ELISA
ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a widely used laboratory technique for detecting and quantifying substances such as proteins, peptides, antibodies, and hormones. ELISA is based on the principle of antigen-antibody interactions and utilizes enzymes for detection.
Here’s how ELISA typically works:
- Coating: The surface of a microtiter plate is coated with a specific antigen or antibody that binds to the target molecule being measured. This can be done by incubating the plate with the target antigen or antibody.
- Blocking: After coating, the plate is washed to remove any unbound molecules, and a blocking agent (such as bovine serum albumin or milk proteins) is added to prevent nonspecific binding of other proteins.
- Primary Antibody Binding: The sample containing the target molecule is added to the plate and allowed to bind to the coated antigen or antibody. If the target molecule is present in the sample, it will bind to the immobilized antigen or antibody.
- Washing: After a suitable incubation period, the plate is washed again to remove any unbound sample components.
- Secondary Antibody Binding: A secondary antibody, which is linked to an enzyme (such as horseradish peroxidase or alkaline phosphatase), is added to the plate. This secondary antibody binds specifically to the primary antibody if it has captured the target molecule.
- Substrate Addition: A substrate specific to the enzyme is added to the plate. In the presence of the enzyme, the substrate undergoes a reaction that produces a detectable signal, typically a color change.
- Signal Detection: The intensity of the signal is measured using a spectrophotometer or a plate reader. The signal intensity is proportional to the amount of target molecule present in the sample.
ELISA is highly sensitive and specific, making it a valuable tool in medical diagnostics, research laboratories, and the pharmaceutical industry. It can be used for various applications, including detecting infectious diseases, monitoring autoimmune disorders, quantifying hormones and cytokines, and screening for allergens. ELISA assays can be performed in different formats, including direct, indirect, sandwich, and competitive ELISA, depending on the specific requirements of the experiment.