AffiELISA Assay
The ELISA (Enzyme-Linked Immunosorbent Assay) is a highly specific biochemical technique used to detect and quantify antigens or antibodies in a sample. The assay utilizes the specificity of antigen-antibody interactions and the enzymatic activity of a linked enzyme to produce a measurable signal.
Key Components
- Coating: A solid-phase (usually a microtiter plate) is coated with a capture antibody or antigen specific to the target molecule.
- Blocking: Non-specific binding sites on the plate are blocked using a blocking buffer to prevent false positives.
- Sample Addition: The sample, containing the target antigen or antibody, is added. The target binds to the capture antibody or antigen on the plate.
- Detection: A secondary antibody or antigen, which is linked to an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), is added. This binds to the target molecule.
- Substrate Addition: A substrate for the enzyme is added. The enzyme-substrate reaction produces a colorimetric, fluorometric, or luminescent signal.
- Detection and Quantification: The intensity of the signal is measured using a spectrophotometer, fluorometer, or luminometer, which correlates with the concentration of the target molecule in the sample.
Types of ELISA
- Direct ELISA: Involves direct conjugation of the enzyme to the primary antibody.
- Indirect ELISA: Utilizes a secondary antibody linked to the enzyme to detect the primary antibody.
- Sandwich ELISA: Involves a capture antibody, target antigen, and a detection antibody linked to an enzyme.
- Competitive ELISA: Measures the amount of target antigen by competing with a labeled antigen for binding sites on a specific antibody.
Applications
ELISA is widely used in clinical diagnostics, research, and quality control to measure biomolecules such as hormones, proteins, and antibodies.
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