Principle, Techniques, and Application of ELISA (Direct and Indirect). IVth Semester UNIT-V. Immunology and Mycology.

Principles, Techniques, and Application of ELISA (direct and indirect)

Enzyme-linked immunosorbent Assay (ELISA) is widely used for the detection and quantification of large molecules like proteins, peptides, antigens, antibodies, hormones, and other molecules in biological samples. However, it is mainly used to detect either antigen or antibody.

Principle: In the ELISA test, antibodies combine with an enzyme by the addition of glutaraldehyde to form a conjugate. Conjugate retains both immunological and enzyme activities. They can be assayed by their ability to break down a substrate. Color change in the substrate due to enzyme activity is assessed visually or by using a spectrophotometer. ELISA tests are used to detect antigens and antibodies. There are two types of ELISA tests.

1. Direct ELISA.

2. Indirect ELISA,

Direct ELISA

Procedure:

• 1. Coating: A known antibody (depending on the assay) is directly adsorbed onto a solid surface, such as a microtiter plate.

• 2. Blocking: Non-specific binding sites on the solid surface are blocked to prevent false positive results,

• 3. Primary Antibody Incubation: The sample containing the antigen is added to the solid surface containing the antibody and incubated. If the antigen is present in the sample, it binds directly to the antibody present on the solid surface.

• 4. Enzyme-Conjugated Secondary Antibody: An enzyme-conjugated secondary antibody that recognizes the primary antibody is added.

• 5. Substrate addition: A substrate specific to the enzyme is added, and a color change occurs if the antigen molecule is present.

Application

• 1. Direct ELISA is often used when a highly specific antibody or antigen is available.

• 2. It is generally quicker and simpler compared to indirect ELISA.

Indirect ELISA

Procedure/Technique

• 1. Coating: Similar to direct ELISA, the solid surface is coated with the antigen of interest.

• 2. Blocking: Non-specific binding sites are blocked.

• 3. Primary Antibody Incubation: The sample is incubated with the coated antigen. The primary antibody binds to the antigen,

• 4. Enzyme-Conjugated Secondary Antibody: A secondary antibody specific to the primary antibody is added. This secondary antibody is conjugated with an enzyme.

• 5. Substrate Addition: A substrate specific to the enzyme is added, and a color change occurs.

Applications

• 1. Indirect ELISA is more versatile and is commonly used when specific antibodies against the target molecule are available.

• 2. It allows the binding of multiple secondary antibodies to a single primary antibody.

Application of ELISA test: ELISA techniques are powerful tools in medical and biological research due to their sensitivity, specificity, and ease of use. They are widely employed in various fields, including clinical diagnostics, pharmaceutical development, and academic research.

• · Clinical diagnostics for diseases such as HIV, hepatitis, and autoimmune disorders,

• · Monitoring of antibody or antigen levels in research and medical laboratories,

Dr Pramila Singh