5 Types of Elisa in Detail & the Principle of Elisa Test

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The term ELISA in full form is an Enzyme linked immuno-sorbitent assay. Amongst the available immunoassays, Elisa is one of the distinguished and widely used types.  It is extensively used due to the advantages like rapidity or speed in experimentation, greater sensitivity and specificity for even small amount of test samples. The reaction is measurable in both qualitative and quantitative terms i.e. qualitatively it measures the specific type of substance while quantitatively it measures quantity of substance present in the given sample respectively. It finds its application in daily basis in clinical diagnosis, biological, food and medical scientific research.

Immunoassays are those assays wherein any anti-gen or anti-body are detected for their presence in a given sample.

        But what is an what is 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 and hence the body tries to reject that 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   etc. into the body and neutralize or destroy the foreign substance containing the antigen.

So in condition of infections or diseases by bacteria and other microbes, their antigens are present and the antibodies directed against these antigens are also formed in the patient body.

Hence Immunoassay 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 bacteria present in patient sputum or blood can be detected by suing suitable antibody or vice versa.

What is Elisa test?

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.

Elisa principle:

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.

Check the video below for ELISA PRINCIPLE

Types of Elisa:

Elisa is broadly of two types as direct Elisa and indirect Elisa. But lets see on over all the Different Types of Elisa

Heterogeneous: Here  the antigens first added is a solid type. i.e. it is fixed to the plate and not in liquid form while next antibodies or antigens added are always in liquid phase.

Homogeneous: Here the antigens first added are also 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 colour reaction with externally added substrate i.e. Elisa reagent.

Ag or Ab + Ab or Ag-(e) −−−−−→ Reaction color..

# Elisa testIndirect 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..

The indirect Elisa protocol: 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 colour 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 colour reaction with substrate added.

Ab  +  Ag  + Ab-(e)  −−−−−→ Reaction color..

#Sand wich elisa principle

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.

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. 

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