ELISA: Principle, Types, Procedure and Application

ELISA is an acronym for Enzyme-Linked Immunosorbent Assay. ELISA is a plate-based assay designed to detect and quantify proteins, antibodies, peptides, and hormones. It is typically formed on a 96 well polystyrene plate.

ELISA is based on antibody/antigen binding. It relies on the specificity and affinity of antibodies to antigens. The binding site of an antibody is called a paratope while that of an antigen is called an epitope. The binding of an antibody paratope to a particular antigenic epitope forms the basis of the immunological specificity.

Principle of ELISA

ELISA is based on antibody/antigen binding. Antibodies specifically bind to antigen with particular epitopes. Enzyme labeled antigens or antibodies are used and the activity of the enzyme is measured calorimetrically. Enzyme activity measured or detected using a chromogenic substrate that changes color when modified by the enzyme.

The commonly used enzymes are horseradish peroxidase with its substrate tetramethylbenzidine (TMB) and alkaline phosphatase with its corresponding substrate p-nitrophenyl phosphate (a disodium salt).

One can determine an antibody by starting with a known antigen or detect an antigen by starting with a known antibody. Antibodies are labeled with an antibody without compromising their

Types of ELISA

1 Direct ELISA

A target protein or antigen (or target antibody) is immobilized on the surface of a microtiter plate and incubated with an enzyme-labeled antibody (or enzyme-labeled antigen/protein). A substrate specific to the enzyme used is added. The color development is measured and it is directly proportional to the amount of antibody or antigen present.

2. Indirect ELISA

It requires the use of two antibodies produced from different animal species that do not cross-react with each other.

A target protein/antigen is immobilized on the microtiter well and incubated with an antibody to target the antigen (primary antibody), followed by a labeled anti species against the primary antibody (secondary antibody), a substrate-specific to the enzyme is added and color development is measured.

3. Sandwich ELISA

An antibody is immobilized on the plate, a sample is added and incubated, a labeled antibody against the antigen added, the substrate is added, color development is measured spectrophotometrically using an Absorbance reader.

4. Competitive ELISA

Antibodies specific for a target protein is immobilized on the surface of a microtiter well and incubated with samples containing the target protein and a known amount of enzyme-labeled target protein. After incubation, the activity of the enzyme is measured. When the antigen level in the sample is lower and the color is lighter.

Competitive ELISA is used when the target antigen is a small molecule, two antibodies cannot simultaneously bind the antigen in a sandwich ELISA hence competitive ELISA is used.  Histamine, pesticides, and dioxin have low molecular weight hence detected bt competitive ELISA.

Illustration of Different types of ELISA; Direct, indirect competitive and the sandwich- mawanga.com
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Illustration of the different types of ELISA.
Photo Attributes: Wikimedia Commons


  • Immobilization of antibody or antigen on the microtiter wells on the plates, This is done at 4 degrees for 15-18 hours. The microtiter plates should be sealed to prevent operation.
  • Wash to remove unbound antibody or antigen using a washing solution.
  • Blocking and incubation at 37 degrees for 1 hour to prevent non-specific binding sites. Bovine serum albumin is the preferred blocking agent.
  • Wash to remove blocking agent
  • Add sample and incubate at 37 degrees for 1 hour
  • Wash to remove non-reactants
  • Add labeled antibodies and secondary antibodies and incubate
  • And substrate and incubate
  • Add stop solution to stop the reaction. concentrated acids are used as stop solutions as they alter ph.
  • Measure the absorbance at 540nm using an ELISA reader.

Advantages of Using ELISA in Sample Analysis

  • It has high sensitivity and specificity with the sandwich and indirect Elisa being more sensitive compared to direct Elisa due to the use of two antibodies for different antigenic epitopes.
  • Easy to perform
  • It is versatile as it can detect both antigen and antibody
  • It is used to detect and quantify the antigen
  • No radiation hazards occur during the labeling or disposal of waste.

Application of ELISA

  • Screening donated blood for evidence of viral contamination with HIV-1, HIV 2, Hepatitis A and B.
  • Measuring hormone levels.
  • Detection of food allergens in the food industry.
  • Detection of tumor markers.

Check your understanding with our clinical chemistry test question!