General overview of ELISA
ELISA stands for enzyme-linked immunoassay. It is commonly used in research and clinical settings to detect molecules of interest in a sample. In brief, the ELISA method detects an antibody-antigen interaction by having an enzyme conjugated to antibody which is stimulated to emit a signal indicating that binding has occurred. If you want to read more of a background about ELISA and also learn about the types of reporters, read our ELISA article. As an example, ELISA can be used to test a person for HIV (human immunodeficiency virus). HIV antigens are attached to the bottom of the wells of the ELISA plate then sample is placed into the wells of the plate. Then some kind of enzyme reporter is used to indicate if antibodies against HIV are present in the sample and are bound to the HIV antigen.
For the indirect and direct elisa, the antigen is applied to the surface of the elisa plate. For sandwich elisa, a capture antibody is attached directly to the surface of the plate. Aside from this difference, the indirect and direct elisa protocols have similarities to the sandwich elisa protocol. There are plenty of blocking and washing steps to avoid non-specific binding, and there are incubation times to allow antibodies and antigens to bind properly. The indirect elisa requires two antibodies—a primary antibody to bind to the antigen, and a secondary antibody conjugated to an enzyme or fluorophore. The direct elisa uses a primary antibody that is directly conjugated to an enzyme or fluorophore. Either way, both of these methods—and indeed every elisa protocol, is a labeled assay. The antibody-antigen binding event cannot be quantified without the presence of the enzyme or fluorophore.
Choosing plates for an elisa protocol
The most commonly used plate is a 96-well polystyrene plate. These are used because they provide many wells for performing replicate side-by-side reactions. Also, proteins passively bind to the polystyrene surface by passive adsorption. Additionally, the plates come as translucent plastic or as dark black plastic. The translucent plates are ideal for colorimetric detection systems that produce a color change that can be quantified by a spectrophotometer. The dark plates are useful for chemiluminescent systems where the light production is detected by a luminometer. They are also used for fluorescent systems, which are quantified by a fluorometer. An elisa can be performed with a plain uncoated 96-well plate, but the benefits of pre-coated plates athat are commercially available is their ability to improve binding specificity, optimize concentrations, and to decrease the overall working time.
Some examples of pre-coated plates:
- protein A/G
- maleic anhydride coated plates improve binding of amino acids rich in primary amines
- maleimide coated plates improve binding of amino acids rich in sulfydryl groups
- Streptavidin or biotinyated plates
- Nickel or copper chelate coatings to preferentially bind histidine-tagged proteins
Basic steps in a direct or indirect elisa protocol
- Optimize concentrations of antigen and antibody:
2. Bind antigen to plate surface:
- can use an uncoated or coated plate
- an overnight incubation at 4°C or a 1 hour incubation at 37°C is standard
3. Wash away unbound antibody and block unbound sites on the plate
- This step is also called “blocking”. It prevents non-specific binding of antibodies to the plate by binding up the space at the bottom of the wells not bound by antigen (or antibody for sandwich ELISA).
- milk is a common blocking agent, but proteins such as BSA, ovalbumin, aprotinin, or others are also used. Animal serum is also a useful blocking agent, but it contains far more proteins and antibodies than single-source protein blocks.
4. Wash away block5. Select and add antibodies and antibody labels. Incubate for at least 1 hour. This is where direct and indirect ELISA differ!
- For direct elisa: The antibody that is added to the wells to bind the antigen is pre-conjugated to the enzyme that will be stimulated to report the binding event.
- For Indirect elisa: a primary antibody is added that is known to bind the antigen. This is followed by a wash step, since we do a wash step after incubations, to remove unbound material. Finally there is an incubation with a secondary antibody which is pre-conjugated to an enzyme like the primary antibody is for direct ELISA. This ELISA is called indirect for this reason; that the presence of a binding reaction is indicated by a secondary molecule not directly bound to the antigen on the plate.
6. Wash away unbound antibody
7. Apply substrate to the wells of the plate
- Alkaline phosphatase (AP) and horseradish peroxidase(HRP) are two commony used enzymes
- AP catalyzes the hydrolysis of phosphate groups from the substrate to produce color or light.
- HRP catalyzes the oxidation of substrates by hydrogen peroxide to produce color or light.
8. Detect the resulting color change, fluroescence, or light production from the interaction of the substrate with the antibody-linked enzymes.
The elisa is the gold standard of immunoassays. ELISA is considered time-consuming because it requires pipetting many wells and includes several wash steps. Some other downfalls of ELISA are that it is label-dependent, expensive, and difficult to make into a multiplex assay (testing for multiple antigens or antibodies at once). Cutting edge research in biosensing and immunoassay development is trending toward label-free assays that do away with enzyme reactions and conjugated fluorophores. Such devices will be compact and will enable multiplex analysis of samples. However, until these devices hit the market, the trusty elisa will be the standard diagnostic tool in every clinical laboratory.
Advantages of Direct or Indirect ELISA
The indirect ELISA will be more sensitive because there is potential for signal amplification with more than one secondary antibody binding the primary antibody. Although the secondary could also potentially bind non-specifically to the antigen which will skew results. The Advantage of the direct ELISA is that it has fewer steps because you only need to incubate with one antibody, cutting out incubation and wash steps associated with the secondary antibody. It is time consuming however, to have to create a unique pre-conjugated primary antibody for each ELISA you need to do. It is also a benefit of indirect ELISA that you can use the secondary antibody for many experiments.