ELISA Test – Principle, Procedure, and Types

ELISA full form is Enzyme-Linked Immunosorbent Assay. It is a widely used ELISA test that helps detect and quantify various substances like proteins, hormones, peptides, and antibodies in a sample, typically blood. This ELISA technique relies on the specific interaction between an antigen and an antibody, making it highly sensitive and specific for diagnosing infections, pregnancy, and other conditions.

When you want to check if your body has produced antibodies against a particular disease agent (antigen), the ELISA procedure becomes a crucial diagnostic tool. It is commonly used in medical labs, research facilities, and even for at-home testing kits in some cases.

Principle of ELISA

The principle of ELISA (also known as ELISA principle) is based on the specific binding of an antigen to its corresponding antibody. Here is a simple breakdown:

1. Antigen-Antibody Interaction: A specific antibody captures the target antigen or a specific antigen captures the target antibody.

2. Enzyme Conjugation: A secondary antibody is linked with an enzyme to detect the presence of the antigen-antibody complex.

3. Substrate Addition: A suitable substrate is introduced, which the enzyme converts into a coloured product.

4. Measurement: The colour change is measured using a spectrophotometer. The intensity of the colour indicates the amount of antigen or antibody present in the sample.

This principle of ELISA ensures high accuracy and sensitivity. The more antigen or antibody you have in your sample, the stronger the colour development.

Types of ELISA

There are three main types you need to understand to get a complete picture of the ELISA principle and procedure:

1. Indirect ELISA

• Used to detect the presence (or concentration) of specific antibodies in a sample.

• The antigen is first coated on the microtitre well.

• The sample (containing the primary antibody) is added. If the antibody is present, it binds to the antigen.

• A secondary, enzyme-linked antibody binds to the primary antibody.

• Upon adding the substrate, the enzyme reacts to produce a visible signal (colour), which can be measured.

2. Sandwich ELISA

• Commonly used to detect the presence (or concentration) of a specific antigen in a sample.

• Antibody is coated onto the microtitre plate.

• The sample (with the suspected antigen) is added. The antigen, if present, will bind to the immobilised antibody.

• A second enzyme-conjugated antibody, which recognises a different epitope on the antigen, is added.

• Substrate is added, leading to a colour change proportional to the amount of antigen present.

3. Competitive ELISA

• Typically used to measure the concentration of an antigen when it’s present in low quantities or to study antigen-antibody affinity.

• The microtitre well is coated with a known antigen.

• The sample (unknown concentration of the same antigen) is mixed with a known amount of antibody, forming an antigen-antibody complex.

• This mixture is transferred to the antigen-coated plate. If the sample contains a high concentration of the antigen, fewer free antibodies remain to bind the plate’s antigen.

• After adding the enzyme-linked secondary antibody, the signal measured is inversely proportional to the antigen concentration in the sample.

ELISA Diagram

A typical ELISA diagram includes a 96-well microtitre plate. Each well can be coated with either antigen or antibody, depending on the type of ELISA you are performing. Then, the other components (primary or secondary antibodies and substrates) are sequentially added. The final colour change indicates the presence and quantity of your target molecule.

ELISA Procedure

While there can be slight variations depending on the type of ELISA, the general ELISA procedure usually involves the following steps:

1. Coating

• A known antibody or antigen is adsorbed onto the solid surface (microtitre plate).

• Excess unbound molecules are washed away using a suitable buffer.

2. Blocking

• Any remaining protein-binding sites on the plate are blocked to prevent non-specific binding. Common blocking agents include BSA (bovine serum albumin) or skimmed milk.

3. Incubation with Sample

• The sample containing the target antigen or antibody is added to the well.

• If the target analyte is present, it will bind to the coated antibody or antigen.

4. Detection Antibody (if required)

• A secondary enzyme-conjugated antibody is added. This secondary antibody will specifically bind the target if it is captured in the well.

5. Washing

• Unbound materials are removed through several wash cycles.

6. Substrate Addition

• A suitable substrate for the enzyme is added. The substrate reacts with the enzyme to produce a coloured or luminescent product.

7. Reading Results

• The intensity of the developed colour or light is measured, often using a spectrophotometer or plate reader.

• Higher absorbance or luminescence indicates a higher concentration of the target molecule in the sample.

Diagnosis with ELISA

The ELISA test plays a vital role in diagnosing various diseases and conditions. Some examples include:

• HIV/AIDS: Detects antibodies against HIV.

• Lyme Disease: Checks for antibodies against Borrelia burgdorferi.

• Ebola: Identifies antibodies specific to the Ebola virus.

• Zika Virus: Detects antibodies formed against Zika infection.

• Rotavirus: Identifies proteins or antibodies related to rotavirus infection.

• Toxoplasmosis: Recognises antibodies against Toxoplasma gondii.

• Pernicious Anaemia: Helps detect intrinsic factor antibodies.

• Carcinoma of Epithelial Cells: Sometimes used to measure tumour markers.

In pregnancy tests, ELISA is used to detect the HCG (Human Chorionic Gonadotropin) protein in blood or urine.

Advantages of ELISA

ELISA offers several benefits over other diagnostic tools:

• High Specificity and Sensitivity: Uses antigen-antibody binding, so results are very accurate.

• Quantitative, Semi-Quantitative, or Qualitative: Measurements can be taken in different formats.

• Simple and Rapid: Procedures are straightforward and can be done relatively quickly.

• No Need for Radioactive Substances: Unlike older assays, it relies on enzymes, making it safer to handle.

• Adaptability: Can be customised for various applications, including medical diagnosis, food safety, and more.

• Cost-Effective: Basic lab equipment (microtitre plate reader, pipettes) is generally sufficient.

Application of ELISA

The application of ELISA extends beyond just diagnosing infectious diseases. It includes:

1. Medical Diagnosis

• Rapid screening and confirmation of several infections and autoimmune disorders.

2. Food Industry

• Detects allergens like peanuts, soy, milk, and gluten in food products to ensure consumer safety.

3. Environmental Monitoring

• Monitors the presence of contaminants (e.g., pesticides) in water, soil, or food.

4. Pharmaceutical and Drug Research

• Measures the concentration of drugs, antibodies, or hormones in various samples to test efficacy or toxicity.

5. Epidemiological Studies

• During disease outbreaks, large-scale ELISA test procedures help track infection rates within populations.

Quick Quiz (with Answers)

1. What does ELISA stand for?

Answer: Enzyme-Linked Immunosorbent Assay.

2. Which type of ELISA is commonly used to detect antibodies in a sample?

Answer: Indirect ELISA.

3. True or False: In Competitive ELISA, the colour intensity is directly proportional to the antigen concentration in the sample.

Answer: False (it is inversely proportional).

4. Name an enzyme commonly linked to the secondary antibody in ELISA.

Answer: Horseradish Peroxidase (HRP) or Alkaline Phosphatase (AP).

5. Which ELISA type is often used to measure cytokines or other proteins at very low concentrations?

Answer: Sandwich ELISA.

Related Topics

• Blood Groups

• Antigens and Immunology

• Vestigial Organs

• Genetic Drift