Immunofluorescence is a technique that uses the binding of antibodies to antigens to detect the presence of substances. It relies on tagging antibodies with fluorescent dyes called fluorophores. When exposed to ultraviolet light, the fluorophore-tagged antibody complexes emit visible light that can be seen using a fluorescent microscope. There are several types of immunofluorescence assays including direct, indirect, and quantitative assays that are used to detect microorganisms, antibodies, and other biomolecules through the antigen-antibody reaction and fluorescent signal produced.

1. IMMUNOFLUORESCENCE
By GROUP II

2. WHAT IS IMMUNOFLUORESCENCE?
• A qualitative observation; uses a fluorescent microscope
• Relies on the principle of antigen (Ag)-antibody (Ab) reaction:
> Ab is “tagged” with a fluorophore or fluorochrome (fluorescent
compound)
> Ab binds to a specific Ag of interest
>Ag-Ab complex emits a green or red light (depending on the
fluorophore used) when an incident light (usually UV light) is introduced
• Introduced by Albert Coons in 1941

3. WHAT IS A FLUOROPHORE
• Organic molecules with a ring structure
• Has a characteristic optimum absorption range
(e.g. Fluorescein isothiocyanate emits a GREEN light when against a BLUE light)
• ABSORBS incident light, CONVERTS it, and EMITS fluorescent energy of longer
wavelength and lower energy

4. EXAMPLES (FLUOROPHORES)
• Fluorescein isothiocyanate – most common; emits green light
• Tetramethylrhodamine – emits red light
• Phycoerythrin
• Europium (β – naphthyl trifluoroacetone)
• Lucifer Yellow VS
• Acridine Orange
• Lissamine
• Calcofluor White

5. WORKING PRINCIPLE
• Employs Ag-Ab reaction
• Ab tagged with a fluorophore is introduced to specimen
• Ab binds to specific Ag
• Specimen is viewed under a UV light with a dark background in a fluorescent
microscope

6. WORKING PRINCIPLE (CONT.)
• Fluorophore absorbs radiant energy and is excited
• Fluorophore goes back to a ground state, emitting light energy of longer
wavelength and lower energy
• Interval of absorption and emission is very short; occurs in nanoseconds

7. PURPOSE OF
IMMUNOFLUORESCENT ASSAYS
• Rapid identification of microorganisms in cell culture or infected tissue, tumor-
specific Ag on neoplastic tissue, transplantation Ag, and CD Ag on T and B cells (via
Cell Flow Cytometry)

10. TYPES OF
IMMUNOFLUORESCENT ASSAYS
1. Direct Immunofluorescence Assay (DIFA)
 Tagged Ab is directly added to an unknown Ag fixed to a slide
 Requires incubation and a wash step
 Ag are seen as bright apple green or orange-yellow against a dark backgrounds
 Suitable for detection of specific Ag in tissue or body fluids
e.g. Legionella pneumophila, Pneumocystis carinii, Chlamydia
trachomatis, respiratory syncytial virus (RSV)

11. TYPES OF
IMMUNOFLUORESCENT ASSAYS
2. Indirect Immunofluorescent Assay
 Specimen with a known Ag is incubated on a solid phase
 Antihuman immunoglobulin (from mouse) tagged with a fluorophore is added
 Specimen Ab binds with antihuman Ab, forming a sandwich and localizing
fluorescence
 Used in Ab identification and detection of Treponema species-specific, antinuclear,
chlamydial, and toxoplasma Ab, and Ab to herpes simplex virus (HSV), Epstein-Barr
virus (EBV), and Cytomegalovirus (CMV)

12. TYPES OF
IMMUNOFLUORESCENT ASSAYS
ADVANTAGE (over DIFA):
a.) Uses only one Ab conjugate (tagged Ab) for different reactions, eliminating the
need for numerous purified, labeled reagent antibodies)
b.) More sensitive; has increased staining property. Multiple molecules bind to each
primary molecule.

13. TYPES OF
IMMUNOFLUORESCENT ASSAYS
3. Microimmunofluorescence
 Detects Ab in patient serum
 Has the same working principle as Indirect Immunofluorescence Assay but employs
Teflon slides with many wells dotted with Ag
 Used for:
a. Serodiagnosis of Q fever, Mediterranean Spotted Fever
b. Detection of IgG, IgA, and IgM Ab to Chlamydia, Toxoplasmosis, epidemic
Typhus, etc.

14. TYPES OF
IMMUNOFLUORESCENT ASSAYS
4. Quantitative Fluorescent Immunoassays (FIAs)
 can be classified as heterogenous or homogenous, depending on the type of
enzymatic immunoassays
 label is fluorescent; can be applied to either Ag or Ab
 Solid-phase heterogenous fluorescent assay is employed for the identification of
Ab to nuclear Ag, Toxoplasma Ag, Rubella Ag, and numerous other virus Ag
 Can also be used to detect important biological compounds
e.g. Cortisol, Progesterone, serum Thyroxine (T4)

15. TYPES OF
IMMUNOFLUORESCENT ASSAYS
 Homogenous FIA require no separation procedure; rapid and simple
• Has only one incubation step and no wash step
• Competitive binding is involved
• Fluorescent label changes as the conjugated Ag binds to specific Ab
• Changes include wavelength emission, rotation freedom, polarity, or dielectric strength
• Amount of fluorescence is proportional to the amount of Ag present; as Ag binding
increases, binding of fluorescent analyte decreases, which gives off more fluorescence

16. TYPES OF
IMMUNOFLUORESCENT ASSAYS
 Fluorescence Polarization Immunoassay (FPIA) – based on the change in
polarization of fluorescent light emitted by the fluorophore of a tagged Ab
• Incident light directed to a specimen is polarized with a lens or a prism; waves are
aligned at one plane
• Labelled molecules bound to an Ab rotates less and emits an increased amount of
polarized light
• Degree of fluorescence of polarization is INVERSELY PROPORTIONAL to concentration
of the analyte
• Limited to small molecules that tumble freely in the solution (< 2000 Daltons)
• Nonspecific binding of tagged Ab to other serum proteins occur, increasing polarization,
and falsely decreasing values

17. TYPES OF
IMMUNOFLUORESCENT ASSAYS
• Used mainly in determining therapeutic drug concentrations and hormone
concentrations
• Requires sophisticated instrumentations; basis for several automated analyzers

19. ADVANTAGE AND DISADVANTAGE OF
FLUORESCENT IMMUNOASSAY
ADVANTAGES DISADVANTAGES
• Has the potential of being highly sensitive and
versatile
• Separation of signal on the tag from the
autofluorescence produced by different
substance in serum
• Methodology is fairly simple • Nonspecific binding causes quenching;
fluorescence generated is changed
• No need to deal with or dispose of hazardous
substances

20. ADVANTAGE AND DISADVANTAGE OF
FLUORESCENT IMMUNOASSAY
• FPIA was introduced to overcome some of the problems
• Requires expensive dedicated instrumentation; limits its use in smaller laboratories

21. REFERENCES:
• Stevens, C. D.. (2010). Clinical Immunology & Serology in Laboratory Medicine, (3rd
ed.). Philadelphia, PA: F.A. Davis Company
• Sridar, R.P.N.. (2006). Immunofluorescence. Retrieved April 11, 2016, from
http://www.microrao.com/micronotes/immunofluorescence.pdf
• Abcam. (n.d.). Direct vs indirect immunofluorescence. Retrieved April 11, 2016, from
http://www.abcam.com/secondary-antibodies/direct-vs-indirect-
immunofluorescence