What are the main purposes of using indirect immunofluorescence in clinical immunology?

Question 1

What is immunofluorescence?

Answer 1

A method that modifies antibodies by adding fluorescent dye to create a probe that can be used to target specific antigens/proteins, which can be on and within the cell. The probe allows the targeted structure to be easily seen in specific wavelengths of light which causes the probe to glow under a microscope

Question 2

Importance of immunofluorescence

Answer 2

Allows the detection and localisation of a wide variety of antigens in different types of tissues of various cell preparations

Question 3

Direct immunofluorescence

Answer 3

Uses a single antibody directed against the antigen of interest and binds to it.
Used for the accurate diagnosis of autoimmune disorders

Question 4

Indirect immunofluorescence

Answer 4

Uses two antibodies. The primary antibody binds to the antigen of interest, after a secondary antibody conjugated to a fluorophore is used to detect the primary antibody.

Question 5

Purpose of indirect immunofluorescence

Answer 5

Purpose is to amplify signal as several secondary antibodies will bind to the primary antibody resulting in an amplified signal

Question 6

Procedure of immunofluorescence

Answer 6

• Slides are prepared for the patient’s tissue which is cut into slices. Several are prepared with different tissue substrates
• The tissue sections are incubated with patient’s serum
• Slides are washed to remove any unbound primary antibodies
  The slides are incubated again with secondary antibodies containing a fluorescent dye
• Each slide is mounted under a coverslip and examined using fluorescence microscopy