ELISA

ELISA or Enzyme-Linked Immuno Sorbent Assay is an immunological techniques to detect the presence of an antigen, antibody, other proteins or glycoproteins in a given biological sample. The basis of this test is the ability of the antibody to bind specifically to a given antigen. (We know how the monoclonal antibodies to a particular antigen is synthesized.)

This involves an antigen (or antibody) coated onto the substrate. The sample containing the antibody (or antigen) is added, which gets immobilised due to its interaction with the coated antigen (or antibody). Finally, the antibodies ( primary or secondary) are added, which are conjugated to a detection system. Detection system consists of an enzyme capable of converting a substrate to a coloured product. The colour change can be used to quantify the amount of antibody (or antigen) present in the sample.

Let’s understand this in a better way!! Let’s start with the components of an ELISA procedure.

Basic ELISA procedure. — Basic ELISA procedure

There are five major components of ELISA, depending on the type of format used all the components may or may not be present.

1. Antigen:

Antigens present in the diagnostic samples are immobilised or coated directly onto the substrate or held by the capture antibody.

In the cases where the antibodies have to be detected from the sample (serum, etc), the antigens are absorbed/ immobilised onto the substrate directly as well.

2. Capture Antibody:

The capture antibody is specific for the sample antigen. It binds and captures the antigen to be detected. The function of the capture antibody is to immobilise the antigen from the sample onto the substrate, and prevent its wash off.

3. Primary Antibody:

The primary antibody is used to detect the presence of the antigen. The primary antibody directly binds the antigen at epitope different than the capture antibody. This antibody can be conjugated to an enzyme or a protein (biotin), which helps in detection.

4. Secondary Antibody:

A secondary antibody specifically binds to untagged primary antibody. It is conjugated with the detection system. A single secondary antibody can be used for a range of primary antibodies, hence surpassing the need to conjugate each primary antibodies with the detection system. Usually this involves having the secondary and primary antibodies from different species (example primary antibody from mouse and secondary antibody from rabbit) and secondary antibody being targeted to the constant region of the primary antibodies.

5. Detection system:

The detection antibody (either primary or secondary antibody) is linked to a detection system. This either involves an enzyme (linked directly or to streptavidin) and a substrate.

The most commonly used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). β-galactosidase, acetylcholinesterase and catalase are also used.

Horseradish peroxidase (HRP) catalyses the conversion of chromogenic or chemiluminescent substrates. There are several different types of HRP substrates.

Chromogenic HRP substrates, become colored after reaction with HRP. Commonly used chromogenic HRP substrates include 3,3′,5,5′-tetramethylbenzidine (TMB) and 2,2′ -azino-di-[3-ethylbenzthiazoline-6-sulfonic acid] (ABTS). Colourless TMB in presence of HRP and H2O2, is oxidised to a blue coloured product (at 650nm).

In a chemiluminescence assay, luminescent HRP substrate emits light when oxidised by HRP. (e.g. Enhanced Chemiluminescence by luminol).

The primary/ secondary antibodies can be tagged with biotin, which has high affinity towards avidin and streptavidin. The streptavidin (or avidin) is in turn is conjugated to enzymes.

The choice of substrate depends upon the required assay sensitivity and the instrumentation available.

There are different types of ELISA, using these components. They are:

1. Direct ELISA:

This is a one step and most basic format. In this format, the antigen from the diagnostic sample is immobilized onto the substrate and detected with an antibody (primary antibody) conjugated to enzyme (detection system).

2. Indirect ELISA

This is a two step method. In this, the antigen is immobilized to the surface. But the primary antibody is not conjugated with the detection molecule, rather a secondary antibody specific for the primary antibody. The detection molecule is conjugated to the secondary antibody.

3. Sandwich ELISA

This has a capture antibody coated onto the substrate. Then the antigen sample is added. If the antigen under investigation is present, the antigen gets immobilised. Then the primary antibody against another epitope of antigen is added, which is linked to the detection system and brings about change in the colour of the substrate. The format may use a secondary antibody as well.

4.Competitive ELISA

In this format the sample antigen competes with a reference antigen for binding the same paratope on the antibody (conjugated with the detection system) present. The sample is incubated with the linked antibodies, this mix is then introduced into the tube with reference antigens coated onto the substrate.

If the sample antigen is present in high concentration, they bind the antibodies, which in turn will be unable to bind the reference antigens coated onto the substrate. The antibodies bound to sample antigens will be washed away and there will not be enough detection system to change the colour of the substrate. Hence the amount of antigen in the sample is inversely proportional to the colour intensity. Therefore higher the concentration of antigen, lighter/ lesser will be colour change.

In some kits, the reference antigens are labelled rather than the antibodies. Hence the labelled antigen and sample antigens compete for interaction with the capture antibody for immobilisation. If the concentration of antigen in sample is low there will be stronger signal and the addition of the detection antibody can be surpassed.

ELISA is been widely used due to its specificity and sensitivity. It is possible to carry out ELISA in 96 well plates, or even 384 plate format. It can be used for quantitative analysis as well. However the prior information about the antigen and antibodies may be required. Nonetheless it has really become quite popular in the medical and biological field.

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