The term ELISA in full form is an Enzyme linked immuno-sorbitent assay.
Besides Western Blotting, Elisa is one of the distinguished and widely used types of immunoassays.
It is extensively used techniques in microbiology due to the advantages like rapidity or speed in experimentation unlike radioimmunoassay.
Further it has greater sensitivity and specificity for even small amount of test samples.
The reaction is measurable in both qualitative and quantitative terms.
Qualitatively it measures the specific type of substance.
While quantitatively, it measures quantity of substance present in the given sample.
It finds its application on daily basis in clinical diagnosis, biological, food and medical research.
Before you go further, you may need to read what is immune reaction for an idea.
Immunoassays are those assays wherein any anti-gen or anti-body are detected for their presence in a given sample.
But what is an antibody (Ab) or anti-gen (Ag) and why and what makes them to be detected ?
“Antigens are any proteinacious chemical (humoral) substances which are present on the surface of body cells and tissues. Every organism or animal has its own set of biochemical identity in the form of its antigens.
When a foreign substance enters into its blood or fluids, it’ s antigens are recognized to be different from that of 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 as antibodies. These antibodies recognize the newly entered antigens of bacteria or virus into the body and destroy them.
So in condition of infections & diseases by bacteria and other microbes, their antigens are recognized. Then immunological reaction triggers and the antibodies are formed, 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 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 patient’s sample are allowed to stick to a polyvinyl plate and then plate is washed to separate antigens or antibodies (if any present) from 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 there by identified.
Here the ezyme’s role is the key to develop color. So we use immobilised enzymes for the purpose to keep them stable.
Check the video below for ELISA PRINCIPLE
Types of Elisa:
There are different methods and protocols in Elisa. But basically Elisa is broadly of two types as direct Elisa and indirect Elisa. But it can be differentiated as in the image below.
Heterogeneous: Here the antigens are first added and is in a solid form. i.e. fixed to the plate and not in liquid form while next antibodies or antigens added are always in liquid phase.
Homogeneous: Here the antigens are first added and are in liquid phase and the next antibodies added on to it are also in liquid phase.
Direct Elisa 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 antibodies sample which is tagged to an 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 anti-body 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 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 other is sandwich Elisa.
Ag or Ab + Ab or Ag +Ag or Ab-(e) −−−−−→ Reaction color.
Normal Indirect Elisa: Here an external antigen of suspected antibody which might be present in patient sample is added to the plate. Then patient sample is added to see that if any suspected antibody is present, it reacts with the external antigen fixed in the plate. Then another antibody is which is enzyme linked is added to react with antibody from sample and produce color on addition of substrate.
Ag + Ab + Ab-(e) −−−−−→ Reaction color..
Sandwich Elisa: 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 plate, over it 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 substrate added.
Ab + Ag + Ab-(e) −−−−−→ Reaction color..
Competitive Elisa: It is not a special type of Elisa but a slight modification to the protocols mentioned in above types of Elisa like Direct, Indirect, Sandwich Elisa types.
Here one more substance (preferably biotinated 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 in between Ag or Ab but nit mere addition of them.
Also you may check out “ELISA: Theory and practice“.
Elisa test cost: Elisa is not cheaper but are cost effective and very reliable in terms of results. Some kits are affordable at minimal cost like US $200 to $ 300 but some can be very expensive like few thousand dollars. So the cost varies on the type of Elisa kit and also the manufacturer.
For instance you can estimate a protein by western blot for 45 sample over 30 days with double the cost than required for Elisa which only can be done in a single day. Even Elisa is quantitative while Western blot is semi quantitative. Elisa method is so faster due to use of elisa plate readers which can estimate approximately 90 samples per test.