Types of ELISA
Enzyme-linked immunosorbent assay (ELISA), also referred to as enzyme immunoassay (EIA) is one of the most common types of immunoassays, which is widely used to either detect the presence of a target antigen or antibody (qualitative ELISA), or quantify the concentration of the target protein in a sample (quantitative ELISA). This approach relies on highly specific antibody-antigen interactions to detect and quantify substances such as peptides, proteins, antibodies and hormones, and can be employed for both diagnostic and research purposes.
To establish an ELISA assay, the antigen must either be directly fixed onto a microplate (96- or 384-wells), or indirectly immobilized via a capture antibody that has been previously coated onto the microplate. Addition of the second antibody, named detection antibody, which has been conjugated to a molecule such as an enzyme or a fluorophore enables the detection process via producing a measurable signal after a period of incubation time.
The specific interaction between antigen and antibody is the main principle of the immunoassay, however based on how it works ELISA is categorized to four different subclasses: Direct, Indirect, Sandwich and Competitive.
Additionally, based on the substrate type and the detection approach ELISA methods can be classified into colorimetric ELISA, fluorescent ELISA (FELISA), fluorescent-linked immunosorbent assay (FLISA), and chemiluminescent ELISA which all have their advantages and disadvantages.
In the direct ELISA, a mixture of an unknown antigen is coated on a microplate and an enzyme-conjugated primary antibody is directly added to bind to the immobilized antigen. Subsequent addition of the substrate produces a visible signal. Hence, the more antigen exists in the sample, the higher the signal is.
The similarity between direct and indirect ELISAs is that in both the antigen is immobilized on a microplate. However, in an indirect ELISA, an unconjugated primary detection antibody is first added to form an antigen-antibody complex. Then, an enzyme-conjugated secondary antibody directed against the host species of the primary antibody is added to amplify the detection step. Addition of the substrate produces a signal proportional to the amount of the antigen bound in the well. In other words, an indirect ELISA uses a secondary enzyme-conjugated antibody to recognize the primary antibody which interacts with the immobilized (coated) antigen. The subsequent enzymatic reaction produces a detectable visible light from substrate.
Unlike the direct and indirect ELISAs, whereby the antigen is directly immobilized on a plate, in sandwich ELISA, the antigen is trapped between the two antibodies referred to as “capture” and “detection” antibodies that recognize different epitopes. First, the capture antibody is immobilized (coated) on the plate, followed by addition of the target antigen. Subsequently the detection antibody either enzyme-conjugated (secondary) or unconjugated (primary) is added. The substrate colour change will be initiated either by secondary enzyme-labeled antibody (direct sandwich ELISA) or by adding enzyme-conjugated anti primary antibody (indirect sandwich ELISA).
In a competitive ELISA, also known as an inhibitory ELISA, the antibody in the sample competes with the enzyme-conjugated antibody (Reference) to bind to the target antigen. The more antibody present in the sample, the less reference conjugated antibody will attach to the antigen, and the weaker visible light signal will be produced after addition of the substrate. Most competitive ELISAs are designed based on direct ELISAs, but they are also compatible with other types of ELISA including indirect and sandwich ELISA.
As mentioned above, ELISA methods can be also categorized to four types of colorimetric, fluorescent, FLISA and chemiluminescent based on their detection approach.
Colorimetric assays, in general, are referred to all assays in which the presence of an analyte results in a measurable visible colour change in reagents. These assays are widely used in biochemistry to detect and quantity the amount of enzymes, specific compounds, antibodies, hormones and many other analytes.
In a colorimetric ELISA, as the most common type of ELISA, the antigen-antibody interaction is determined using a conjugated enzyme to change the color of substrate in a visible range. Hence, the higher intensity of the color is an indication of the enhanced enzymatic reaction due to the presence of more antigen or antibody in the sample. It is worth noting that direct correlation of the generated color intensity with increasing amount of antigen or antibody is only relevant in direct, indirect and sandwich ELISAs; whereas, in competitive ELISAs, the higher intensity of the color inversely represents the lower concentration of the antigen or antibody in the sample.
Fluorescent ELISA (FELISA)
In a fluorescent ELISA (FELISA), the interaction of an antigen-antibody is detected based on the intensity of the conjugated enzyme which change the substrate to a fluorescent signal producing substance. In other words, it uses the same approach as in a colorimetric ELISA except for the application of a fluorescent-producing substrate.
Being independent of any enzymatic reaction, FLISA or fluorescent-linked immunosorbent assay is theoretically different from ELISA, as it uses a fluorescent-conjugated detection antibody to quantify the amount of a target. However, the principle of FLISA is still similar to ELISA whereby an increased antibody-antigen binding generates a higher fluorescent signal proportional to the amount of the analyte in the sample.
In a chemiluminescent ELISA, similar to colorimetric and fluorescent ELISAs, the interaction of an antigen-antibody complex is determined via the enzymatic reaction of the conjugated secondary/detection antibody. However, here the chemical reaction converts a substrate to a reaction product that emits a photon of light instead of developing a particular color or a fluorescent signal.