Enzyme-linked immunosorbent assay (ELISA) test

The enzyme-linked immunosorbent assay (ELISA) is a biochemical technique used to detect and quantify the presence of antigens (such as proteins, peptides, or hormones) or antibodies in a sample. It is one of the most widely used immunoassays in diagnostic laboratories, biomedical research, and clinical testing, owing to its high sensitivity, specificity, and adaptability. The method relies on the interaction between antigens and antibodies, which are linked to an enzyme that produces a measurable colour change upon reaction with a suitable substrate.

Principle of the ELISA Test

The ELISA test is based on the specific antigen–antibody interaction, where an enzyme conjugated to either the antigen or antibody catalyses a colour-producing reaction. When the enzyme reacts with its substrate, it generates a detectable signal, usually in the form of a colour change, which can be measured spectrophotometrically.
The basic steps of ELISA involve:

1. Immobilisation of antigen or antibody on a solid surface, typically a microtitration plate (commonly a 96-well plate made of polystyrene).
2. Binding of the corresponding antibody or antigen from the test sample to form an immune complex.
3. Addition of enzyme-linked secondary antibody, which binds to the antigen–antibody complex.
4. Substrate reaction, where the enzyme converts a colourless substrate (chromogen) into a coloured product.
5. Measurement of the optical density (OD) of the colour developed, which is directly proportional to the concentration of the target molecule in the sample.

Common enzymes used include horseradish peroxidase (HRP) and alkaline phosphatase (ALP), with substrates such as tetramethylbenzidine (TMB) or p-nitrophenyl phosphate (pNPP).

Types of ELISA

There are four main types of ELISA tests, each differing in design, sensitivity, and application.
1. Direct ELISA

• In this method, the antigen is immobilised on the microplate surface.
• An enzyme-labelled antibody specific to the antigen is applied directly.
• After washing to remove unbound antibody, the substrate is added, producing a measurable colour.
• Advantages: Simple, rapid, and involves fewer steps.
• Disadvantages: Lower sensitivity and flexibility; no signal amplification since only one antibody is used.

2. Indirect ELISA

• The antigen is first coated on the microplate.
• The primary antibody (from the sample or test serum) binds to the antigen.
• A secondary antibody conjugated with an enzyme is then added to bind to the primary antibody.
• The signal intensity increases because multiple secondary antibodies can bind to one primary antibody, enhancing sensitivity.
• Advantages: Higher sensitivity and greater versatility; cost-effective as enzyme-labelled secondary antibodies are widely available.
• Disadvantages: Possibility of cross-reactivity between antibodies.

3. Sandwich ELISA

• Two antibodies specific to different epitopes of the same antigen are used.
• The capture antibody is immobilised on the microplate and binds the antigen from the test sample.
• A detection antibody (also antigen-specific) is then added, forming a “sandwich” structure (capture antibody–antigen–detection antibody).
• The detection antibody is either enzyme-labelled or followed by an enzyme-conjugated secondary antibody.
• Advantages: Extremely sensitive and specific; ideal for detecting complex samples such as serum or plasma.
• Disadvantages: Requires highly specific antibody pairs and optimisation.

4. Competitive ELISA

• The test antigen competes with a labelled antigen for binding sites on a specific antibody.
• The amount of colour produced is inversely proportional to the antigen concentration in the sample.
• Useful for detecting small molecules (e.g. hormones, drugs, and toxins) that cannot bind two antibodies simultaneously.
• Advantages: Suitable for low-molecular-weight compounds and detection of antigen or antibody in complex mixtures.
• Disadvantages: More complex to interpret and may have lower sensitivity than sandwich ELISA.

Procedure Outline

A general ELISA procedure follows these steps:

1. Coating: The microplate wells are coated with either antigen or capture antibody and incubated to allow attachment.
2. Blocking: Unbound sites are blocked with a blocking buffer (commonly bovine serum albumin or casein) to prevent non-specific binding.
3. Incubation: The sample containing the target analyte is added and allowed to bind.
4. Washing: Excess material is removed using a buffer solution (such as phosphate-buffered saline with Tween-20).
5. Detection: Enzyme-linked secondary antibody is introduced, binding to the captured antigen or antibody.
6. Substrate Addition: The enzyme-specific substrate is added, resulting in colour development.
7. Measurement: The intensity of colour is read using an ELISA reader (spectrophotometer) at a specific wavelength (often 450 nm).

Applications of ELISA

ELISA has a wide range of applications in clinical diagnostics, biological research, and industrial monitoring.

• Medical diagnostics:
  • Detection of infectious diseases, including HIV, hepatitis B and C, COVID-19, and syphilis.
  • Measurement of hormone levels such as insulin, hCG, or thyroid hormones.
  • Detection of tumour markers like prostate-specific antigen (PSA).
• Immunology and research:
  • Quantification of cytokines, antibodies, or antigens in cell culture supernatants or serum samples.
  • Evaluation of immune responses in vaccine development.
• Food and environmental safety:
  • Detection of food allergens, pesticide residues, and bacterial toxins (e.g. aflatoxins or botulinum toxin).
  • Monitoring contamination in water or industrial environments.

Advantages of ELISA

• High sensitivity and specificity due to antigen–antibody recognition.
• Quantitative and qualitative analysis capability.
• Ease of automation for large-scale testing.
• Relatively low cost and use of minimal reagents.
• Rapid results compared to culture-based assays.
• Safe handling, as the process does not require radioactive materials.

Limitations of ELISA

• False positives or negatives may occur due to cross-reactivity or improper washing.
• Enzyme activity may be affected by temperature or pH variations.
• Requires highly specific antibodies for accurate results.
• Limited detection of low-abundance molecules without signal amplification.
• End-point measurement may not always reflect real-time dynamics of antigen–antibody interaction.