Immunofluorescence (IF) Test

Immunofluorescence (IF) Test

1. Dr. P. Saranraj
Head
Department of Microbiology
Sacred Heart College (Autonomous)
Tirupattur –635 601
Tamil Nadu, India
Mobile: [phone]; E.mail:
[email protected]
IMMUNOFLUORESCENCE
TEST

2. IMMUNOFLUORESCENCE TEST
In1944,AlbertCoonsshowedthatantibodies
couldbelabeledwithmoleculesthathavethe
propertyofFluorescence.
Fluorescentmoleculesabsorblightofone
wavelength(excitation)andemitlightof
anotherwavelength(emission).
Ifantibodymoleculesaretaggedwitha
fluorescentdye,orfluorochrome,immune
complexescontainingthesefluorescently
labeledantibodies(FA)canbedetectedby
coloredlightemissionwhenexcitedbylightof
theappropriatewavelength.
Antibodymoleculesboundtoantigensincellsor
tissuesectionscanbeviewedwitha

3. Fluorescent Dyes used in
Immunofluoresecence Test
Fluorescein-anorganicdyethatisthemost
widelyusedlabelforImmunofluorescence
procedures,absorbsbluelight(490nm)and
emitsanintenseyellow-greenfluorescence
(517nm).
Rhodamine–anorganicdye,absorbsinthe
yellow-greenrange(515nm)andemitsadeep
redfluorescence(546nm).
Phycoerythrin-anefficientabsorberoflight
(~30-foldgreaterthanFluorescein)andabrilliant
emitterofredfluorescence,stimulatingitswide
useasalabelforImmunofluorescence.

4. Direct and Indirect
Immunofluoresecence Test
DirectImmunofluoresecenceTest-Usesa
singleantibodythatisconjugateddirectlyto
aFluorescentdye(Fluorescein).Theantibody
recognizesthetargetmolecule,bindstoitand
theconjugatedfluorescentdyecanbedetected
bytheFluorescentMicroscope.
IndirectImmunofluoresecenceTest-Usestwo
antibodies.APrimaryantibodywhichrecognizes
thetargetbiomoleculesandbindstoitanda
SecondaryantibodyconjugatedtoaFluorescent
dye,whichrecognizesandbindstotheprimary
antibodyandindirectlylocalizesthetargetfor
detectionbytheFluorescentmicroscope.

5. Direct Immunofluoresecence Assay

6. Indirect Immunofluoresecence Assay

7. Applications of Immunofluoresecence
Immunofluorescencehasbeenappliedto
identifyanumberofsub-populationsof
Lymphocytes,notablytheCD4,CD8andT-
cellsubpopulations.
Thetechniqueisalsosuitableforidentifying
bacterialspecies,detectingAg-Abcomplexes
inautoimmune diseaseanddetecting
complementcomponentsintissues.
Localizationofhormonesandothercellular
productsstainedinsitu.
Localizationofantigensintissuesectionsorin
sub-cellularcompartments.