Immunoassays comes under bioanalytical methods where the quantity of antigen depends on the antigen antibody reaction. When these immunoanalytical reagents (antigen and antibody) are mixed and incubated, the antigen binds to the antibody forming an immune complex.1 Enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) are most common types of assays which are used as diagnostic tools in medicine and as quality control measures in various industries and for the detection of specific antigens or antibodies in a collected sample. These two procedures have common basic principles and modifications of radioimmunoassay (RIA) method. RIA was first described by Berson and Yalow in the year 1960. Due to certain limitations associated with the principle of radioactivity, RIA assays were modified by replacing the radioisotope with an enzyme, thus creating the new methods of Enzyme immunoassay and ELISA.2
ELISA is a basic method of immunoassay, to detect and measure antibodies in the given sample of blood. P. Perlmann and E. Engvall developed this test3 as a substitute for certain radioimmunoassay tests in 1974, and slowly it has replaced the western blot test. The ELISA test is a versatile test when compared to other complicated tests and medical professionals can easily do it.
Applications of ELISA
Principle of ELISA
ELISA test is carried out in 96-well polystyrene plates. The principle behind ELISA test is that specific antibodies bind to the target antigen and detect the presence of antigens and amount of antigenic load present in the given sample. In order to increase the sensitivity and accuracy of the test, antibodies with high affinity should be coated on the plate. This test also provides the information regarding the concentration of antigen and antibody in the given sample.
Enzymes used in ELISA
Many enzymes were proposed but most commonly used is
Horse radishperoxidase followed by
An ELISA test most commonly used to diagnose
Most commonly in detection of HIV antigen in AIDS
Various infections of bacteria and virus
Oral cancer lesions such as Squamous cell carcinoma
For GMO (Genetically modified organism)
Quality control of Vaccines
In Screening of new born children for unknown antigens
In clinical research
Results may not be absolute.
Availability of antibody not possible all the time.
Concentration not clear.
False positivity due to long standing primary antibody or non specific binding of antibody or antigen in the given sample.
Advantages of ELISA:5
Simple procedure and easy to do.
As it involves two antibodies, test results in an accurate diagnosis.
Helps to find diagnosis in complicated cases as antigen is not required to get purified to detect.
As direct and indirect analysis methods are involved, it is highly responsive.
Rapid test, yields results quickly.
Not a complicated method as it does not involve the presence of radioactive materials and large amounts of organic solvents.
Low cost-effective as reagents are of low cost.
Equipment is inexpensive and widely used method compared to others.
Finest technique and expensive to prepare antibody.
To obtain a specific antibody, culture cell media are required.
Plasma constituents may affect the activity of enzyme in the sample.
More chances of false positive or negative results.
Instability of antibody in the sample.
Storage and transport in cold media is required
Kits are commercially available but not cheap.
Test is specific to particular type of antigen and can’t detect other antigens in the sample.
Types of ELISA tests4
ELISA tests have been categorized as
In this method, the antigen directly attaches to the enzyme labelled antibody. Most commonly used to assess the affinity and specificity of antibody and to in inhibitory interactions.
Time- consuming and expensive as each primary antibody has to be labeled for specific antigen.
Formation of cell smear.
Immunoreactivity is reduced as it is enzyme linked.
Choice of primary antibody label to choose from one experiment to another is less.
Minimal signal amplification.
Potential high background.
In this method, the antigen is coated to a multi-well plate and is detected in two stages. In the first step, an unlabeled primary antibody, which is specific for the antigen, is applied to the plate followed by an enzyme-labeled secondary antibody which will bound to the first antibody. Most commonly used for measuring endogenous antibodies.
Wide variety of labeled secondary antibodies are available commercially.
Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection.
Maximum immunoreactivity of the primary antibody is retained because it is not labeled.
Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for amplification of signal.
In this method, Antibody is coated on the microtitre well and it require the use of antibody pairs which are already matched, and each antibody is specific for different antigens. A first antibody (known as capture antibody) is coated to the wells and then the given sample added to the well. A second antibody (known as detection antibody) follows this step in order to measure the concentration of the sample. This method is used to determine the concentration of analyte in the given sample.
Competitive ELISA (Inhibition ELISA)
In this method, antigen is coated to the Microtiter well and the antigen-antibody complex is added to it. After that the well is then washed to remove any unbounded antibodies. The enzyme-conjugated secondary antibody specific for the isotype of primary antibody is added to determine the amount of primary antibody present in the well. The concentration is then determined by spectrophotometry. It is generally used to determine the concentrations of a small molecules and hormones.
ELISA Data Interpretation
The ELISA assay yields three different types of data output and it is interpreted in relation to standard curve i.e serial dilution of a known and purified antigen.
Quantitative: To calculate the concentrations of antigen in various samples
Qualitative: To know a particular antigen is present in the given sample or not and it is compared to a blank well containing no antigen or an unrelated control antigen.
Semi-Quantitative: To compare the relative levels of antigen in given sample as the intensity of signal will vary directly with the concentration of the antigen in the given sample.
Recent Modifications in ELISA
Multiple and portable ELISA
It is a new technique which uses a multicatcher device with 8 or 12 immunosorbent protruding pins on a central stick that can be immersed in a collected sample. The washings and incubation with enzyme-conjugated antigens and chromogens are performed by dipping the pins in prefilled microwells with the known reagents.2
This method uses advanced techniques like flow cytometry, fluorescence, chemiluminescence, or electrochemiluminescence for the determination of multiple cytokines expression and to assess the levels of more than one protein in the given sample.
ELISA test is an innovation in biomedical research field as it gives most accurate results. It has many advantages over other methods in terms of sensitivity, specificity and in terms of cost. It detects almost all types of biological molecules in the given sample at very low concentrations and quantities. ELISA remains an important tool in both clinical and basic research, as well as in clinical diagnostics although it has its own disadvantages and limitations.