Overview on ELISA: Its History, Principle, Application and Limitation

ELISA (Enzyme-Linked Immunosorbent Assay) is one of the common laboratory techniques used to measure the concentration of antibiotics or antigens. It is a popular technique in molecular biology and has revolutionized the field of immunology. ELISA, simply, works on the principle of antigen-antibody reaction. It is used in many areas of research and drug discovery on a wide variety of samples. Cell lysates, blood samples, food items are most analysed for specific substances of interest using ELISA.

1.1 History of ELISA

In 1942, Albert H. Coons along with his colleagues introduced the concept of ‘immunofluorescence’. They labelled antibodies with a fluorescent dye and used it to identify antigens in tissue sections. Concept of radioimmunoassay was brought forward by Rosalyn Sussman and Solomon Berson in 1960 but due to potential health hazards presented by radioactivity, the idea was subdued. Later, in 1971, Eva Engvall and Peter Perlman invented the ELISA test, to seek out the presence of hormone or viruses. In the subsequent years different approaches to ELISA test were devised, such as: the Indirect ELISA method (1975) the competitive ELISA method (1976) and Sandwich ELISA method (1977). In 1985, ELISA test was recognized as the first screening test commonly approved for HIV.¹ In the recent years, more specifically during the global COVID-19 pandemic, it emerged as a wide topic of discussion due to its role in testing the antibodies of COVID-19.

1.2 Principle of ELISA

An antibody (Ab) reacts with an antigen (Ag) in a highly specific manner. An antibody only reacts with the particular determinant or region of an antigen for which it is specific and produces Ab-Ag complex. Factors such as amount of Ab-Ag complex formed or the rate of its formation determines the quantity of the antigen or antibody involved in the reaction.²

^{Fig 1: Steps involved in ELISA test. Source: OpenLearn}

1.3 Types of ELISA test

Different approaches to the ELISA testing have been introduced over time. Some of the most prominent methods of ELISA testing are as follows:

Direct ELISA

The antigen of interest is immobilized on the surface of the test tube or microliter well. Specially constructed ELISA plates are available for processing large number of test samples. An antibody specific to the antigen is added and allowed to interact with the absorbed antigen.² The molecules which do not undergo any reaction are washed away leaving behind the Ab-Ag complex. These steps are referred to as primary reaction.

During the secondary reaction, an anti-immunoglobin (Anti-Ig) is added to the vessel and allowed to interact with the Ab-Ag complex. Anti-Ig binds to antibody component of Ab-Ag complex. Anti-Ag is linked to a conjugate enzyme molecule. The unreacted Anti-Ig is washed away. Then, the substrate of the enzyme is added along with the necessary reagents to develop colour because of the enzymatic activity. The concentration of the enzyme is directly proportional to colour intensity. Thus, colour intensity is used to determine the quantity of antigen or antibody. For more accuracy, a computerized ELISA reader is used to measure the colour intensity.² This approach of ELISA is often referred to as direct antigen coating (DAC) ELISA.

This approach is useful in qualitative assay of specific antibody present in the antiserum used for primary reaction.

Indirect ELISA

It is a two-step ELISA test that involves two binding processes of primary antibody and labelled secondary antibody. In this approach, the microliter plates are incubated with specific antigens. The remaining protein binding sites are blocked by addition of blocking buffer. The primary antibody without label is added, and the plate is incubated at 37°C. Later, the unbound antibodies are removed by washing.² An enzyme-conjugated secondary antibody is added to the Ag-Ab complex. It recognizes and binds to the primary antibody. Unbound enzyme-linked antibodies are completely removed from the plate by washing. Finally, the substrate is added which reacts with pre-existing enzyme resulting in colour formation.

Sandwich ELISA

In this approach, an unlabelled antibody specific to the antigen of interest is added and allowed to react. Then, the antigen is added and allowed to react with the immobilized antibody forming Ag-Ab complex.² A second antibody specific to the antigen and labelled with the enzyme is added to the mixture. The new antibody reacts with the Ag-Ab complex. The unreacted antibodies are washed out and enzyme substrate is added. Then, colour change is measured. This approach is mainly used for the rapid qualitative assay of specific antigen of interest.

1.4 Enzymes used in ELISA test

Generally, horseradish peroxidase, alkaline phosphatase, B-galactosidase, Lacto-peroxidase, etc are most used for labelling of antibodies. Among these enzymes, horseradish peroxidase is the most popular one. If peroxidase is present, the substrate Hydrogen peroxide (H₂0₂) is converted into water and O₂ in the presence of diaminobenzidine or 4-chloronaphthol. Diaminobenzidine is oxidized and gives dark brown colour. 4-chloronaphthol is oxidized to produce purple colour.²

1.5 Application of ELISA

It can detect antibodies produced by the body in response to pathogen. It is used to diagnose diseases such as HIV/AIDS, Hepatitis B and C, Lyme disease, Syphilis etc.
It can be used to diagnose autoimmune diseases such as Lupus, Rheumatoid, Arthritis, multiple Sclerosis, etc.
ELISA can also be used to diagnose certain type of cancer such as prostate cancer, ovarian cancer, etc.
It can be used to detect and identify allergens (pollen, dust mites, food).
It is used to monitor drug levels in the blood. This helps to detect drug abuse.
It is also used in analysis of hormones, vitamins, metabolites, diagnostic markers (Follicle stimulating hormone, Insulin, Testosterone, Vitamin B12).

1.6 Limitation of ELISA test

ELISA bears the probability of giving false positive or false negative result. False positives may occur due to non-specific binding of antibodies or cross-reactivity with similar antigens. False negatives may occur when the antigen or antibody concentration is too low to be detected.
Traditional ELISA may prove to be time consuming. Multiple steps involved such as incubation and washing may even take several hours.
Component in biological sample (like blood or serum) may interfere with Ab-Ag binding or enzyme activity. This decreases the accuracy of result.
ELISA requires stable environmental parameters (especially temperature) for accurate results. It is less suitable for field used without proper equipment.