ELISA stands for Enzyme-Linked Immunosorbent Assay, and it is a widely used laboratory technique in biology and immunology to detect and quantify the presence of specific substances, such as proteins, peptides, antibodies, hormones, and antigens, in a sample. ELISA is based on the principle of antigen-antibody interactions.
Here's how the ELISA process typically works:
1. Coating: The first step involves immobilizing the antigen (or antibody) of interest onto the surface of a solid support, such as a microtiter plate or a membrane.
2. Blocking: To prevent any non-specific binding, the next step involves blocking the remaining uncoated surface sites on the solid support with a blocking agent, commonly bovine serum albumin (BSA) or milk proteins.
3. Incubation: The sample (containing the analyte of interest) is added to the coated and blocked wells. If the analyte is present, it will bind to the immobilized capture molecule (e.g., an antibody) on the surface.
4. Washing: After an appropriate incubation period to allow binding to occur, the unbound components are washed away to remove any non-specifically bound substances.
5. Detection: In this step, a detection antibody is added. This detection antibody specifically binds to the target analyte, creating a "sandwich" complex (capture antibody - analyte - detection antibody) if the analyte is present in the sample.
6. Second Washing: Any unbound detection antibody is washed away.
7. Enzyme Conjugation: The detection antibody is linked to an enzyme (such as horseradish peroxidase or alkaline phosphatase). This enzyme will later catalyze a reaction to produce a measurable signal.
8. Substrate Addition: A substrate specific to the enzyme is added to the wells. If the enzyme is present (i.e., the "sandwich" complex has formed), the enzyme will catalyze a reaction with the substrate, producing a detectable signal, usually a color change.
9. Signal Measurement: The intensity of the signal is measured using a spectrophotometer or a plate reader. The amount of signal generated is proportional to the concentration of the analyte in the sample.
ELISAs are commonly used in research, clinical diagnostics, and quality control in various industries, including medicine, biotechnology, and agriculture. They are particularly valuable for detecting antibodies in diseases, such as HIV, detecting antigens in infectious agents like bacteria or viruses, and quantifying specific proteins in biological samples. ELISA's sensitivity, specificity, and ease of use have made it one of the most widely adopted immunoassay techniques.
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