Immunofluorescence Techniques Overview

Immunofluorescence

• Immunofluorescence is a technique visualizing specific antigens.
• It involves binding a specific antibody to the antigen.
• The antibody is chemically conjugated with a fluorescent dye.
• Fluorochromes absorb short UV light wavelengths and emit longer wavelengths (e.g., visible green light).
• Examples of fluorochromes include fluorescein isothiocyanate (FITC) which emits green light, and tetramethylrhodamine which emits red light.
• This technique can make viral plaques more visible.

Principle of Immunofluorescence (IF)

• The principle of IF relies on the specific antigen-antibody interaction.
• A fluorescently labeled antibody binds to its corresponding antigen.
• When exposed to ultraviolet (UV) light, the fluorescent dye emits visible light.
• This allows visualization of the antigen-antibody complex.

Requirements for Immunofluorescence

• A slide is needed.
• A tagged antibody is required.
• A fluorescent microscope is essential.
• Specimens are needed.

Methods of Immunofluorescence Assay

• There are two types: Direct and Indirect.
• Direct IF Assay (DIFA):
  • A single fluorescently labeled primary antibody binds directly to the target antigen.
  • Sample preparation involves fixing tissue sections or cells to a slide.
  • A fluorescently labelled antibody is added that is specific to the target antigen.
  • The antibody binds to the antigen if it's present.
  • The slide is examined under a fluorescence microscope. A positive reaction confirms the presence of the target antigen.
• Indirect IF Assay (IDIFA):
  • Detects antibodies in a sample (e.g., serum).
  • A specific antigen is coated on a slide.
  • The patient's sample is applied to the slide.
  • If the target antibodies are present, they bind to the antigen.
  • A fluorescent secondary antibody that binds to the patient's antibodies is added.
  • The slide is examined under a fluorescence microscope. The presence of fluorescence confirms the presence of the specific antibodies in the sample.

Results of Immunofluorescence

• Confocal images can detect and visualize phosphorylated AKT.
• This example shows the results in cardiomyocytes infected with adenovirus.

Comparison of Direct and Indirect Immunofluorescence

• Direct:
  • Detects antigens using a primary antibody conjugated to a fluorescent dye.
  • Single step process.
  • Highly specific but limited to the labeled antibody.
  • Lower sensitivity.
  • Used for pathology to detect tissue-bound antigens.
  • Faster and cheaper than indirect.
• Indirect:
  • Detects antibodies in a sample.
  • Uses a secondary antibody labeled with a fluorescent dye to detect binding of primary antibody to antigen.
  • Two step process (antigen, then labeled secondary antibody).
  • More versatile, using one secondary antibody for different primary antibodies.
  • Higher sensitivity due to signal amplification.
  • Used in serological tests, autoimmune studies.
  • More time-consuming and expensive.