Last Updated on March 17, 2025
Enzyme-linked immunosorbent assay or ELISA is a commonly used immunological biochemical assay. It is also called EIA or enzyme immunoassay.
It is used to detect and measure antibodies, antigens, hormones, peptides, and proteins in blood or other biological samples. Similar to other types of immunoassays, ELISA uses antibodies to detect a particular antigen using highly specific antibody-antigen reactions.
It plays an important role in diagnostic medicine and drug screening.
The ELISA technique was conceptualized and developed by Peter Perlmann and Eva Engvall at Stockholm University, Sweden in 1971.
Read more about Antibodies and Their Role in Body Defence
Principle of ELISA
It is based on the principle of antigen-antibody complex formation.
The antigen is immobilized or coated on a solid surface. This is done either directly or through the use of a capture antibody which itself is immobilized on the surface.
The antigen then combines with a detection antibody which is conjugated with a molecule (eg. enzyme) that can be easily detected.
The conjugated enzyme acts on a specific substrate called chromogenic substrate and generates a colored reaction product (chromogenic detection).
The colored product thus formed can be quantitatively or qualitatively read using an ELISA plate reader.
An ELISA assay is performed in a multi-well plate (96- or 384-wells), which provides the solid surface required to immobilize the antigen. Immobilization of the analytes helps in separating the antigen from the rest of the components present in the sample. Due to this feature, ELISA is one of the easiest and highly sensitive assays to be performed on a large number of samples simultaneously.
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Procedure
Coat plate with antigen/ antibody
↓ wash
Add blocking buffer
↓ wash
Add test sample
↓ wash
Add enzyme-conjugated antibody
↓ wash
Add substrate
↓
Add stop solution
↓
Read absorbance at 450 nm.
The substrate is converted by the enzyme to form a colored product, which can be measured by spectrophotometry.
The most commonly used conjugated enzymes include Horseradish peroxidase (HRP) and alkaline phosphatase.
Commonly used substrates include OPD (o-phenylenediamine dihydrochloride), TMB (3,3′,5,5′-tetramethylbenzidine), ABTS (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) and PNPP (p-Nitrophenyl Phosphate).
Types of ELISA
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Direct ELISA
The antigen is attached to the surface of the wells of the microtitre plate. This antigen is detected with an antibody specific for the antigen The antibody is directly conjugated to the enzyme.
Advantages
- Faster than other ELISA – has fewer steps
- No cross-reactivity from secondary antibody
Disadvantages
- Higher background noise – because all proteins in the sample, including the target protein, will bind to the surface
- Less flexible – each target protein needs a specific conjugated primary antibody
- No signal amplification – reduces sensitivity
Ideal when analyzing the immune response to an antigen.
Indirect ELISA
It uses a two-step process for the detection of an antibody in a sample. A primary antibody specific for the antigen binds to the antigen, and a labeled secondary antibody against the primary antibody binds to the primary antibody for detection.
- The antigen is coated to the microtitre wells
- A sample containing the antibodies is added to the antigen-coated wells for binding with the antigen.
- The free primary antibodies are washed away.
- The antigen-antibody complex is detected by adding a secondary antibody conjugated with an enzyme that can bind with the primary antibody.
- After washing all the free secondary antibodies, an enzyme-specific substrate is added which gives a colored product that can then be measured.
Advantages
- High sensitivity – several secondary antibodies can bind the primary antibody
- Greater flexibility – different primary antibodies can be used with a single labeled secondary antibody
Disadvantages
- Possibility of cross-reactivity from the secondary antibody
- Longer procedure than direct ELISA – additional incubation step for secondary antibody needed
Sandwich ELISA
It is the most commonly used type. It helps to detect the presence of antigen in a sample. It requires two antibodies (matched antibody pairs) specific for different epitopes of the antigen. One of the antibodies is coated on the surface of the wells of the microtitre plate and used as a capture antibody to facilitate the attachment of the antigen. The secondary antibody then binds to another epitope of the antigen and helps to detect the antigen.
- The antibody is coated to the wells of the microtitre plate.
- The sample containing the antigen is added to the well.
- The free antigens are washed away.
- An enzyme-linked secondary antibody, which binds to another epitope on the antigen is then added.
- After washing away any free secondary antibodies, an enzyme-specific substrate is added which forms a colored product, that can be measured.
