope, a small linear or three dimensional sequence of amino acids found on an antigen, and a matching antibody binding site.
The antibodies used in an ELISA can be either monoclonal (derived from unique antibody producing cells called hybridomas and capable of specific binding to a single unique epitope) or polyclonal (a pool of antibodies purified from animal sera that are capable of binding to multiple epitopes).
There are four basic ELISA formats, allowing for a certain amount of flexibility which can be adjusted based on the antibodies available, the results required, or the complexity of the samples.
It is possible to use both monoclonals and polyclonals in an ELISA; however, polyclonals are more typically used for the secondary detection layer in indirect ELISAs, while monoclonal antibodies are more typically used for capture or primary detection of the antigen.
The ELISA provides a wealth of information in its simplest formats, but it can also be used in more complex versions to provide enhanced signal, more precise results, or if certain reagents are not available. The four typical ELISA formats are described briefly below. The end result for all the ELISAs is shown in figure 3, a single well, or a series of wells in a multiwall dish, with color intensity varying in proportion to the amount of antigen/analyte in the original sample.
An antigen coated to a multiwell plate is detected by an antibody that has been directly conjugated to an enzyme. This can also be reversed, with an antibody coated to the plate and a labeled antigen used for detection, but the second option is less common.
This type of ELISA has two main advantages:
Antigen coated to a polystyrene multiwell plate is detected in two stages or layers. First an unlabeled primary antibody, which is specific for the antigen, is applied. Next, an enzyme-labeled secondary antibody is bound to the first antibody. The secondary antibody is usually an anti-species antibody and is often polyclonal.
This method has several advantages:
Sandwich ELISAs typically require the use of matched antibody pairs, where each antibody is specific for a different, non-overlapping part (epitope) of the antigen molecule.
The first antibody, termed the capture antibody, is coated to the polystyrene plate. Next, the analyte or sample solution is added to the well.
A second antibody layer, the detection antibody, follows this step in order to measure the concentration of the analyte.
Polyclonals can also be used for capture and/or detection in a sandwich ELISA provided that variability is present in the polyclonal to alow for both capture and detection of the analyte through different epitopes.
If the detection antibody is conjugated to an enzyme, then the assay is called a direct sandwich ELISA. If the detection antibody is unlabeled, then a second detection antibody will be needed resulting in an indirect sandwich ELISA.
This type of assay has several advantages:
This is the most complex ELISA, and is used to measure the concentration of an antigen (or antibody) in a sample by observing interference in an expected signal output. Hence, it is also referred to as an inhibition ELISA. It can be based upon any of the above ELISA formats, direct, indirect, or sandwich, and as a result it offers maximum flexibility in set up.
It is most often used when only one antibody is available to the antigen of interest or when the analyte is small, i.e. a hapten, and cannot be bound by two different antibodies.
A simple example of a competitive ELISA is shown in figure 7. In this case samples are added to an ELISA plate containing a known bound antigen. After coating, blocking, and washing steps, unknown samples are added the plate. Detection then follows pretty much as with other ELISA formats. If the antigen in the sample is identical to the plate-adsorbed antigen, then there will be competition for the detection antibody between the bound and free antigen. If there is a high concentration of antigen in the sample, then there will be a significant reduction in signal output of the assay. Conversely, if there is little antigen in the sample, there will be minimal reduction in signal.
Therefore, with a competition ELISA, one is actually measuring antigen concentration by noting the extent of the signal reduction. If the detection antibody is labeled, then this would be a direct competition ELISA and if unlabeled, then this would be an indirect competition ELISA.
For further examples of competition ELISAs, and a thorough explanation of this technique, please refer to The ELISA Guidebook (Crowther 2001).
Figure 7. Competition ELISA. Bound and free antigen compete for binding to a labeled detection antibody.
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