Educational Information
What is the principle of ELISA?
ELISA, or Enzyme-Linked Immunosorbent Assay, is a widely used laboratory technique that allows for the detection and quantification of proteins, hormones, antibodies, and other biomolecules in a sample. The principle of ELISA is based on the specific binding interactions between antigens and antibodies, combined with an enzyme-linked detection system.
Here are the key steps and principles involved in an ELISA:
1. Coating the Plate: The wells of a microplate (usually made of polystyrene) are coated with a specific antigen or antibody. If the goal is to detect an antigen, the wells are coated with antibodies specific to that antigen. If the goal is to detect antibodies, the wells are coated with the corresponding antigen.
2. Blocking: After the wells are coated, a blocking solution is added to cover any unoccupied sites on the plate surface. This step prevents non-specific binding of detection antibodies or other reagents in subsequent steps.
3. Sample Addition: The sample containing the target analyte (e.g., serum, plasma, or other biological fluid) is added to the wells. If the analyte is present, it will bind to the coated antibodies (or antigens) in the wells.
4. Detection Antibody: A secondary antibody that is specific to the target analyte and is conjugated to an enzyme (such as horseradish peroxidase or alkaline phosphatase) is added. This antibody binds to the target analyte, forming a complex.
5. Substrate Addition: A substrate for the enzyme linked to the detection antibody is added. The enzyme catalyzes a reaction with the substrate, leading to a detectable signal, often a color change.
6. Signal Measurement: The intensity of the color change (or other measurable signal) is proportional to the amount of target analyte in the sample. This signal can be quantified using a spectrophotometer or other detection methods.
7. Data Interpretation: A standard curve is often generated using known concentrations of the analyte, allowing for the quantification of the analyte in unknown samples based on the measured signal.
Overall, ELISA is a powerful and versatile technique due to its specificity, sensitivity, and ability to process multiple samples simultaneously. It is commonly used in clinical diagnostics, research, and quality control in various fields.