Advantages
- High sensitivity – much more sensitive as compared to direct or indirect ELISA
- High specificity – two antibodies are involved in capture and detection
- Flexibility – both direct and indirect methods can be used
Disadvantages
- Finding two antibodies against the same target that recognize different epitopes and work well together can be difficult.
- Cross-reactivity may occur between the capture and detection antibodies.
Ideal for the analysis of complex samples, as the antigen does not need to be purified before testing.
Competitive ELISA
It is also known as inhibition ELISA. It helps to detect the presence of an antigen in a sample by detection of signal interference.
The sample antigen competes with a reference antigen for binding to a specific amount of labeled antibodies. The reference antigen is pre-coated on a multi-well plate and the sample is pre-incubated with labeled antibody and added to the wells. Depending on the amount of antigen in the sample, more or less free antibodies will be available to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the signal.
- The microtitre wells are coated with the reference antigen.
- Antibodies are incubated in a solution along with the sample (which contains the antigen to be detected)
- The solution containing antigen-antibody complex is added to the microtitre wells.
- The well is washed to remove any unbound antibodies.
- The enzyme-linked secondary antibody is added which will attach to the reference antigens present in the well.
- If the concentration of antigen in the sample is high, less number of free antibodies will be available to bind with the reference antigen (that is coated in the well). Thus less reference antigen will be detected and a weaker signal will be created.
Advantage
- No sample processing is required
- High sensitivity – it gives a stronger signal when the sample contains a lesser amount of antigen
- High consistency – as results are less variable
- High flexibility – it can be based on direct, indirect, or sandwich ELISA
Disadvantages
Similar to base ELISA – as each ELISA technique can be adapted to the competitive technique
Ideal when only one antibody is available for the antigen of interest. It is also ideal for detecting small antigens that cannot be bound by two different antibodies (as required in a sandwich assay).
Applications of ELISA
Diagnosing diseases
It is a useful tool to detect various kinds of infectious diseases by detecting either the microbial antigen or the antibodies present in the body in response to the infection. These include:
- Detection of mycobacterium antibodies in tuberculosis
- Detection of HIV antibodies present in the blood
- Detection of hepatitis B and C markers in blood
- Detection of rotavirus in feces
- Detection of enterotoxin of E. coli in feces
- Detection of other diseases such as dengue, West Nile fever, Newcastle disease virus, Lyme disease, Chagas disease, influenza, leishmaniasis, etc
- Detection of SARS-CoV-2 antibodies in blood samples
Besides infectious diseases, it may also be used to diagnose various other diseases or conditions
- Detection of hormone human chorionic gonadotrophin (hCG) in urine – Pregnancy test
- Detection of antibodies in celiac disease
- Detection of parietal cell antibodies in pernicious anemia
- Biomarkers for detecting cancer such as breast and ovarian cancer
- Detection of platelet antibodies in serum to identify diseases such as idiopathic thrombocytopenic purpura (ITP) and systemic lupus erythematosus (SLE)
Read more about Home Pregnancy Test – Principle, Procedure, Accuracy, and Interpretation
Food industry
It is used in the food industry to detecting various food allergens, such as milk, peanuts, eggs, etc. This is important for legally required labeling of the ingredients present in a food item.
Toxicology
ELISA can be used as a quick presumptive screen for certain drugs. The concentration of illicit drugs such as cannabinoids, amphetamines, opiates, cocaine, benzodiazepines, methadone, etc can be determined in urine samples.
Advantages Of ELISA
- It is a quick test.
- It is easier to perform.
- Has no radiation hazards as compared to other assays which require the presence of radioactive materials.
- The test has high sensitivity and specificity.
- A large number of tests can be performed at one time. This makes it suitable for use in surveillance and blood transfusion departments.
- Reagents are cheap with a long shelf-life.
- Provides both quantitative and qualitative results.
- Can be performed on almost all biological samples like plasma, serum, urine, cell extracts, etc.
Disadvantages
- Only monoclonal antibodies can be used as match pairs.
- Monoclonal antibodies are expensive as compared to polyclonal antibodies.
- Negative controls may indicate a positive result if the blocking solution is ineffective.
- The enzyme-substrate reaction is short-term and hence the color needs to be read immediately.
References
- Engvall E. “Enzyme-linked immunosorbent assay, Elisa”. The Journal of Immunology. 1972-11-22. 109 (1): 129–135.
- Crowther J.R. “Chapter 2: Basic Principles of ELISA”. ELISA: Theory and Practice. Methods in Molecular Biology. 1995. 42. Humana Press. pp. 35–62.
