ELISA is a technology used to identify antigens present in body fluid or build up artificially. This technology is based on the principle that antigen (Ag) binds to its specific antibody (Ab) only. Every antigen (Ag) have some specific receptors those identify and bind to specific antibody (Ab) like a key for a lock. To identify presence of antigen we build a plate coated with specific antibody against the antigen to find, add up antigen to find from the group of antigen. If specific type of antigen present in the titer it will bind with the antibody present in the plate. This coupling can be proved by the action of enzyme or colour developing agent those will prove the binding o Ag-Ab complex. So it is named as enzyme linked immunosorbent assay.
By using ELISA we can identify antigen, antibody, glycoprotein in biological samples. Some common test those can be carried out by ELISA include HIV diagnosis, pregnancy test, and measurement of cytokines.
Types of ELISA – direct, indirect and sandwich ELISA
In direct ELISA technique we use antigen coated plate (to find antibody) and pour antibody titer over the plate. Comparable antibody bind with the antigen present in the plate and makeup a complex. This complex can be detected either directly (labelled primary antibody) or indirectly (such as labelled secondary antibody).
Process of direct ELISA
- Antigen (Ag) is immobilised on well of polystyrene plate via passive adsorption.
- Enzyme linked antibody (Ab) is added to the well and allow to bind directly to the coated antigen.
- A respective enzyme substrate is added to the well which on reaction with the enzyme already bound to the antibody (Ab) develop colour. This colour production is the confirmation of Ag-Ab reaction on the polystyrene plate well.
Advantages of direct ELISA
• Less steps are required, making this ELISA method simple and efficient. And minimising user errors.
• Cross-reactivity of antibody is eliminated.
Disadvantages of direct ELISA
• Primary antibody must be labelled individually, which is time consuming and raise the coast, i.e. expensive.
• Signal amplification does not take place in direct ELISA.
• While labelling primary antibody with enzyme, it may adversely effect its immune reactivity.
There are two steps in indirect ELISA. First step involved binding of primary antibody and second step involve binding of labelled antibody. During the process, primary antibody is first incubated with the antigen then secondary antibody is added and again incubate to complete immunological reaction.
Process of indirect ELISA
- Micro-well plates are coated with the antigens.
- Samples with antibody (primary antibody) is added and washed up to remove any excess amount.
- Now enzymes linked secondary antibody is added to the micro-well and incubated.
- Now substrate is added which react with the enzyme present in secondary antibody and develop colour. This is the indication of binding antigen-antibody reaction.
Advantages of indirect ELISA
• Indirect ELISA delivers more flexibility since different primary antibodies can be used with same enzyme linked secondary antibody.
• Indirect ELISA is highly sensitive. Since the use of enzyme linked secondary antibody make signal amplification.
Disadvantages of indirect ELISA
• Indirect ELISA protocols is more complex then direct ELISA.
• Enzyme linked secondary antibody (Ab) may show cross reaction with antigen this may cause inaccurate result.
It is called a sandwich, because it uses two antibody. One is captured antibody and other is target antibody. The antigen is bound within two antibody. Like any other ELISA, purpose of sandwich ELISA is also to find the target antibody. To do this antigen is bound over the captured antibody on the well of plates, and then susceptible antibody solution is poured and allowed to agglutinate.
Steps of sandwich ELISA
- Coat a well of plate with captured antibody.
- Add the sample in which target antigen is present. Incubate the entire plate to allow agglutination reaction. After that wash well softly to remove any extra antigen.
- Now add up detection antibody label conjugate. Incubate the well plate and was to remove any extra antibody conjugate.
- Like any other ELISA technique, add substrate to the well, and observe for the change in colour.
Advantages of sandwich ELISA
• It is highly flexible, as detection method can be direct and indirect.
• Sandwich ELISA technique is highly sensitive and specific, because it uses two antibody against same antigen.
Disadvantages of sandwich ELISA
• The antigen under test must be large enough to allow to bind two antibodies.
• It is not always possible to have pairs of antibody against same antigen.
Competitive ELISA is ideal to detect antigen of small size or to the antigen those are in low concentration.
Application of ELISA
ELISA is one of the revolutionary technique used in biomedical science. It is basically used for the detection of presence of specific antibody in the serum. Other uses of ELISA can be as follow:
- to find the presence of specific antigen or antibody in a sample.
- In food industry, ELISA is used to detect any food allergens present.
- ELISA can be used to determine the concentration o serum antibody in a virus test.
- During COVID-19, rapid eating kits are used to determine the presence of antibodies in the blood sample.