The enzyme-linked immunosorbent assay (ELISA) is a commonly used analytical biochemistry assay, first described by Engvall and Perlmann in 1971. The assay uses a solid-phase type of enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured. ELISA has been used as a diagnostic tool in medicine, plant pathology, and biotechnology, as well as a quality control check in various industries.
In the most simple form of an ELISA, antigens from the sample to be tested are attached to a surface. Then, a matching antibody is applied over the surface so it can bind the antigen. This antibody is linked to an enzyme and then any unbound antibodies are removed. In the final step, a substance containing the enzyme’s substrate is added. If there was binding the subsequent reaction produces a detectable signal, most commonly a color change.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a “sandwich” ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly “immuno” assays, though the name carried the original “immuno” because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified. In between the washes, only the ligand and its specific binding counterparts remain specifically bound or “immunosorbed” by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g., a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase, which is part of the plate, and so are not easily reusable.
The four major types of ELISA are indirect, direct, sandwich and competitive.
Direct ELISA
These are considered the simplest form of ELISA. They are referred to as direct as the use of only one antibody is required for the detection process. The primary detection antibody is directly labelled with a conjugated enzyme producing a colour change when substrate is added. No secondary antibody is required. When testing for the antigen of interest within a sample a blocking agent must also be used such as BSA to block off any other potential binding sites. Direct ELISAs are recognised to reduce cross reactivity between other antibodies as only one is used. However as each of the primary antibodies used within these assays need to be labelled with enzyme this can increase the cost of the assay and the time required to produce them. With each new assay a new detection antibody would need to be labelled. Direct ELISAs are recognised to have a lower sensitivity with the signal produced from the assay less amplified when compared to indirect ELISAs however they have a much faster detection speed as only one step for detection is required.
Indirect ELISA
Indirect ELISAs require the use of two antibodies during the detection stage. This involves a two step process, increasing the time involved in carrying out the assay. The antigen of interest is first coated on to the plate , the primary antibody will then be introduced to the wells. The primary antibody will specifically bind to the antigen of interest if present in the wells. Following this the secondary antibody will be introduced, this secondary antibody will have been previously labelled with an enzyme for detection. The secondary antibody will bind to the primary antibody which will in turn be bound to the antigen. The concentration of antigen can then be determined depending on a colour change observed. The more antibody bound to antigen the greater the colour change as less will be removed following the wash cycles. The benefits to this process can include a higher sensitivity as more than one labelled antibody can bind to the primary antibody. Flexibility may also be increased as more than one secondary detection antibody can be used with a single primary detection antibody. There may also be a reduction in cost to perform this assay as only one type of antibody will need to be labelled.
Competitive ELISA
Most often used to determine small molecules such as lipids, hormones and small peptides. Larger molecules may also be detected however larger concentrations would be required.
Competitive ELISAs follow the principle that the sample antigen of interest and an enzyme conjugated version of the same antigen will compete with each other for limited numbers of specific antibody binding sites. Another method can be the antibody competes for target sites on the bound antigen pre coated to the plate. Labelled antibody will compete for binding sites with the antibody within the sample.
When detecting the concentration of antibody in a sample the lower the signal produced from the assay the higher the level of specific antibody within the sample. The known labelled antibody will compete for binding with the antigen pre coated to the wells, if more of the antibody in the sample is able to bind this will result in more of the labelled antibody being removed following the wash cycles. For labelled antigen this follows the same principle.
Sandwich
Sandwich ELISAs tend to be the most readily recognized, and as the name suggests the antigen of interest will be sandwiched between two antibodies, these assay may either be direct or indirect.
As the antigen is sandwiched between two antibodies the antigen of interest is usually required to be at least 20aa in length. This ensures that the antibodies used are able to bind to different epitopes of the antigen and will not disrupt the binding of each of the antibodies used. The capture antibody is the antibody used to absorb the target antigen onto the wells of the assay. Once the sample has been added the detection antibody may then be introduced to the wells, binding to the target antigen if present in the wells. When carrying out a sandwich ELISA it is important that the antibodies used are matched pairs. Matched pairs refers to antibodies being specifically tested together to ensure that they bind to different epitopes of an antigen. This prevents the chance of the antibodies binding to the same site or recognising each other. The detection antibody within the sandwich ELISA may be enzyme conjugated or a secondary antibody may be added that is enzyme conjugated. Enzymes used within the detection process more often include HRP (horse radish peroxidase) or AP (alkaline phosphatase). The final step in the process is the addition of the substrate. The substrate added will react with the enzyme conjugated antibody to produce a reaction that often involves a colour change, the release of light or fluorescence. The signal strength can be detected using specific absorbency readers. A stop solution must be added during this procedure to halt the reaction over a specified period of time otherwise the signal will become saturated/ degrade.
Sandwich ELISAs are beneficial when it is suspected that the samples may have low levels of antigen present. This is due to the capture antibody only binding to the antigen of interest. The other proteins within the sample that have not bound to the capture antibody will be subsequently washed away.
Qualitative or Quantitative?
ELISAs may be run as either qualitative or quantitative. Qualitative results provide either a positive or negative result for the sample. The cut off between positive and negative will be determined by the end user. In quantitative ELISAs however the amount of coloured product observed will be directly proportional to the amount of enzyme linked antibody that has bound to the primary antibody in turn bound to the antigen. The concentration of antigen/antibody within a sample can be determined from standards used which have known amounts of antibody or antigen. A standard curve is produced from serial dilutions of the standard and the absorbency values they produce. Using the graph the concentration of the antibody/antigens can be determined when taking into account their absorbency values.