The term ‘ELISA’ in full form is an Enzyme-linked Immuno-sorbent assay. It is one of the widely used immunoassays besides the Western Blotting technique.
The ELISA tests are of different types like
- Direct Elisa
- Indirect Elisa and
- Sandwich Elisa.
This ELISA test is extensively used in clinical diagnosis for healthcare. medical research and microbiological research due to its advantages like
- rapidity of experimentation
- small sample size
- Specificity and
- a large number of samples
that can be tested at once, unlike western-blot or radioimmunoassay.
Further, the ELISA reaction can be measured in both qualitative and quantitative terms.
Qualitatively one can identify the specific substance present in the sample. While quantitatively, the amount of substance present in the given sample can be known.
Before you dig further, you must understand what is an immune reaction for an idea.
Immune reactions and Immunoassays
A reaction that occurs between an antigen and antibody leading to an antigen-antibody complex formation is called an immune reaction.
But now what is an antibody (Ab) and antigen (Ag)?
“Antigens are proteinaceous substances that are present on the surface of body cells and tissues. Every organism or animal has its own set of biochemical identities in the form of its antigens.
When a foreign substance enters into its blood, the body’s immune cells recognize the antigen. These antigens are different from that of the body’ s own antigens. Hence the body triggers an immune reaction to arrest the foreign particle and also destroy it.”
For this, it produces a special set of substances called antibodies. These antibodies recognize the entered antigens like that of bacteria or viruses into the body and destroy them.
So in the condition of infections by bacteria and other microbes, their antigens are recognized. Then immunological reaction triggers and the antibodies are formed, which are directed against these antigens. So in the body of the patient antibodies are and even antigens (in severe conditions) are present.
Hence Immunoassays are used to recognize or detect the type of antigen inflicting the body. For example, if a person is inflicted with hepatitis, the antigens of that virus are present in patient serum. They can be detected by taking the blood sample and using a suitable antibody by Elisa. Even vice-verse i.e. detecting antibodies is possible.
It is also a type of immunoassay aimed to detect the antigen or antibodies present in an inflicted individual for proper diagnosis and further treatment.
The antigens or antibodies present in the patient’s sample are allowed to stick to a polyvinyl plate and then the plate is washed to separate antigens or antibodies (if any present) from the remaining sample components. To this plate, a corresponding second antibody or second antigen is added to get fixed to the already adhered first antigen in the plate. To this added second antigen or second antibody an enzyme is also tagged so that, when a suitable substrate is added, the enzyme reacts with it to produce a color. This color produced is measurable as a function or quantity of antigens or antibodies present in the given sample and thereby identified.
Types of Elisa
There are different methods in Elisa protocols. But, basically, there are of two distinct types as the
- Direct Elisa
- Indirect Elisa.
But in a detailed sense, they can be differentiated as shown in the image below.
Here the antigens are first added and are in a solid form. i.e. fixed to the plate and not in liquid form while the next antibodies or antigens added are always in the liquid phase.
Here the antigens are first added and are in the liquid phase and the next antibodies added on to it are also in the liquid phase.
It is one wherein there is only one set of antigens and one set of antibodies to react. In this ELISA method, antigens from the patient sample fixed to the Elisa plates are made to react with an antibody sample which is tagged to a marker enzyme. I.e. directly to the antigen in the test an enzyme-linked antibody is added to produce a color reaction with externally added substrate i.e. Elisa reagent.
Ag or Ab + Ab or Ag-(e) −−−−−→ Reaction color.
Indirect Elisa principle
HIV Elisa Test is done using this principle. This has a difference to the Direct Elisa in that one more additional antibody is added in the reaction. To the antigen (fixed to Elisa plate) an antibody is added. Again secondary antigen is added which is enzyme-linked. This requirement is due to the reason that sometimes in patients antigen of the disease-causing agent may not be present but a corresponding antibody is available in the patient sample. which can be traced. These are of two types like normal Indirect Elisa and the other is a sandwich, Elisa.
Ag or Ab + Ab or Ag +Ag or Ab-(e) −−−−−→ Reaction color.
Normal Indirect Elisa
Here an external antigen of a suspected antibody that might be present in the patient sample is added to the plate. The patient sample is added to see that if any suspected antibody is present, it reacts with the external antigen fixed on the plate. Then another antibody is which is enzyme-linked is added to react with antibodies from the sample and produce color on the addition of substrate.
Ag + Ab + Ab-(e) −−−−−→ Reaction color..
This is also an indirect type of Elisa. The only difference in this ELISA principle is that, just like a sandwich, in between two antibodies an antigen is present just a seen in the figure below. The antibody at bottom fixes to the surface of the plate, over its antigen is fixed onto which one more antibody (junction) is attached. On this junction antibody, an enzyme-linked antibody is present to give the color reaction with the substrate added.
Ab + Ag + Ab-(e) −−−−−→ Reaction color..
It is not a special type of Elisa but a slight modification to the protocols mentioned in the above types of Elisa like Direct, Indirect, Sandwich Elisa types.
Here one more substance (preferably biotinylated substance) is added to compete with Ab, Ag to bind to the already added Ag, Ab during the reaction. The intention of adding this competitor substance is to prevent unnecessary binding of Ab or Ag and see that the binding is only due to greater affinity between Ag or Ab but not the mere addition of them.
Also, you may check out “ELISA: Theory and practice“.
It finds its application on a daily basis in clinical diagnosis, biological, food, and medical research.