What is an ELISA (enzyme-linked immunosorbent assay)?
ELISA (enzyme-linked immunosorbent assay) is a type of plate-based assay procedure that’s created for identifying and quantifying soluble materials like peptides, proteins, antibodies, and hormones. Another name is an enzyme immunoassay (EIA), which is also used to represent the same technology.
In an ELISA technique, the antigen (target macromolecule) is immobilized on a solid surface (microplate) and then, it’s complexed with an antibody that’s linked to a reporter enzyme. Detection is possible by evaluating the action of the reporter enzyme by incubation with a suitable substrate to generate a measurable product. The common essential element of an ELISA is a highly specific antibody-antigen interaction.
The enzyme-linked immunosorbent assay (ELISA) is a type of immunological assay. It is commonly used to identify the antibodies, antigens, proteins, and glycoproteins in biological samples.
Some examples involve;
• The analysis of HIV infection
• For pregnancy test
• It is also used in the measurement of cytokines or soluble receptors in cell supernatant or serum.
ELISA assays are usually performed in ninety-six well plates, providing multiple samples to identify in a single experiment. These plates require special absorbent plates (e.g. NUNC Immuno plates) to ensure the antibody or antigen attach to the surface. Each ELISA contains a specific antigen, and kits for a variety of antigens are generally available.
Although nowadays, many modifications of ELISA have been developed and used in different circumstances, and they all depend on the same basic elements:
• Coating/capture: It includes direct or indirect immobilization of antigens to the surface of polystyrene microplate wells.
• Plate blocking: It includes the addition of irrelevant protein or another molecule to cover all unsaturated surface-binding sites of the microplate wells.
• Probing/detection: It includes the incubation with antigen-specific antibodies that affinity bind to the antigens.
• Signal measurement: It is used for the detection of the signal generated via the direct or secondary tag on the specific antibody.
The enzymes which are used in labels are following;
• Horseradish peroxidase (HRP)
• Alkaline phosphatase (AP)
• Acetylcholinesterase, and
A wide selection of substrates is prepared commercially for making ELISA with an HRP or AP conjugate. The choice of substrate depends upon the required assay sensitivity and the instrumentation available for signal detection (spectrophotometer, fluorometer, or luminometer).
ELISA formats–direct, indirect, and sandwich ELISA
There are various types of ELISA formats such as;
• Direct ELISA,
• Indirect ELISA,
• Sandwich methods and a detection system.
The First and important step is the immobilization of the antigen which is accomplished by either direct adsorption to the assay plate or indirectly via a capture antibody that has been attached to the plate. Then, the antigen is detected directly (labeled primary antibody) or indirectly (such as a labeled secondary antibody).
The most extensively used ELISA assay format is the sandwich ELISA assay, which indirectly immobilizes and indirectly detects the presence of the target antigen.
In this procedure, the antigen is immobilized to the well plate and ensures that the antigen is attached to the surface and then detected with the antibody which is specific to that antigen. The antibody is directly linked to HRP or other detection molecules.
Indirect ELISA is a type of ELISA that includes a two-step method for detection.
The first step includes the primary antibody which is specific for the antigen and binds to the target.
The second includes a labeled secondary antibody that is against the host species of the primary antibody and binds to the primary antibody for further detection.
In a serum sample, when we replace the serum with a primary antibody, then this technique is very useful for the detection of the specific antibodies in the sample.
It is the most generally used format. This is called sandwich ELISA because the sample which is used for measurement is bound between two primary antibodies and each identifies a different epitope of the antigen that is the capture antibody and is coated on the surface of the well plate to facilitate the immobilization of the antigen. and other is the detection antibody which conjugates and facilitates the detection of the antigen.
The sandwich ELISA is a very sensitive technique and has very high specificity.
It is also called the inhibition ELISA or competitive immunoassay. This technique includes the concentration of an antigen by detection of signal interference.
The sample antigen meets a reference antigen for binding to a particular amount of labeled antibodies. The reference antigen is pre-coated on a well plate and the sample is pre-incubated with labeled antibody and mixed to the wells. Depending on the quantity of antigen in the sample, more or less free antibodies will be ready to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.
Some competitive ELISA kits use labeled antigens rather the labeled antibody. The labeled antigen and the sample competes for the binding with the primary antibody. If the quantity of antigen is less in the sample, then the signal due to more labeled antigen in the well is stronger.
Advantages of ELISA technique
• It is a highly sensitive technique and very high specificity.
• It is used for the detection of antigens at the picogram level in a very accurate manner because of the use of specific antibodies.
• It is a high throughput procedure because the commercial ELISA kits have ninety-six well plates but the assay can be easily adapted to three hundred eighty-four well plates.
• The protocols are very easy to follow and it is very easy to perform and involves only a little hands-on time.
• This technique is quantitative in nature.
• It can be used for examining the concentration of antigen in a biological sample.
• It is used to test the various types of a biological sample such as;
- Cellular and Tissue extracts
- Urine, and,
- Saliva, etc.
Disadvantages of ELISA technique
• In this technique, the detection is based on antigen and antibody reactions and therefore readout must be obtained in a short period.
• The information is very limited to the presence of the antigen in the biological sample.
Application of ELISA
• It is used for cancer screening to drug and pregnancy testing.
• It is used for the detection of platelet antibodies.
• It is used to identify food allergens.
• It is a type of immunological assay which is used to measure the antibodies, antigens, proteins, and glycoproteins in biological samples.