Avidin-Biotin mediated assay, Immunohistochemistry.

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Avidin-Biotin Mediated Assay and Immunohistochemistry (IHC) are two techniques used in the field of molecular biology and histology to detect and visualize specific proteins or antigens within tissue samples. These techniques are based on the principles of binding specificity and signal amplification:

  1. Avidin-Biotin Mediated Assay:
    • Principle: This assay relies on the strong and highly specific binding between avidin (or streptavidin) and biotin. Avidin is a protein found in egg whites, and streptavidin is a bacterial protein with similar properties. Biotin is a small molecule that can be conjugated to antibodies or other molecules of interest.
    • Procedure:
      • A target-specific antibody is conjugated to biotin.
      • The biotinylated antibody is then added to the sample, where it binds to the target antigen.
      • Avidin or streptavidin, which has been coupled to a reporter molecule (e.g., enzyme or fluorophore), is introduced. Multiple avidin/streptavidin molecules can bind to each biotin molecule, leading to signal amplification.
      • After a washing step to remove unbound components, the signal is detected using the reporter molecule, which generates a measurable signal.
    • Applications: Avidin-Biotin Mediated Assays are used in various techniques, including Western blotting, ELISA, and immunohistochemistry. They provide signal amplification and are valuable for detecting low-abundance antigens.
  1. Immunohistochemistry (IHC):
    • Principle: IHC is a technique used to visualize the distribution and localization of specific proteins or antigens within tissue sections. It relies on the specific binding of antibodies to target antigens in fixed and embedded tissue samples.
    • Procedure:
      • Tissue sections are prepared, fixed, and embedded in paraffin or other appropriate materials.
      • Antigen retrieval may be performed to expose masked epitopes.
      • Sections are incubated with a primary antibody that specifically recognizes the target antigen.
      • After washing away unbound antibodies, a secondary antibody is applied. This secondary antibody is conjugated to a label, such as an enzyme (e.g., horseradish peroxidase) or a fluorophore.
      • Visualization occurs when a substrate is added for enzyme-linked IHC, or when the sections are directly observed for fluorescent IHC.
      • Counterstains, like hematoxylin, may be used to visualize cellular structures and provide context.
    • Applications: IHC is widely used in pathology and research to study protein expression and localization in tissues. It is valuable for diagnosing diseases, characterizing tumor markers, and understanding tissue biology.

Both avidin-biotin mediated assays and immunohistochemistry are powerful techniques for the detection and visualization of specific proteins or antigens, making them essential tools in molecular biology, histopathology, and biomedical research.

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