ELISA – The Enzyme Linked Immunosorbent Assay

The ELISA or enzyme-linked immunosorbent assay is a quantitative immunological procedure where the interaction of an antibody with an antigen is monitored using an enzyme catalysed reaction.

It is sometimes called the EIA or enzyme immunoassay and belongs to a class of assays relying on immunodetection.

The term ELISA was first used by Engvall and Perlma in 1971. It came to prominence as a screening test for HIV because of its high sensitivity. That sensitivity is such it is used to detect antigens or antibodies in solution between ng/mL and picogram (pg)/mL quantities. It also has the benefit of being relatively rapid and simple to use.

Overview

The method described is one that describes the indirect ELISA but it serves as a good description of what happens in an ELISA.

1. The antigen of interest is bound to a plastic surface.
2. It is recognised by a specific antibody.
3. The antibody is recognised by a second antibody which has an enzyme attached.
4. The attached enzyme reacts with colourless substrates (chromogen) to produce coloured products which are detected. The presence of a coloured product is directly linked to the presence of  antigen bound to the plastic surface.

Equipment

The plastic surface is  a microwell plate which is a flat bottomed polystyrene plate consisting of 8 by 12 small wells. Each holds 350 microlitres.

A multipipette which is an 8-channel 100 microlitre pipette for adding solutions to each 8 cell row of the microwell plate.

A washing device is designed to clean equipment of any  extraneous material. It is a useful device if the material is highly infectious and must be collected for subsequent sterilisation.

A microplate washer is an automatic microwell plate washer which removes material during the assay. It is designed to reduce low carry-over of contaminated material.

A plate reader is a necessity. Good examples include the BioTek Synergy™ HT plate reader (Winooski, Vt., USA).

Typical Reagents

1. A coating buffer. Typically 0.01M phosphate buffer and 0.15 M sodium chloride. Usually called PBS.
2. A dilution and wash buffer which is PBS with added Tween 20 to 0.1%w/w.
3. Blocking buffer. A solution of bovine serum albumin (BSA)
4. Enzyme. Always horseradish peroxidase (HRPO)
5. chromogenic substrates – trimethyl benzidine (TMB)
6. Stop solution – 0.5M sulphuric acid.

Types of ELISA

The different types of ELISA are based on the range of detection methods. 

1. The colourimetric ELISA. As already described, the immobilised antigen is bound to the microwell plate. A primary antibody is added which fixes to the antigen whilst excess antibody is washed away. A labeled secondary antibody is added to which is bound an enzyme. Substrate is added which binds to the secondary antibody. A coloured substrate is generated which is detected as a signal and the amount of signal is quantified. 
2. The Chemiluminescent ELISA. In this method the process is exactly the same up to the point where the substrate produces chemiluminescence as opposed to a coloured product.

The Indirect ELISA

This is the method most of us are familiar with. It’s used to detect an antigen and is an effective method for assessing degrees of response by the human body to an infection. It is primarily used for the HIV Infection test.

In the indirect ELISA, the unlabelled primary antibody is targeted by a labelled secondary antibody. 

Very often the labelled secondary antibody is directed against all antibodies of a given species such as a rat or mouse. It can this be used with a wide variety of primary antibodies.

The process involves fixing the antigen of interest to a plate. The blocking buffer is added followed by a suitable primary antibody. Then a secondary antibody with HRPO attached is added to recognise and bind to the primary antibody. Then the TMB substrate is added and converted to a detectable form.

The Direct ELISA

In this method, the antigen is bound to the plate and an enzyme conjugated antibody is added. There is no secondary antibody to be added.

Sandwich – ELISA

In this assay, an antibody is added to the well. The antigen to be measured is then added. An enzyme-conjugated secondary antibody is added  which binds to the antigen. A chromogen is added and the colour measured.

Competition-ELISA

A method where there is competition between antibodies for one antigen or by two antigens for one antibody.

The Main Benefits 

• rapidity – at least 90 samples can be tested in 2 to 3 hours
• sensitivity – up to 10 picograms/mL are detected
• highly specific so can work with dirty samples that are contaminated
• lots of samples are processed almost simultaneously
• uses a colorimetric test – observed easily and measured using a spectrophotometer.
• Can analyse antibodies and antigens
• Samll sample sizes used from between 10 microlitres and 100 microlitres.