ELISA test principle procedure types

Here you will learn about “what is ELISA test principle procedure types”

ELISA or Enzyme-linked immunosorbent Assay is a widely used biochemical technique that plays a crucial role in the detection and quantification of proteins, peptides, antibodies, and hormones.

What is the principle of ELISA test

It involves immobilizing the antigen of interest onto a solid surface, such as a microtiter plate. Subsequently, an enzyme-linked antibody is added, forming a complex with the antigen.

Procedure of ELISA test step by step

1. Coating
   The first step involves coating a solid surface (typically a microtiter plate) with the antigen of interest. This can be achieved by directly adsorbing the antigen or by using a capture antibody that specifically binds to the antigen.
2. Blocking
   To prevent non-specific binding, the coated surface is treated with a blocking agent, such as bovine serum albumin (BSA) or non-fat milk. This ensures that only the target antigen or antibody interacts with the surface.
3. Incubation with Sample
   The test sample, which may contain the antigen or antibody of interest, is added to the coated and blocked wells.
   • NOTE: If the target substance is present, it will bind to the immobilized molecule on the surface.
4. Washing
   Unbound substances are removed by washing the plate, ensuring that only specific interactions are retained.
5. Enzyme-Linked Antibody Addition
   An enzyme-linked antibody, specific to the target antigen or antibody, is added. This forms a complex with the bound antigen.
6. Second Washing
   Excess enzyme-linked antibodies are removed by washing the plate again.
7. Substrate Addition
   A substrate for the enzyme is added, and the enzyme catalyzes a reaction that produces a detectable signal.
   • Most commonly used substrates include chromogenic substances that result in a color change.
8. Signal Measurement
   The intensity of the developed color is measured spectrophotometrically, and the concentration of the target substance is determined by comparing it to a standard curve.

Types of ELISA (Direct, Indirect, Sandwich, Competitive)

Direct ELISA

In this type, the antigen is directly immobilized on the solid surface. The enzyme-linked antibody is then added to detect the bound antigen.

Indirect ELISA

In this type, the antigen (Ig) is immobilized, and an unlabeled primary antibody is added. A secondary enzyme-linked antibody, specific to the primary antibody, is then introduced.

Sandwich ELISA

This type utilizes two antibodies – one to capture the antigen and another enzyme-linked antibody to detect it. It is highly sensitive and commonly used for quantifying antigens.

Competitive ELISA

In competitive ELISA, a known amount of enzyme-labeled antigen competes with the sample antigen for binding to a limited amount of immobilized antibody.