IMMUNOFLUORESCENCEBy GROUP II

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 thefluorophore used) when an incident light (usually UV light) is

introduced• Introduced by Albert Coons in 1941

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

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

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

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

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)

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)

TYPES OFIMMUNOFLUORESCENT 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)

TYPES OFIMMUNOFLUORESCENT 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.

TYPES OFIMMUNOFLUORESCENT 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.

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 compoundse.g. Cortisol, Progesterone, serum Thyroxine (T4)

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

TYPES OFIMMUNOFLUORESCENT 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

TYPES OFIMMUNOFLUORESCENT ASSAYS

• Used mainly in determining therapeutic drug concentrations and hormone concentrations

• Requires sophisticated instrumentations; basis for several automated analyzers

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

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

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