Intro to medical laboratory technology

1. Immunofluorescent Labeling
Techniques
and how it can detect rabies
infections
ERIN TAYMAN
MELISSA WARD

2. Why use immunofluorescence?
 Enables the laboratory technician to
find a specified protein as well as its
abundance. (Immunofluorescence
Method, 2001)
 This technique is important in
diagnostic medicine in that it can be
used to differentiate cell types. (Kemp,
2002)
 Allows intracellular structures to be
seen. (Kemp, 2002)
Figure 1. Immunofluorescence
micrograph. (Osborn, 2002)

3. Other uses for immunofluorescence
 Originally, fluorescence microscopy itself was a powerful technique to visualize
the location of particular molecules in the cell.
 “Live-cell imaging for the molecular dynamics” (Matsuzaki, et al, 2007)
 “The analyses of the activity or status of signal transduction by visualizing the
binding of proteins with other molecules or their folding in itself by fluorescence
resonance energy transfer (FRET) microscopy.” (Matsuzaki, et al, 2007)
 “The analyses of movement and trafficking of particular proteins by
fluorescence recovery after photobleaching (FRAP) techniques.” (Matsuzaki,
et al, 2007)

4. How is it done?
 An antibody must first be made in reference to a chosen
protein. (Kemp, 2002)
 In order for the antibody to be able to enter the cell, the
cell must be made permeable. This is done through one
of several fixation processes. (Immunofluorescent
Labeling, n.d.)
 The newly made antibody then binds with the chosen
antigen. (Kemp, 2002)

5. The Methods
 There are three methods to choose from
when performing immunofluorescence
(Turgeon, 2015).
 Direct Immunofluorescent assay
 Inhibition Immunofluorescence assay
 Indirect Immunofluorescence assay

6. Figure 2. Comparison of methods. (Song, n.d.)
Direct Immunofluorescent assay
This technique is used mainly to
find antigen-antibody reactions.
This is done by staining the
primary antibody with a fluorescent
dye. (Song, n.d.)
Indirect Immunofluorescent
assay
As implied by the name, this
technique is done by staining the
secondary antibody, which is
indirectly bound to the antigen.
(Song, n.d.)

7. The Methods, cont.
 Inhibition Immunofluorescent assay
Used to confirm the “specificity of the fluorescent
antibody technique.” (Turgeon, 2015)

8. Clinical Applications
 The immunofluorescent labeling techniques are used with an epi-
fluorescent microscope. (Estridge & Reynolds, 2012)
 The fluorescent dye allows microorganisms to be viewed under this
specialized microscope. (Estridge & Reynolds, 2012)
 PCP (Pneumocystis jiroveci pneumonia)and Cryptosporidium are two
examples of organisms that are detected with direct
immunofluorescent labeling techniques. These are opportunistic
infections that can take advantage of the compromised immune
systems of HIV positive patients. (Benson et al, n.d.)

9. Rabies Diagnosis
 Immunofluorescence can also be used to diagnose rabies.
 Immunofluorescence, using skin biopsies, is one of the most
reliable tests to diagnose rabies in humans. (Audry, et al,
1998)
 It has also been known to give results 2 or more days before
clinical symptoms are shown in animals. (Bell, J.F., Blenden,
D.C., Tsao, A.T., & Umoh, J.U., 1983)

10. Figure 3. Positive dFA test. (CDC, 2011)
►A dFA (Direct Fluorescent Antibody)
test using nervous tissue.
►The fluorescent green is the labeled
antibody bound to the rabies
antigen. (CDC, 2011)

11. Bibliography
 Audry, L., Bourhy, H., Caroff, C., Crepin, P., Gacoin, A., & Rotivel, Y. (1998) Intravitam Diagnosis of Human Rabies by PCR Using Saliva and Cerebrospinal Fluid. NCBI,
National Library of Medicine. Retrieved September 14, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC104702/
 Bell, J.F., Blenden, D.C., Tsao, A.T., & Umoh, J.U. (1983). Immunofluorescent Examination of the Skin of Rabies-Infected Animals as a Means of Early Detection of Rabies
Virus Antigen. Journal of Clinical Microbiology, 18(3), 631. Retrieved September 14, 2015 from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC270866/?page=1
 Benson, Constance A., et al (n.d.) Treating Opportunistic Infections Among HIV-Infected Adults and Adolescents. Recommendations from CDC, the National Institutes of
Health, and the HIV Medicine Association/Infectious Diseases Society of America. Retrieved October 10, 2015 from
https://aidsinfo.nih.gov/contentfiles/TreatmentofOI_AA000875.pdf
 Campbell, A. (2001). Immunofluorescence Labeling Method. Retrived from http://www.bio.davidson.edu/genomics/method/IMF.html
 Centers for Disease Control and Prevention. (2011). Direct Fluorescent Antibody Test. Retrieved September 14, 2015, from
http://www.cdc.gov/rabies/diagnosis/direct_fluorescent_antibody.html
 Estridge, Barbara H., & Reynolds, Anna P. (2012) Basic Clinical Laboratory Techniques; Sixth Edition. Delmar Cengage Learning.
 Immunofluorescence Labeling of Cells. (2015). Retrieved from http://www.sigmaaldrich.com/life-science/cell-biology/antibodies/antibodies-
application/protocols/immunofluorescence.html#procedure
 Kemp, M. (2002) Science in Culture. Nature, 417, 23. Retrieved from http://web.a.ebscohost.com/ehost/pdfviewer/pdfviewer?sid=8b4273a8-4a32-4c4a-9cb4-
2b65ef37fda4%40sessionmgr4005&vid=6&hid=4201
 Song, W. (n.d.) Immunofluorescence Microscopy. Retrieved from http://www.life.umd.edu/classroom/bsci423/song/Lab9.html
 Suzuki, T., Matsuzaki, T., Hagiwara, H., Aoki, T., & Takata, K. (2007, December 12). Recent Advances in Fluorescent Labeling Techniques for Fluorescence Microscopy.
Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2156041/
 Turgeon, M.L. (2015). Linné & Ringsrud’s Clinical Laboratory Science: Concepts, Procedures, and Clinical Applications. Elsevier