- Received March 14, 2020
- Accepted February 12, 2021
- Publication April 02, 2021
- Visibility 8 Views
- Downloads 0 Downloads
- DOI 10.18231/j.ijmr.2021.003
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CrossMark
- Citation
Enzyme linked immunosorbantassay- lab diagnosis: A review
Introduction
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 test[3] 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
To detect the presence of unknown antigens and antibodies in the given sample.
To detect potential food allergens in the food industry.
To determine the concentration of antibody in the serum in viral diseases.
To track the spread of diseases during endemic cases.
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.
ELISA Procedure:[4]
Enzymes used in ELISA
Many enzymes were proposed but most commonly used is
Horse radishperoxidase followed by
Alkaline phosphatase
B-Galactosidase
Lactoperoxidase
Tetra Methylbenzidine
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
Tumor markers
Proteins
Hormones
Risks
There are few risks associated with this test. These include:
Cross Infections
Low blood pressure
Bruising
Excess Bleeding
Limitations:[2]
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.
False negativity.
Advantages of ELISA:[5]
Simple procedure and easy to do.
As it involves two antibodies, test results in an accurate diagnosis.
High specificity.
High sensitivity.
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.
Disadvantages[5]
Labor-intensive.
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 tests[4]
ELISA tests have been categorized as
Direct ELISA
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.
Advantages
Procedure is rapid and fast as only one antibody is involved.
Chances of cross-reactivity is less as no secondary antibody
Short protocol.
Disadvantages
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.
Low sensitivity.
Potential high background.
Indirect ELISA
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.
Advantages
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.
Disadvantages
Formation of cell smear.
Cross-reactivity.
An extra incubation step is required in the procedure.
Sandwich ELISA
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.
Advantages
High specificity and sensitivity.
Suitable for complex samples.
Flexibility.
Minimal sample purification needed.
Disadvantages
Must use “matched pair” primary and secondary antibodies.
Time consuming and expensive.
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.
Advantages
Minimal sample purification needed.
Used to measure large range of antigens in a sample.
Used for small antigens.
Low variability.
Disadvantages
Low specificity so cannot be used in dilute samples.
Requires a conjugated antigen.
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.
Reverse ELISA
It does not require traditional wells. It leaves the antigens suspended in the test fluid.
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]
Advantages
Inexpensive.
Readily available kits.
Used to screen large population.
Do not require skilled personnel or laboratory equipment.
Used for the detection of antigens in various infections, bacterial toxins, oncologic markers and screening of drugs.
Multiplex ELISA
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 Kits
These are readily available kits which contains pre-coated polystyrene plates, detection antibodies, and usually all of the chemicals required to perform an ELISA test. These kits are easier to use for detecting molecules or performing ligand-binding assays successfully.
Conclusion
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.
Source of Funding
None.
Conflict of Interest
The authors declare that there is no conflict of interest.
References
- IA Darwish. Immunoassay Methods and their Applications in Pharmaceutical Analysis: Basic Methodology and Recent Advances. Int J Biomed Sci 2006. [Google Scholar]
- SD Gan, KR Patel. Enzyme Immunoassay and Enzyme-Linked Immunosorbent Assay. J Invest Dermatol 2013. [Google Scholar] [Crossref]
- A Bald. ELISA- A Mini Review-Research and Reviews. J Pharm Anal 2016. [Google Scholar]
- . Boster antibody and ELISA Experts. ELISA fundamental principle, how ELISA works - immunoassays| boster. . [Google Scholar]
- S Sakamoto, W Putalun, WS Vimolmangkang, Y Phoolcharoen, H Shoyama, S Tanaka. Enzyme-linked immunosorbent assay for the quantitative/qualitative analysis of plant secondary metabolite. J Nat Med 2018. [Google Scholar]
- Introduction
- Applications of ELISA
- Principle of ELISA
- Limitations:[2]
- Advantages of ELISA:[5]
- Disadvantages[5]
- Types of ELISA tests[4]
- Direct ELISA
- Indirect ELISA
- Sandwich ELISA
- Competitive ELISA (Inhibition ELISA)
- ELISA Data Interpretation
- Recent Modifications in ELISA
- Conclusion
- Source of Funding
- Conflict of Interest