ELISA is known as Enzyme-Linked Immunosorbent Assay because :
- The antigen/antibody which adsorbed on the plastic surface is known as sorbent.
- The antigen recognized by the antibody is known as immuno.
- And this antibody is recognized by enzyme attached antibody which is known as enzyme-linked.
- The substrate produces a product which is usually colored on the reaction with the enzyme.
It is a plate-based assay technique. It is widely used for the detection of a variety of antigens and antibodies. It is a diagnostic tool in the field of biotechnology and medicine.
History of ELISA
Peter Perlman from Netherland and Eva Engvall from Sweden discovered ELISA independently at the same time ad published their papers that synthesized this knowledge into the method to perform ELISA.
The terminology used in ELISA
- Solid-phase – The microtitre plate well having 8*12 well used as a solid phase in ELISA.
- Adsorption- It is a process of adding an antigen /antibody and diluted with a buffer so it can be attached to the solid phase at the time of incubation.
- Washing- The process of flooding and emptying of wells with a buffered solution for the separation of bound reagents from unbound reagents.
- Antigen – It is the molecule that evokes the production of antibodies when antigen introduced into the body.
- Antibodies – It is a protein that is produced in response to antigenic stimuli.
- Enzyme conjugate – It is an enzyme that is linked irreversibly to an antibody .e.g. Horse – reddish peroxidase (HRPO) and alkaline phosphate (AP).
The principal of ELISA
On the basis of basic immunology response, the ELISA is based on the lock and key concept where antigen act as a key and antibody as a lock.
The basic principle of an ELISA is binding of antigen-antibody in the presence of enzyme which produces enzyme-linked antibody and then this enzyme-linked antibody reacts with the substrate which produces color and this color will be analyzed in a calorimeter to gain the particular quantity of that particular antigen.
The procedure used in ELISA
- Take a polystyrene plate which provides a surface for the attachment of antigen.
- Add antigen/sample in this plate then antigen will be attached on the surface.
- Wash the microtitre plate by using a buffer such as Tris phosphate buffer and Detergent- tween 20 (0.01-0.05%.) to remove the other substances and proteins except for antigens.
- Add enzyme-linked antibody to the microtitre plate.
- Wash the plate with buffers
- Now add specified substrate which will react with enzyme-linked with antibody
- Enzyme and substrate reactions produce a blue color product.
- Then this color product will be used in the analysis of the amount of that particular antigen
Types of Elisa
A.) Non-competitive ELISA
- Direct ELISA- In this type of essay, a primary labeled antibody is used which reacts with antigen directly. It can be performed directly with an immobilized antigen on an assay plate. It is not widely used but most common in immunohistochemical staining of cells ad tissues.
- Add a sample of known antigen to a surface of a microtitre plate.
- Then add enzyme-linked the primary antibody to the plate.
- Wash the plate with a buffer then antibody-antigen complexes remain attached only.
- Add substrate to evoke the chromogenic signal.
- It is a quick methodology as only one antibody is used in this assay and
- The cross activity of the secondary antibody is eliminated.
- The labeling of the primary antibody is time-consuming.
- Immunoreactivity of primary antibody may be reduced.
- Indirect ELISA – In this type of assay unlabelled primary antibody is used for the conjugation with a secondary labelled antibody. Secondary antibody ha specificity fr primary antibody.
- Add antigen to the microtitre plate
- Wash with buffer.
- Now add suitable primary antibody
- Now add secondary antibody (HPRO) which recognizes and binds t primary antibody.
- Then add TMB substrate which produces a colored product which will be detectable form fo a particular antigen.
- The labeled antibody is available in a wide variety of commercially.
- It is versatile
- Immunoreactivity is not affected by labeling o primary antibody.
- Sensitivity is increased.
- Sandwich ELISA- This type of ELISA used for the detection of antigens like tumor markers, hormones, serum proteins. The antigen in the sample became immobilized on the reaction with the captured antibody. A labeled antibody added to the plate and Enzyme HRPO added which binds with a labelled antibody. Then TMB is added and converted by HRPO y colors product.
- Competitive ELISA – There are two specific antibodies i.e. one conjugated with enzyme and the other one is present in serum. They both compete with each other for the same antigen. The ag-ab complex bound is inversely proportional to the concentration of antigen present in the sample.
Advantages of ELISA
- The reagents are cheap and have a long shelf life.
- It is very specific and sensitive.
- It is easy to perform
- It can be used in the detection of a variety of infections.
- Its types of equipment are easily available
Disadvantages of ELISA
- Enzyme activity measurement can be more complex than radioisotopes.
- Plasma consistent may affect enzyme activity.
- Kits are expensive
- It is very specific to a particular antigen.
Applications of ELISA
- It is used in the screening of blood on the time of donation for the evidence of viral contamination by-
- Hepatitis B
- Hepatitis C
- It is also used in the measuring of various hormones like HCG, LH, TSH, T3, T4, etc.
- It is used for the detection of various sexually transmitted diseases and infections.
- It also used to detection of allergens in food ad house dust.