ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY)

PRERNA FOR IAS

ELISA (ENZYME-LINKED IMMUNOSORBENT ASSAY)

1. Definition of ELISA

ELISA (Enzyme-Linked Immunosorbent Assay) is a highly sensitive laboratory technique used to detect and measure specific antigens (Ag) or antibodies (Ab) in blood, serum, plasma, or other body fluids. It is based on the specific interaction between an antigen and an antibody. ELISA is widely used in medical diagnostics, research laboratories, and biotechnology. It helps diagnose infectious diseases, hormonal disorders, autoimmune diseases, and certain cancers. Because of its high sensitivity, specificity, and accuracy, ELISA can detect even very small amounts of target molecules, making it one of the most reliable and commonly used immunological diagnostic methods worldwide.

2. Principle of ELISA

ELISA works on the principle of the specific binding between an antigen and its corresponding antibody. One of these components is attached to a solid surface, usually a microplate well. An enzyme-linked antibody is then added, which binds specifically to the target antigen or antibody. After washing away unbound substances, a substrate is added. The enzyme reacts with the substrate and produces a visible color change. The intensity of the color is directly proportional to the amount of antigen or antibody present in the sample. This color is measured using a spectrophotometer, providing accurate quantitative or qualitative results.

3. Direct ELISA

Direct ELISA is the simplest type of ELISA used to detect antigens. In this method, the antigen is first attached directly to the microplate surface. An enzyme-labeled primary antibody is then added, which binds specifically to the antigen. After washing away excess antibody, a substrate is added. The enzyme reacts with the substrate, producing a measurable color change. The color intensity indicates the amount of antigen present in the sample. Direct ELISA is quick, simple, and requires fewer reagents. However, it is less sensitive than other ELISA methods because it uses only one antibody for detection.

4. Indirect ELISA

Indirect ELISA is mainly used to detect antibodies in a patient's sample. First, a known antigen is coated onto the microplate. The patient's serum is added, allowing any specific antibodies to bind to the antigen. After washing, an enzyme-linked secondary antibody is added, which binds to the patient's antibody. Finally, a substrate is added to produce a color reaction. The intensity of the color reflects the amount of antibody present. Indirect ELISA is more sensitive than Direct ELISA because multiple secondary antibodies can bind to each primary antibody, amplifying the signal and improving detection accuracy.

5. Sandwich ELISA