5 Types of Elisa Tests | Their Differences and Principle Explained

Elisa is a highly specific immunological test that produces quick results for large-scale screening. It is of the following types

1. Direct Elisa
2. Indirect Elisa
3. Sandwich Elisa.
4. Competitive Elisa.

The Enzyme-linked immunosorbent assay is abbreviated as ELISA.

It is one of the most widely used immunoassays besides the Western Blotting technique or radioimmunoassay.

This test is extensively used in clinical diagnosis, medical research, and microbiological research due to advantages like

• Rapid experimentation
• Requirement of Small sample size
• Highly Specific for the pathogen under question
• Supports a large number of samples
• Used for both qualitative and quantitative analysis.

Elisa principle:

Before diving into the types, one needs to know how it works.

In brief, an antigen present in the patient’s sample is made to react with an antibody stuck onto a polyvinyl plate.

The plate is then washed to discard unbound antigens to prevent noise.

Then, a corresponding secondary antibody conjugated with an enzyme is added to the plate and allowed to incubate so that this new antibody binds to the existing plate.

The plate is again washed, and a substrate corresponding to the conjugated enzyme is added to produce a colored product.

The color produced is measured and identified as a function of antigens in the given patient’s sample.

Elisa types in detail.

Direct Elisa

Here, the antigen (sample) of the patient is added to bind to the plate well.

Then, the plate is washed, and an antibody tagged with a marker enzyme is added.

After the incubation for a brief period, the wells are washed to remove unbound antibodies.

A substrate suitable for an enzyme producing a colored product is added.

After the color develops, the sample is identified and even quantified with a spectrometer.

It is one wherein there is only one set of antigens and one set of antibodies to react.

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

Thus, in direct ELISA, one set of antigens reacts with one set of antibodies for detection.

Indirect Elisa principle

This is different from the Direct Elisa in that one more antibody is added to the reaction. An antibody is added to the antigen (fixed to the Elisa plate). Again, a secondary antigen, enzyme-linked, is added. This requirement is because sometimes, an antigen of the disease-causing agent may not be present in the patients. Still, a corresponding antibody is available in the patient sample, which can be traced. There are two types: normal Indirect Elisa and the other is sandwich Elisa.

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

HIV Elisa Test is done using this principle.

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 enzyme-linked antibody is added to react with antibodies from the sample and produce color with the 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, as seen in the figure below. The antibody at the bottom fixes to the surface of the plate, and over its antigen, it is fixed onto which one more antibody (junction) is attached. An enzyme-linked antibody is present on this junction antibody to give the color reaction with the 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 the above types of Elisa, like Direct, Indirect, and Sandwich Elisa types.

Here, one more substance (preferably a 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.

It finds its application on a daily basis in clinical diagnosis, biological, food, and medical research.

Besides the above, one can even classify it based on the state of substances used, like

• Heterogeneous type
• Homogeneous type

Heterogeneous type

Here, the solid-form antigens are first added and allowed to be fixed on the plate. The next antibodies or antigens in the liquid state are added.

Homogeneous type

Here, the antigens in the liquid phase are first added, and then antibodies in the liquid phase are added to react.

Compare ELISA with western blot.