Overview- Basic ELISA Procedure
- Antibodies and Antigens in ELISA
- Four typical ELISA formats
- ELISA Detection Options
- ELISA Results
- Sensitivity
Overview
The enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique which is used to measure the concentration of an analyte (usually antibodies or antigens) in solution. The basic ELISA, or enzyme immunoassay (EIA), is distinguished from other antibody-based assays because separation of specific and non-specific interactions occurs via serial binding to a solid surface, usually a polystyrene multiwell plate, and because quantitative results can be achieved. The steps of the ELISA result in a colored end product which correlates to the amount of analyte present in the original sample.
ELISAs are quick and simple to carry out, and since they are designed to rapidly handle a large numbers of samples in parallel, they are a very popular choice for the evaluation of various research and diagnostic targets. Figure 1 shows a typical ELISA result.
ELISAs were first developed in the early 1970s as a replacement for radioimmunoassays. They remain in wide use in their original format and in expanded formats with modifications that allow for multiple analytes per well, highly sensitive readouts, and direct cell-based output.
Basic ELISA Procedure
ELISAs begin with a coating step, where the first layer - either an antigen or an antibody - is adsorbed to a polystyrene 96 well plate. (Adsorption is the passive attachment of a liquid to a solid surface creating a thin film.) Coating is followed by blocking and detection steps as shown in the simple schematic diagram below.
Since the assay uses surface binding for separation, several washes are repeated between each ELISA step to remove unbound materials. During this process it is essential that excess liquid is removed in order to prevent the dilution of the solutions added in the next stage. For greatest consistency specialized plate washers are used.
ELISAs can be quite complex, including various intervening steps and the ability to measure protein concentrations in heterogeneous samples such as blood. The most complex and varying step in the overall process is detection, where multiple layers of antibodies can be used to amplify signal.
Figure 2. ELISA overview flowchart and schematic.
Antibodies and Antigens in 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.
Four Typical ELISA Formats
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.
Direct ELISA
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:
- It is faster, since fewer steps are required
- It is less prone to error, since there are fewer steps and reagents
Indirect ELISA
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:
- Increased sensitivity, since more than one labeled antibody is bound per primary antibody
- Flexibility, since different primary detection antibodies can be used with a single labeled secondary antibody
- Cost savings, since fewer labeled antibodies are required
Sandwich ELISA
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 indirectsandwich ELISA.
This type of assay has several advantages:
- High specificity, since two antibodies are used the antigen/analyte is specifically captured and detected
- Suitable for complex samples, since the antigen does not require purification prior to measurement
- Flexibility and sensitivity, since both direct and indirect detection methods can be used
Competition or Inhibition ELISA
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.
ELISA Detection Options
ELISAs, by definition, take advantage of an enzymatic label to produce a detectible signal that is directly correlated to the binding of antibody to an antigen. There are a few different types of enzymes and enzyme substrates that are typically used for ELISAs and a few slightly different methods for incorporating the enzyme step into the process. The final assay signal is measured with a spectrophotometric or fluorescent plate reader (depending upon the substrate chosen).
One aspect of ELISA terminology that often leads to confusion is the variability in the way the terms direct and indirect are applied. We will adhere to the use of these terms as they apply to the detection portion of the assay as indicated below:
Direct detection
Antibodies are directly labeled with alkaline phosphatase (AP) or horseradish peroxidase (HRP); this is the most common ELISA detection strategy. HRP and AP substrates typically produce a colorimetric output that is read by a spectophotometer. Detection can also occur by fluorescently-labeled antibodies [here the assay is usually termed a fluorescence-linked immunosorbent assay (FLISA)]
Indirect detection
Antibodies are coupled to biotin, followed by a streptavidin-conjugated enzyme step; this is most common.
Additionally, it is possible to use unlabeled primary antibodies followed by enzyme-coupled or biotinylated secondary antibodies. If the secondary antibody is biotinylated, then a tertiary step is required for detection. In this case treatment with the streptavidin-enzyme conjugate, followed by an appropriate substrate.
ELISA Results
The ELISA assay yields three different types of data output:
1) Quantitative:
ELISA data can be interpreted in comparison to a standard curve (a serial dilution of a known, purified antigen) in order to precisely calculate the concentrations of antigen in various samples.
2) Qualitative:
ELISAs can also be used to achieve a yes or no answer indicating whether a particular antigen is present in a sample, as compared to a blank well containing no antigen or an unrelated control antigen.
3) Semi-quantitative:
ELISAs can be used to compare the relative levels of antigen in assay samples, since the intensity of signal will vary directly with antigen concentration.
ELISA data is typically graphed with optical density vs log concentration to produce a sigmoidal curve as shown in Figure 8. Known concentrations of antigen are used to produce a standard curve and then this data is used to measure the concentration of unknown samples by comparison to the linear portion of the standard curve. This can be done directly on the graph or with curve fitting software which is typically found on ELISA plate readers.
Figure 8. A typical ELISA standard curve.
Sensitivity
ELISAs are one of the most sensitive immunoassays available. The typical detection range for an ELISA is 0.1 to 1 fmole or 0.01 ng to 0.1 ng, with sensitivity dependent upon the particular characteristics of the antibody –antigen interaction. In addition, some substrates such as those yielding enhanced chemiluminescent or fluorescent signal, can be used to improve results. As mentioned earlier, indirect detection will produce higher levels of signal and should therefore be more sensitive. However, it can also cause higher background signal thus reducing net specific signal levels.
